Article 1

Anthocyanins of açaí applied as a colorimetric indicator of milk 2

spoilage: a study using agar-agar and cellulose acetate as solid 3

support to be applied in packaging 4

Samiris Côcco Teixeira1, Taila Veloso de Oliveira1 , Lais Fernanda Batista1, Rafael Resende Assis Silva1, Matheus 5
de Paula Lopes2, Alane Rafaela Costa Ribeiro1, Thaís Caroline Buttow Rigolon3, Paulo César Stringheta3, Nilda de 6
Fátima Ferreira Soares1 7

1 Food Packaging Laboratory, Department of Food Technology, 8

Universidade Federal de Viçosa, Viçosa, Brazil. 9
2 Department of Food Technology, Federal University of Espírito Santo, Alegre, Brazil 10
3 Laboratory of Pigments and Bioactive Compounds, Federal University of Viçosa, Viçosa, Brazil. 11
* Correspondence: samiristeixeira@gmail.com; Tel.: +55 28 999010521 12

Abstract: Food that is still fit for consumption is wasted in the domestic environment every day, so 13
the development of technologies for food packaging in order to monitor the quality of the products 14
in real time is being developed. Highly perishable milk is currently one of the products that suffers 15
most from this waste due to its short shelf life. Therefore, Active Use By Date (AUBD) indicators 16
have been shown to discriminate between fresh and spoiled milk. Colorimetric indicators undergo 17
Citation: Teixeira, S.C.; de Oliveira, characteristic changes in their chemical structure, causing abrupt color changes, and the Chromatic 18
T.; Batista, L.F.; Silva, R.R.A.; de
Changes in Red (CCR) method has been used to quantify this change. Among the polymeric mate- 19
Paula Lopes, M.; Ribeiro, A.R.C.;
rials studied to function as solid support are cellulose acetate (CA) and agar-agar (AA). The AA 20
Rigolon, T.C.B.; Stringheta, P.C.; de
colorimetric indicator proved to be more suitable as a solid support due to its ability to maintain 21
Fátima Ferreira Soares, N. Anthocya-
the color change properties of the anthocyanin and its high colorimetric performance. The technique 22
nins of açaí applied as a colorimetric
indicator of milk spoilage: a study
was shown to be capable of indicating, in real time, changes in milk quality. 23
using agar-agar and cellulose acetate
as solid support to be applied in Keywords: Polymers; Shelf-Life; Food. 24
packaging. Polysaccharides 2022, 3, x. 25
https://doi.org/10.3390/xxxxx

Academic Editor(s):
1. Introduction 26
Received: date
For effective food distribution, important factors must be taken into consideration, 27
Accepted: date
such as the effects of processing, compliance with regulations that aim to ensure safety 28
Published: date
and quality, and consumer attitudes towards the product. As a consequence of the supply 29
Publisher’s Note: MDPI stays neu- chain not being adapted to these factors, it is estimated that approximately one-third of 30
tral with regard to jurisdictional all food produced for human consumption is lost or wasted [1]. 31
claims in published maps and institu- Thus, the use of technology can be useful in establishing a positive interaction be- 32
tional affiliations.
tween the consumer and the product, through the use of colorimetric indicators in smart 33
packaging has gained notoriety due to the simplicity and ease of communication with the 34
consumer [2–4]. Thus, the development of colorimetric indicators from biodegradable 35
Copyright: © 2022 by the authors. polymers has gained prominence. Biodegradable polymers are natural materials that 36
Submitted for possible open access when associated with substances capable of acting as colorimetric indicators, can integrate 37
publication under the terms and con- smart packaging to detect changes due to spoilage in foods [5–7]. Natural dyes such as 38
ditions of the Creative Commons At-
anthocyanins are sensitive to pH changes and widely used as food freshness indicators 39
tribution (CC BY) license (https://cre-
[3]. 40
ativecommons.org/licenses/by/4.0/).
Anthocyanins are non-toxic water-soluble pigments found in fruits and flowers [8]. 41
Indicators added anthocyanins can provide rapid qualitative information through visual 42
colorimetric changes caused by the structural change of the pigment. Thus, to indicate the 43
freshness of low shelf-life, nutrient-rich food products promoting food safety and quality 44

Polysaccharides 2022, 3, Firstpage–Lastpage. https://doi.org/10.3390/xxxxx www.mdpi.com/journal/polysaccharides

Polysaccharides 2022, 3, FOR PEER REVIEW 2

through their monitoring during the distribution chain and storage conditions, active use- 45
by date indicators have been developed [4,9–12]. 46
Active use-by date (AUBD) is a concept that is gaining prominence in research re- 47
lated to the development of food packaging technologies. Active use-by date or freshness 48
indicators can discriminate between fresh and spoiled foods, in products such as pasteur- 49
ized milk, by quantifying the color change by the Chromatic Changes in Red (CCR) 50
method [13]. The objective of this research is the development of a new colorimetric indi- 51
cator for the determination of milk freshness and the investigation of the influence of dif- 52
ferent polymeric matrices on color changes, using for this purpose, extract of acai antho- 53
cyanins incorporated into cellulose acetate and agar-agar polymeric matrices, to assist in 54
monitoring the quality of this product. 55

2. Materials and Methods 56

2.1. Materials 57
Cellulose acetate (GS = 2.5; MM = 2,024,000 g.mol-1). Acetone was supplied by Rho- 58
dia Solvay Group (Santo André, SP, Brazil). Lactic acid solution (≥85%), NaOH and agar- 59
agar were purchased from Sigma-Aldrich (Saint Louis, USA). The pasteurized milk was 60
purchased from the local trade in the city of Viçosa, Minas Gerais. 61

2.2. Açaí (Euterpe oleraceae Mart.) fruit acquisition 62

Açaí (Euterpe oleraceae Mart.) fruits were collected in three cities in Pará State, Brazil, 63
namely: Abaetetuba (Latitude: 1° 43' 42.1" S and Longitude: 48° 52' 11.5" W), Ilha das On- 64
ças Latitude: 1° 26' 34.4" S and Longitude: 48° 33' 10.5" W) and Cametá (Latitude: 2° 14' 65
21.8" S and Longitude: 49° 29' 54.3" W). The fruits were harvested, selected, washed and 66
had their seeds removed. The remaining parts (peel and mesocarp) were ground in a 67
blender with the addition of distilled water for 3 minutes at a ratio of 3:1 fruit/water. The 68
resulting homogeneous pulp was frozen in a deep freezer at -60 °C + 2 ºC until the moment 69
of analysis and used as raw material for the whole experiment. 70

2.3. The acquisition of the crude phenolic extract 71

The obtained crude phenolic extract was made according to Rocha et al. [14]. After 72
this period (session 2.2), the mixture was vacuum filtered in a Buechner funnel. Subse- 73
quently, the extracts were concentrated under vacuum using a rotary evaporator (IKA RV 74
10 digital) at a maximum temperature of 50 °C + 2 °C to evaporate the solvent (alcohol) to 75
a total soluble solids content (TSS) of approximately 7 ºBrix. The soluble solids content 76
(ºBrix) was obtained by direct reading in a Leica refractometer, model AR 200 (New York, 77
USA). This crude phenolic extract was stored in an amber jar under freezing in the ul- 78
trafreezer (approximately -60 °C) until analysis. 79

2.4. Characterization of the obtained extracts 80

2.4.1. Determination of total anthocyanins 81
The determination of total anthocyanins in acai extracts was performed by the single 82
pH method described by Fuleki & Francis [15]. The absorbance of the diluted solution of 83
the acai extract was performed in a UV-VIS spectrophotometer (Shimadzu 1601Pc) at a 84
wavelength of 535 nm. The results were expressed as mg of anthocyanins per 100 g of acai 85
pulp. 86

2.4.2. Total Phenolic Determination 87

The determination of total phenolics in acai extracts was performed according to the 88
methodology described by [16]. The absorbance of the diluted acai extracts was measured 89
at 760 nm using a Shimadzu (Kyoto, Japan) UV-VIS 1601Pc spectrophotometer. The 90
Polysaccharides 2022, 3, FOR PEER REVIEW 3

results were expressed as mg of gallic acid equivalent per mL of extract obtained (mg 91
EAG/100 g of acai pulp). 92

2.4.3. Colorimetric response of the extract to lactic acid 93

The chromatic characteristics of the anthocyanin extract (1%), adjusted to pH 7.0, 6.0, 94
5.0, 4.0, 3.0, and 2.0, were analyzed in the presence of lactic acid using an ultraviolet-visible 95
spectrophotometer (UV-2600, Shimadzu, Japan) over a wavelength range of 200 to 800 96
nm. 97

2.5. Film Preparation 98

2.5.1. Cellulose Acetate Films 99
A polymeric dispersion of CA in acetone at a ratio of 1:10 (w/v) was prepared. The 100
dispersion was sealed and kept at rest for 24h. After the elapsed time, 5 mL of anthocyanin 101
extract was added, followed by manual homogenization for 2 min. The final polymeric 102
dispersion was poured into 100 mm Petri dishes and solvent evaporation was performed 103
at room temperature (25 °C + 2 °C). The films were stored under vacuum in polyeth- 104
ylene/nylon packaging until the time of use. 105

2.5.2. Agar-agar Films 106

A 4% by weight agar-agar solution was dissolved in deionized water under constant 107
stirring at 60 °C + 2 ºC and mixed in 5 mg/L anthocyanin extract. Then, the dispersion was 108
poured into a 100 mm diameter petri dish, where it remained for solvent evaporation at 109
25 °C + 2 °C. The anthocyanin film was removed from the petri dish and cut into 10 mm 110
diameter circles. The films were stored under vacuum in polyethylene/nylon packaging 111
until use. 112

2.6. Scanning electron microscopy (SEM) 113

Micrographs of the films were obtained using a Scanning Electron Microscope 114
(Model TM3000, Hitachi Hi-Tech). Samples of each film, with dimensions of (0.2 x 0.5 115
cm2), were fixed on stubs with the aid of tweezers on a conductive double-sided carbon 116
tape (20 to 30 nm) (17). The electron accelerating voltage was used in automatic mode. The 117
magnification of the images obtained was 800x. 118
119

2.7. Colorimetric response of the film to lactic acid 120

The chromatic characteristics of the anthocyanin-incorporated films were analyzed 121
upon insertion into lactic acid solution adjusted to pH 4.0, 5.5 and 6.8 by adding NaOH 122
and lactic acid. After 15 minutes of exposure under the above conditions, the materials 123
were removed and photographed for digital colorimetric analysis. 124
125

2.8. Colorimetric Response of Milk Samples to Lactic Acid 126

The ability of anthocyanin films to discriminate between fresh and spoiled milk by a 127
color change was analyzed under the presence of lactic acid in milk at pH 6.8, 6.0, 5.5, 5.0, 128
4.5, and 4.0. The anthocyanin films were then submerged in 5 mL of the pH-adjusted milk. 129
After 15 minutes, the materials were removed and photographed for digital colorimetric 130
analysis. 131

2.9. Acidity and pH Analysis 132

Milk acidity was measured by the titration method, in which 10 mL of milk and 20 133
mL of sterile purified water were titrated with 0.1 mol/L NaOH at 25 °C + 2 °C, using 134
Polysaccharides 2022, 3, FOR PEER REVIEW 4

alcoholic phenolphthalein as a color indicator. The pHs were determined by adding 100 135
mL of water to 10 mL of milk, natural or pH-adjusted, and stirred to suspend milk parti- 136
cles. The tests were performed in triplicate. 137

2.10. Colorimetric analysis 138

The coordinates of the films were determined by digital colorimetric technique that 139
quantified the red chromatic change, according to the methodology Weston et al. [13]. 140
Images were captured using a smartphone combined with a 12 MP primary camera (f/1.8, 141
28mm, 1/3") under standardized conditions (working distance of 20 cm, horizontal illumi- 142
nation, and white background). Image-J software was used to interpret the images (35 x 143
35 pixels are). Red intensity (r) was calculated using the additive color system (R-red, G- 144
green, and B-blue), as represented by the following equation: 145

𝑅
𝑟= (1)
(𝑅 + 𝐺 + 𝐵)
In addition, the red chromaticity (CCR) of the samples was calculated using this 146
equation: 147

(𝑟𝑠𝑎𝑚𝑝𝑙𝑒 − 𝑟0 )
𝐶𝐶𝑅 = (2)
𝑟𝑚𝑎𝑥 − 𝑟0
Therefore, the CCR is determined by comparing the intensity of the red color under 148
exposure of the sensors with the natural pH of the milk (r0), about 6.6 - 6.8, and in the acid 149
phase (rsample), under the presence of lactic acid. In addition, rmax is the anthocyanin extract 150
at pH 2, a condition that provides the highest red intensity values. 151

2.11. Statistical Analysis 152

The data were analyzed by the Analysis of Variance (ANOVA) technique, and for 153
each response a regression curve was fitted. The statistical parameters of coefficient of 154
determination (R²) and lack of fit (Faj) were obtained (with significance of 5% probability) 155
when possible, taking into account the factors evaluated. All statistical analyses were per- 156
formed with the Minitab statistical program, version 17 and OriginPro 8.5. 157

3. Results and Discussion 158

3.1. Red chromatic changes for the acai anthocyanins extract solution 159
The açaí extract was obtained from three different municipalities in Pará state, Brazil 160
(Abaetetuba, Ilha das Onças and Cametá) and the values of total anthocyanins determined 161
were 76.13±14.77; 46.88±25.98 e 20.,3±6.70 respectively. Thus, for this study, we used the 162
third county extract, in which the concentration of anthocyanins determined was higher 163
than those, from different fruit sources, reported by several authors, such as 53.74 ± 1.75 164
mg in purple cabbage [13], 37.26 mg in radish [18] and 106 mg in blackberry [19). 165
The chromatic capacity of anthocyanin extracts was analyzed using a new technique 166
called chromatic change in red (CCR), based on the RGB values of sample images [13]. 167
According to Weston et al. [13], the chromatic transition of anthocyanins is usually moni- 168
tored by spectroscopic techniques, but it is limited to transparent solutions and therefore 169
this is not effective for various materials. Therefore, for visual detection and quantification 170
of the color change of anthocyanins, it may be possible to determine the red chromatic 171
response of the indicator using a smartphone app, which is an interesting technology to 172
gather real-time information on food conditions, very accessible, low cost and with excel- 173
lent accuracy [20). 174
As shown Figure 1A, the AE pH 2.0 solution exhibited a red color. As the pH in- 175
creases, the solution color changed to pink (pH 2.0), light yellow (pH 5.0), brown (pH 6.0) 176
and dark blue a (pH 7.0). The color changes can be attributed to the structural changes of 177
Polysaccharides 2022, 3, FOR PEER REVIEW 5

the anthocyanins present in acai, which are cyanidin 3-O-glucoside and cyanidin 3-O-ru- 178
tinoside [21). It can be observed through Fig. 1B, CCR decreased linearly (R² = 0.821) with 179
the increment of pH values after acidification with lactic acid. The anthocyanin extract can 180
be applied for incorporation into solid matrices to indicate milk freshness, since its color- 181
imetric changes were evident. 182

183
Figure 1. Photographs of the anthocyanin solution showing the colorimetric response to lactic acid 184
at different pH levels (A). Quantification of the color change of anthocyanin in response to lactic 185
acid as a function of the reduction in chromaticity in terms of CCR (B). 186

To achieve the ultimate goal of the study, the development of an active wearable date 187
using the different properties of CA and Agar was carried out to choose the optimal pol- 188
ymeric matrix that provides a visual colorimetric change of the sensor due to milk pH 189
modification. 190

3.2. Red Color Change Sensor on Cellulose Acetate (CA) 191

The color change and CCR of the CA indicator at different pH values are shown in 192
Figure 2 (A, B), respectively. Cellulose acetate is a biodegradable polymer that comes from 193
the acetylation of cellulose, the most abundant natural polymer, its use as a film making 194
material has been widely reported [17,22–26). 195
Polysaccharides 2022, 3, FOR PEER REVIEW 6

196
Figure 2. Photographs of the CA sensors with anthocyanins showing the colorimetric response to 197
lactic acid at different pH levels (A). Quantification of the color change of the AC sensor with an- 198
thocyanins in response to lactic acid by chromaticity in terms of R (CCR) (B). 199

The images of the CA colorimetric indicators, which were captured with the 200
smartphone camera and interpreted from Image-J software data, showed no relevant 201
changes in the case of pH changes. The CCR values started at 18,1320,576 at pH 4.5, with 202
a maximum point of 17.3440.148 at 5.5, which decreased slightly to 13.1640.054 at pH 6.8. 203
The slight color change of the CA sensors can be explained by analysis of the SEM 204
images (Session 3.4). The microscopic images show that there was a possible immobiliza- 205
tion of the anthocyanin molecules in the CA polymer matrix. Therefore, it shows no po- 206
tential for color change. This may have been triggered by the hydrolysis reaction of the 207
CA molecules, which leads to the release of acetyl groups, which may make the CA mol- 208
ecule reactive with the existing anthocyanin molecule. Several studies have reported that 209
hydrolysis of cellulose acetate can occur under different pH and temperature conditions. 210
The rate of hydrolysis can occur at different temperatures, including 23°C, and at different 211
pH values [27]. Due to the immobilization of the anthocyanin compounds in the polymer 212
matrix, there is no interaction with the water contained in the lactic acid solution, since 213
CA has a hydrophobic character compared to AA. Since the color change mechanism de- 214
pends on the pH-controlled ionization of the anthocyanin molecule, the system needs to 215
be hydrated [13,28,29]. 216

3.3. Red chromatic sensor changes Agar-Agar (CCR-AA) 217

Natural polymers, such as agar-agar, are biodegradable due to their chemical com- 218
position being predominantly composed of carbon and oxygen, unlike petroleum-derived 219
polymers, where they have predominantly carbon-carbon bonds [30). Agar-agar is a pol- 220
ysaccharide that constitutes the main structural component in the cell walls of algal spe- 221
cies belonging to the class Rhodophyceae (red algae). It is composed mainly of agarose 222
and agaropectin. The gelling fraction, AA, consists of repeating units of alternating groups 223
of β-D-galactopyranose and 3,6-anhydro-α-l- galactopyranose. Agaropectin has a similar 224
structure to agarose, but contains 5-10% sulfated esters and other residues, such as meth- 225
oxyl groups and pyruvic acid. 226
In contrast to the sensors made with CA, the AA sensors (Fig. 3A) showed similar 227
CCR values at pH 4.5 to 6.0 and markedly different at pH 6.8, at which the CCR values 228
dropped (Fig. 3B). Furthermore, the chromatic properties of the AA sensor determined in 229
Polysaccharides 2022, 3, FOR PEER REVIEW 7

this study can distinguish fresh milk from spoiled milk through the color transition from 230
brown to red. This fact can be attributed to the increased hydration caused by the polymer 231
matrix, which is composed of a polysaccharide, as it retains a large amount of water, thus 232
preserving the non-ionized species of anthocyanin molecules that have the potential to 233
change color [Weston et al., 2020). 234

235
Figure 3. Photographs of the AA sensors with anthocyanins showing the colorimetric response to 236
lactic acid at different pH levels (A). Quantification of the color change of the AA sensor with an- 237
thocyanins in response to lactic acid by chromaticity in terms of R (CCR) (B). 238

3.4. Scanning electron microscopy (SEM) 239

Figure 4 presents the SEM images of the fractured surfaces and cross sections of the 240
AA and CA sensors. Notably, it is possible to observe that both the agar-agar and cellulose 241
acetate films have agglomerates that may be associated with not fully dispersed com- 242
pounds, stemming from the production method. On the other hand, it is evident the pres- 243
ence of particles that are attributed to anthocyanins in CA films, which has a semi- 244
rounded shape without the formation of agglomerates, which occurs by the low interac- 245
tion between the components of anthocyanins, mostly hydrophilic, and CA having high 246
hydrophobicity [31). 247
Polysaccharides 2022, 3, FOR PEER REVIEW 8

248
Figure 4. SEM of the AA and CA sensors. The left micrograph shows the surface of the sensors and 249
the right micrograph illustrates the film cross section. All magnifications are 800×. 250

On the contrary, in AA films it was not possible to detect the presence of anthocya- 251
nins, reported previously in CA films, suggesting the successful incorporation of the ex- 252
tract, to which may be due to the interactions between the phenolic hydroxyl groups of 253
anthocyanins with the hydroxyl groups of the agar-agar, forming a semi continuous ma- 254
trix, this behavior was also reported in the development of active films of fish gelatin, 255
developed by Uranga et al. [9]. The cross-sectional surface of the films shows the presence 256
of particulates, more evident in the CA films, and the AA film, shows a smooth structure, 257
with small insoluble particles, absence of bubbles or grooves. 258

3.5. Detection of spoilage in milk 259

The Normative Instruction No. 76, of November 26, 2018, provides that raw refriger- 260
ated milk must present titratable acidity values between 0.14 and 0.18 grams of lactic 261
acid/100 mL of milk. To validate the sensors as a function of changes in acidity, the meth- 262
odology of the Manual of Official Methods of Analysis of Foods of Animal Origin, and 263
the results were expressed in Table 1[32). 264
Polysaccharides 2022, 3, FOR PEER REVIEW 9

Fresh milk showed a titratable acidity of 0.174 g/100g of lactic acid, which increased 265
to 0.3075 g/100g of lactic acid, suggesting deterioration of the milk. Higher concentrations 266
of lactic acid completely spoiled the milk, leading to pH change. The colorimetric indica- 267
tors, produced with cellulose acetate, did not differ the CCR values, remaining pink in 268
color, regardless of pH values (Figure 7A and Figure 8A). The colorimetric indicators, 269
made with agar-agar, showed a greater color change, from greenish blue to pink, when 270
the pH of the milk decreased from pH 6.8 to 6.0, being suitable to detect changes in pH of 271
milk (Figure 7B and Figure 8B). 272
273

274
Figure 7. The color behavior of the CA (A) and AA (B) sensors under milk acidification by the ad- 275
dition of lactic acid (excess milk was removed for better visualization of color changes when cap- 276
turing the photo). 277

278
Figure 8. Red chromatic shift index for the CA (A) and AA (B) sensor. 279

Table 1. Milk pH and acidity values. 280

pH grams of lactic acid /100mL

4.5 0.69768
5 0.54162
5.5 0.3672
Polysaccharides 2022, 3, FOR PEER REVIEW 10

6 0.30753
6.8 0.17442

4. Conclusions 281
This study provides a comparative study between solid supports for the develop- 282
ment of pH-sensitive colorimetric indicators incorporating anthocyanins extract into agar- 283
agar or acetate cellulose, which can be used to detect the freshness of numerous foods. 284
The incorporation step into polymeric matrices was proven by SEM analysis. The devel- 285
oped agar-agar colorimetric indicator can be used to evaluate the freshness of milk, since 286
there was a synchronicity between the color change and the increase in pH. Of the mate- 287
rials examined, agar-agar is cited as the ideal material with the best colorimetric perfor- 288
mance due to its hydrophilic character. The development of AUBD will reduce food waste 289
by decreasing the proliferation of greenhouse gases. Further work will be conducted to 290
make the effectiveness of the sensors more sensitive to minimal pH changes. 291

Author Contributions: Conceptualization, S.C.T., L.F.B., A.R.C.R., R.R.A.S. and T.V.O.; investiga- 292
tion, S.C.T.; resources, M.P.L and L.F.B.; data curation, S.C.T.; writing—original draft preparation, 293
S.C.T.; writing—review and editing, L.F.B., A.R.C.R., T.V.O. and M.P.L; supervision, A.J.D.S. and 294
C.V.; project administration, S.C.T. and T.V.O.; funding acquisition, T.C.B.R., P.C.S. and N.F.F.S. All 295
authors have read and agreed to the published version of the manuscript. 296

Funding: This work was supported by the Laboratory of Pigments and Bioactive Compounds, Fed- 297
eral University of Viçosa, Brazil; and the Department of Food Technology, Federal University of 298
Viçosa, Brazil. 299

Acknowledgments: The authors would like to thank CAPES, CNPq and FAPEMIG for financial 300
support. 301

Conflicts of Interest: The authors declare no conflict of interest. 302

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