Accepted Manuscript

pH-sensitive films containing anthocyanins extracted from black bean

seed coat and red cabbage

Luciana Prietto, Taiane Correa Mirapalhete, Vnia Zanella Pinto, Jessica

Fernanda Hoffmann, Nathan Levien Vanier, Loong-Tak Lim, Alvaro Renato
Guerra Dias, Elessandra da Rosa Zavareze

PII: S0023-6438(17)30151-2 DOI:

10.1016/j.lwt.2017.03.006
Reference: YFSTL 6079

To appear in: LWT - Food Science and Technology

Received Date: 19 August 2016

Revised Date: 16 February 2017
Accepted Date: 6 March 2017

Please cite this article as: Prietto, L., Mirapalhete, T.C., Pinto, V.Z., Hoffmann, J.F., Vanier,
N.L., Lim, L.-T., Guerra Dias, A.R., da Rosa Zavareze, E., pH-sensitive films containing
anthocyanins extracted from black bean seed coat and red cabbage, LWT - Food Science
and Technology (2017), doi: 10.1016/ j.lwt.2017.03.006.

This is a PDF file of an unedited manuscript that has been accepted for publication. As a
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 ACCEPTED MANUSCRIPT
1 pH-sensitive films containing anthocyanins extracted from black bean seed coat

2 and red cabbage

a* a a
4 Luciana Prietto , Taiane Correa Mirapalhete , Vnia Zanella Pinto , Jessica Fernanda
a a b a
5 Hoffmann , Nathan Levien Vanier , Loong-Tak Lim , Alvaro Renato Guerra Dias ,
a
6 Elessandra da Rosa Zavareze
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a
8 Department of Agroindustrial Science and Technology, Federal University of Pelotas,

9 96010-900, Pelotas, Brazil.

b
10 Department of Food Science, University of Guelph, Guelph, ON N1G2W1, Canada.
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12 * Corresponding author. Tel.: +55-53-32757284. Fax: +55-53-32757284.

13 E-mail address: lucianaprietto@gmail.com (L. Prietto)

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26 Abstract

27 The aim of this study was to develop pH-sensitive films based on corn starch and

28 anthocyanins extracted from black bean seed coat and red cabbage. The pH-sensitive

29 films were developed from solvent casting of polymer solutions containing corn starch,

30 glycerol, and anthocyanin extract (from red cabbage or black bean) prepared at pH 5.

31 The color of films changed from pink to purple and blue, as a function of the pH. The

32 pH-sensitive films were evaluated by their morphological, chemical, physical,

33 mechanical and thermal properties. In addition, the stability was evaluated during 28

34 days of storage (presence and absence of light; with and without cooling). The pH-

35 sensitive films with red cabbage anthocyanins showed a higher stability than that with

36 black bean anthocyanins when stored at room temperature and exposed to light. Both

37 pH-sensitive films exhibited greater color stability when stored under refrigeration as

38 compared to storage at room temperature.

39

40 Keywords: Intelligent packaging, color stability, light, pH, temperature.

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41 1. Introduction

42 Many intelligent packaging systems are capable of providing consumers with

43 quality information in real time for packaged food products (Rukchon et al., 2014). For

44 example, colorimetric pH indicator has been exploited in intelligent packaging due to its

45 easy to use, low cost, and well-characterize properties. It can be integrated into food

46 packaging structures to monitor the changes in acidic and basic components in food

47 products (e.g., CO2, organic acids, amines, ammonia), allowing consumers to check the

48 quality of the food as the indicator changes color (Silva-Pereira et al., 2015). The use of

49 natural polymers for the development of packaging has been widely investigated due to

50 their biodegradability and reduction of the accumulation of waste in the environment.

51 Corn starch is an interesting alternative for the development of packages because it is a

52 low cost polymer, widely distributed in nature, involves a simple process of obtaining,

53 being biodegradable, besides being able to be transformed into a thermoplastic material

54 in the presence of a plasticizer, with application of thermal and mechanical energy,

55 forming thin, flexible, transparent films, besides allowing the incorporation of

56 substances with specific properties such as anthocyanins.

57 Natural pigments such as anthocyanins can be added to biodegradable starch

58 films to provide the desirable functional properties of a pH indicator. Anthocyanins are

59 secondary metabolites widely distributed in fruits and vegetables (e.g., red cabbage,

60 sweet potato, bean husk, grapes), making them a promising source of natural indicator

61 that covered a broad color spectrum as a function of pH (Ananga et al., 2013). Several

62 studies investigated the use of biopolymers and anthocyanins for the production of pH

63 indicators. For example, Veiga-Santos et al. (2011) developed biodegradable pH

64 indicator films based on cassava starch plasticized with sucrose and invert sugar

65 containing grape and spinach extracts as sources of anthocyanin and chlorophyll,

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66 respectively. They reported that indicators containing the anthocyanin extract exhibited

67 greater color spectrum, and are more efficient for pH monitoring in comparison with

68 those containing chlorophyll or a mixture thereof. Silva-Pereira et al. (2015) developed

69 a pH indicator for monitoring the deterioration of fresh fish fillets, consisting of

70 chitosan, corn starch, and red cabbage extract. The indicator films exhibited optical and

71 morphological properties sensitive to the changes in pH as the product, suggesting that

72 the indicator was potentially useful as a quality indicator.

73 Anthocyanins derived from various sources may exhibit different intensity and

74 color stability due to their inherent differences in chemical structures (Ananga et al.,

75 2013). Studies related to the evaluation of time, temperature, and light effects on the

76 stability of pH indicators, especially those developed with anthocyanins, are limited.

77 The aim of this study was to develop a pH-sensitive film based on corn starch and

78 anthocyanins extracted from black bean seed coats and red cabbage leaves. The films

79 were evaluated for chemical structure, and their morphological, physical, mechanical

80 and thermal properties. The color stability of pH-sensitive films was evaluated during

81 28 days of storage at 4 C and 25 C under dark andunder light.

82

83 2. Material and methods

84 2.1 Material

85 Commercial corn starch (amylose 31%; supplier Unilever) was used for the

86 preparation of the films. Black beans and red cabbage, purchased in local market in the

87 city of Pelotas, RS, Brazil, were used for the anthocyanin extraction. The seed coats of

88 black beans were manually separated with aid of a scalpel. The red cabbage was cut

89 manually with a knife. Subsequently the samples of black bean seed coat and purple

90 cabbage, individually, were frozen with liquid nitrogen and milled in a ball mill

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91 (Marconi, MA 350, Brazil) to obtain a powder for subsequent extraction of the

92 anthocyanins (Davis et al., 2001). The pelargonidin standard was purchased from

93 Sigma-Aldrich, with 90% purity. The eluent was filtered with a nylon filter of 0.45

94 m.

95

96 2.2 Extraction of anthocyanins

97 The extraction and quantification of anthocyanins were carried out according to

98 the method described by Francis (1982). In falcon tubes, 1 g of black bean seed coat

99 sample or 10 g of red cabbage sample was added, 30 mL of acidified ethanol (85 mL of

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100 ethanol P.A. and 15 mL of 1.5 mol.L HCl) was added and submitted to constant

101 agitation (Phoenix, AP-22, Brazil) for 1 h. The supernatant was separated manually and

102 20 mL of acidified ethanol was added again in the sample, being subjected to constant

103 agitation. After the extraction, the solvent fractions were pooled, filtered and the

104 spectrophotometer readings (Jenway, 6705, England) were run at 525 nm for

105 quantification. The extracts of anthocyanins were diluted in acidified ethanol until
-1
106 reaching a concentration of 0.07 mg.mL , and then used to elaborate pH-sensitive

107 films.

108

109 2.3 Identification and relative quantification of anthocyanins

110 Liquid chromatography-mass spectrometry (LC-MS) analysis was performed on

111 a UFLC system (Ultra Fast Liquid Chromatograph Prominence, Shimadzu, Japan),

112 consisting of a degasser, a binary pump, an autosampler, and a temperature-controlled

113 column compartment. The mass spectrometer (Bruker micrOTOF Impacto HD, Bruker

114 Daltonics, Bremen, Germany) has a dual electrospray ionization source, with positive

115 ionization detection mode. Mobile phase A was consisted of 0.1% formic acid in water,

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116 while mobile phase B consisted of 0.1% formic acid in acetonitrile. Mobile phase

117 gradient used was as follows: 0 min - 5% B; 4 min - 80% B; 6 min - 80% B; 7 min -

118 15% B; and 15 min - 15% B. The equilibration time between successive runs was 5
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119 min. Other operating parameters were as followed: flow rate 0.4 mL.min ; injection

120 volume 1 L; column temperature 35 C.

121 MS analysis was carried out in positive ionization mode with spectra acquired

122 over a mass range of m/z 50 to 1200. The operating parameters were: capillary voltage
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123 4.0 kV; drying gas temperature 180 C; drying gas f low rate 8.0 L.min ; nebulizing gas

124 pressure 2 bar; collision RF 150 Vpp; transfer time 70 s; and pre-pulse storage 5 s.

125 Furthermore, automatic MS/MS experiments were performed by adjusting the collision

126 energy values as follows: m/z 100, 15 eV; m/z 500, 35 eV; and m/z 1000, 50 eV, using

127 nitrogen as the collision gas. The MS data was processed by Data Analysis Software 4.0

128 (Bruker Daltonics, Bremen, Germany), which provided a list of possible elemental

129 formulas.

130 The anthocyanins of the black bean seed coat and red cabbage were
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131 characterized by their mass spectra UV/Vis fragmentation patterns MS UV/Vis (220-

132 800 nm), and compared with data from the database (Metlin, MassBank, Kegg). The

133 calibration curve was developed by adding pelargonidin standard into the sample matrix

134 at different concentrations. Anthocyanins present in the samples were quantified using

135 the peak area of pelargonidin and its concentration.

136

137 2.4 Preparation of the pH-sensitive films

138 The pH-sensitive films were prepared by a casting technique, according to the

139 methodology described by Silva-Pereira et al. (2015), with some modifications. The

140 film solution was prepared from 3 g of commercial corn starch, 0.9 g glycerol and 80

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141 mL of distilled water. The film solution was subjected to heating in a water bath

142 (Fisatam, 550, So Paulo, Brazil) at 85 C for 15 m in. After cooling to 40 C 20 mL of

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143 anthocyanin extract at a concentration of 0.07 mg.mL (previously determined in

144 spectrophotometer) was added, followed by adjusting the solution to pH 5 using a

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145 NaOH 1 mol.L solution. The extract concetration used in this work was defined

146 according to preliminary test results. Subsequently the solution was homogenized in an

147 Ultra Turrax (IKA, T18 Basic, Germany) at 14,000 rpm for 10 min. Next, 20 g of the

148 film-forming solution was spread on a rimmed acrylic dish, 9 cm in diameter, and dried

149 in a convective air oven (Ethik, 420TD, Goinia, Br azil) at 30 C for approximately 16

150 h. The films were then conditioned at room temperature (16 C) and 60% RH for 48 h

151 before testing. The control film was prepared under the same conditions, except that the

152 anthocyanin extract was replaced by acidified ethanol.

153

154 2.5 Morphology

155 The morphologies of the surface and the cross section of the pH-sensitive films

156 were evaluated by a scanning electron microscope (Jeol, JSM-6610LV, USA), under an

157 accelerating voltage of 10 kV at 500 x magnification.

158

159 2.6 Solubility, thickness and mechanical properties

160 The solubility of the pH-sensitive films was determined according to Gontard et

161 al. (1994). The films were cut in a circle with a diameter of 2.5 cm and kept in an oven

162 at 105 C for 24 h. The samples were then immersed in 50 mL of distilled water in

163 falcon tubes and subjected to constant stirring on a horizontal shaker table at 175 rpm

164 and a temperature of 25 C for a period of 24 h. Af ter the resulting films were removed

165 from the tubes, and dried at 105 C until constant weight. The solubility was expressed

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166 in terms of solubilized mass, through the relation between the initial and final mass of

167 the films. Film thickness (mm) for each film was determined by taking ten

168 measurements using a digital micrometer. Mechanical properties of the film (tensile

169 strength and percent elongation) were measured using a texturometer (TA.XTPlus

170 Texture Analyzer, Stable Micro Systems) according to ASTM D882 method (ASTM,

171 1995).

172

173 2.7 Thermogravimetric properties

174 The thermogravimetric properties of the pH-sensitive films were evaluated using

175 a thermogravimetric analyzer (TGA TA-60WS, Shimadzu, Kyoto, Japan). Film samples

176 (6 to 10 mg) were weighed in a platinum dish and subjected to heating up to 600 C at a
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177 heating rate of 10 C.min , under constant nitrogen purge at 50 mL.min . An empty

178 platinum dish was used as a reference.

179

180 2.8 Color spectrum of the pH-sensitive films exposed to different pHs

181 The color spectrum of the films according to the different pHs was determined using a

182 colorimeter (MINOLTA, CR 400, Japan), at five random locations of the films. The

183 films were placed on a white standard background before measurement. Results were

184 expressed in terms of L* value (lightness; 0 is pure black and 100 is pure white),

185 chroma a* (- is green and + is red), and chroma b * (- is blue and + is yellow). The films

186 were exposed to different pH by cutting each one into 8 cm-diameter circles and

187 dividing each circle into 3 parts. Then, the parts were immersed in buffers with a pH

188 ranging from 1 to 10 and dried in an oven with forced air at 30 C for 15 min. The

189 change in color (E) of the film exposed at different pHs was calculated in comparison

190 to the film color at a pH 5 (as produced) using the Equation 1. In this study, the use of

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191 pH 5 was used to represent the pH close to food to which this film could be used as an

192 indication of quality, that is, for the development of intelligent packaging through pH

193 monitoring (future work).

= [

194 + + ]
Eq. (1)
195 L* = L* L 0*

196 a* = a* a 0*

197 b* = b* b 0*

198 Where E is change in color; L*, a* and b* are the color attributes of the films in each

199 pH value; and L0*, a0* and b0* are the color parameters of the reference sample.

200

201 2.9 Color stability

202 The pH-sensitive films were evaluated for their color stability as a function of

203 time, temperature, and lighting. The films prepared initially in pH 5 were exposed in

204 buffers with pH ranging from 1 to 10, dried in an oven with forced air at 30 C for 15

205 min and stored for 28 days at room temperature and under refrigeration, both with and

206 without an incidence of light. The color parameters of the films were monitored every

207 two days. The color change of the films during storage was calculated (Eq. 1) and

208 compared with the initial color of the films on the first day of storage.

209

210 2.10 Statistical analysis

211 Data were analyzed using an analysis of variance (ANOVA). Comparisons

212 between the means were analyzed using the Tukey test with a 5% significance level.

213

214 3. Results and discussion

215 3.1 Identification and relative quantification of anthocyanin compounds

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216 Seven anthocyanin structures were identified in the black bean seed coat extract

217 and six in the red cabbage extract (Table 1). Delphinidin-3-O-glucoside was the

218 predominant structure in the black bean extract, representing 40% of the total

219 anthocyanin content, while petunidin-3-O-glycoside, malvidin-3-O-glucoside, and

220 pelargonidin accounted for 29, 24, and 6%, respectively (Table 1). Other compounds

221 identified in the black bean extract were lower than 1%. These results are in agreement

222 with literature data, wherein delphinidin-3-glycoside is the main anthocyanin present in

223 beans (Choung et al., 2003).

224 In the red cabbage extract, pelargonidin was the largest component detected,

225 representing 71% of the total anthocyanin, followed by cyanidin-3.5-O-diglycoside

226 (14%), cyanidin-3-O-glycoside (7%) and cyanidin-3- (sinapoyl) (sinapoyl) -

227 diglycoside-5-glycoside (5%) (Table 1). According to Wiczkowski et al. (2013), the

228 basic anthocyanin identified in red cabbage are cyanidin-3.5-diglycosides, where the

229 glycoside moieties can be nonacylated, monoacylated, or diacylated. The sinapic

230 phenolic acids, ferulic, caffeic and -coumaric are the main acids linked to these

231 structures. The difference between the results observed in this study, as compared to the

232 literature, can be attributed to the fact that the estimation of the concentration of

233 anthocyanins in the present study was based on the spectral pattern of the pelargonidin.

234 In the present study both the relative quantification by pelargonidin and the peak area

235 observed for each compound in the HPLC-MS analysis were presented in Table 1. Only

236 the red cabbage extract showed the presence of acylated molecules (cyanidin-3-

237 (sinapoyl) (sinapoyl)-diglycoside-5-glycoside).

238 The sum of the anthocyanins quantified in the black bean sample (440,4 g/g) was

239 approximately 3 times higher than those quantified in the samples of red cabbage (146,9

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240 g/g). This will imply a greater amount of cabbage sample needed for the development

241 of the pH indicator films.

242

243 3.2 Morphology

244 The morphologies of the surface and cross section of the pH-sensitive films, with

245 and without black bean and red cabbage anthocyanins, are shown in Figure 1. The film

246 with the red cabbage anthocyanins (Figure 1E) showed a rough surface. By contrast,

247 films with black bean anthocyanins showed more homogenous surface and cross section

248 morphologies than other film treatments (Figures 1C and 1D, respectively). The

249 roughness and unevenness of the films (Figures 1E and 1F) can be attributed to a lower

250 interaction of the anthocyanin compounds with starch and glycerol constituents.

251 Luchese et al. (2015) developed films with pinho starch and jabuticaba flour, as the

252 anthocyanin source, and observed films of roughened surfaces. However, they reported

253 that the surface roughness did not influence in the color variation of the film as a

254 function of the pH.

255

256 3.3 Solubility, thickness, and mechanical properties

257 The incorporation of anthocyanins in pH-sensitive films increased their

258 solubility in water, attributable to an increase in the number of hydrophilic sites

259 available for water absorption after interactions between the starch and anthocyanins.

260 The pH-sensitive film with black bean anthocyanins had a higher solubility compared to

261 the film containing red cabbage anthocyanin (Table 2). According to Borkowski et al.

262 (2005), the glycosylation of anthocyanins increases the water solubility, whereas

263 acylation decreases the solubility. Therefore, even though diglucoside compounds are

264 present among the anthocyanins found in red cabbage, while monoglucoside

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265 compounds are predominant in the black beans anthocyanins, we theorized that the red

266 cabbage anthocyanins are acylated, and hence are responsible for their lower water

267 solubility observed. Because pH-sensitive films have shown high solubility in water, a

268 strategy for their application in the development of intelligent packaging is to avoid

269 direct contact with food if it has a high amount of water, such as meat. In this way, it

270 can be fixed to the top of the package and only contact with volatile compounds

271 released by the product.

272 The thickness of the films increased with the addition of anthocyanins (Table 2).

273 The thickness of the films is directly proportional to the concentration of solids in the

274 formulation. Thus, the presence of anthocyanins would have contributed to the increase

275 in film thickness. The addition of black bean anthocyanins reduced the tensile strength

276 of the films compared to the control film while the addition of the red cabbage

277 anthocyanins did not significantly affect this property (Table 2). The tensile strength

278 was inversely proportional to solubility, possibly due to the plasticization effect of

279 sorbed water and/or the anthocyanin molecules that contributed to the lowering of

280 tensile strength. Moreover, when the film is formed only with starch, after the

281 gelatinization, the molecules form a three-dimensional network organized with

282 intermolecular interactions. However, the presence of anthocyanins can weaken the

283 intermolecular interactions and thus affect the mechanical properties of the films.

284 Pereira et al. (2015) developed a temperature-time indicator film with chitosan,

285 poly(vinyl alcohol), and red cabbage anthocyanins. They reported that the tensile

286 strength of the anthocyanin-containing films decreased when compared to the control

287 film, showing that interactions between anthocyanins and polymers influence the film

288 resistance. The elongation of the films was not affected by the presence of

289 anthocyanins.

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