Halochromic inks applied on cardboard for food spoilage monitorization

Citation: Leite, L.; Boticas, I.; Navarro, M.; Nobre, L.; Bessa, J.; Cunha, F.; Neves, P.; Fangueiro, R. Halochromic Inks Applied on Cardboard for Food Spoilage Monitorization. Materials 2022,15, 6431. https://doi.org/10.3390/ ma15186431 Academic Editors: Ezgi Pulatsu and Zhilong Yu Received: 26 July 2022 Accepted: 12 September 2022 Published: 16 September 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). materials Article Halochromic Inks Applied on Cardboard for Food Spoilage Monitorization Liliana Leite 1,2, Inês Boticas 1,2, Miguel Navarro 1,2, Luís Nobre 1,2 , João Bessa 1,2,* , Fernando Cunha 1,2 , Pedro Neves 3and Raúl Fangueiro 1,2 1Centre for Textile Science and Technology (2C2T), University of Minho, 4800-058 Guimaraes, Portugal 2Fibrenamics-Institute of Innovation on Fiber-based Materials and Composites, University of Minho, 4800-058 Guimaraes, Portugal 3JoséNeves & Cia., Lda., Parque Industrial de Ponte 1ª Fase, Lote F, nº 277, 4801-911 Guimaraes, Portugal *Correspondence: [email protected] Abstract: Control of food spoilage is a critical concern in the current world scenario, not only to ensure the quality and safety of food but also to avoid the generation of food waste. This paper evaluates a dual-sensor strategy using six different pH indicators stamped on cardboard for the detection of spoilage in three different foods: beef, salmon, and strawberries. After function validation and formulation optimizations in the laboratory, the halochromic sensors methyl orange and bromocresol purple 2% (w/v) were stamped on cardboard and, in contact with the previously mentioned foods, were able to produce an easily perceptible signal for spoilage by changing color. Additionally, when it comes to mechanical characterization the inks showed high abrasion (>100 cycles) and adhesion resistance (>91%). Keywords: smart materials; intelligent packaging; halochromic inks; food packaging; food quality; food spoilage 1. Introduction One of the most concerning facts in today’s society is the quick population growth that has taken place in recent years. It is estimated that by the year 2050, the world population will reach 9.7 billion [ 1 ]. In line with the previous indicator, the food demand has increased 50% in the last 50 years, and it is expected to increase from 70 to 110% by 2050 [ 2 ]. Associated with this problem, there are numerous sub-problems that have a negative impact on general welfare and the environment, such as food waste and spoilage. According to the UNEP Food Waste Index 2021, around 931 million tons of food waste were produced in 2019 [3]. Furthermore, in 2016, the Food and Agriculture Organization of the United Nations stated that 70% of the total food waste in the EU arises in the household, food service, and retail sectors, and the remaining 30% in the production and processing sectors [ 4 ]. Food storage and delivery to supermarkets have increased in popularity. During those processes, due to the time and conditions in which they are handled, transported, and stored, the food products begin to dehydrate, deteriorate, and lose their color, appearance, taste, and more importantly their nutritional value [ 5 ]. The packaging process and type of package used are key points in the conservation and preservation of food quality for a longer period of time, and therefore in the decrease in food spoilage [ 6 , 7 ]. The EFSA defines intelligent packaging as materials/articles that “monitor the condition of packaged food or the environment surrounding the food” [ 8 ]. It is a way to reduce food waste, by extending the shelf life, monitoring freshness, and exhibiting information about its quality [ 6 ]. In addition, at the consumer level, there is a constant demand for information, namely the product history, its location, or the conditions to which the product has been subjected [ 6 ]. Among intelligent packaging, there are three common technologies, namely data carriers, indicators, and sensors [ 9 ]. Indicators determine the presence or absence of Materials 2022,15, 6431. https://doi.org/10.3390/ma15186431 https://www.mdpi.com/journal/materials Materials 2022,15, 6431 2 of 15 a given substance, its concentration, and/or the extent of a given reaction. Additionally, they are able to show all these variations through direct changes, such as color [ 10 ]. One critical point in the assessment of food spoilage is the presence of microorganisms and the degradation of food quality. This control can be performed indirectly, i.e., by detecting bacterial growth products, or by detecting variations in the conditions of the medium resulting from the presence of pathogens, such as changes in pH, temperature variations, or water activity. In the situations previously mentioned, the presence of microorganisms and degradation of food quality, there is a common factor: the variation of pH in the food and/or its surroundings. For example, during the spoiling process of meat and fish, there can be an increase in total volatile basic nitrogen (TVBN), which leads to a more basic pH inside the package [ 11 , 12 ]. A research study conducted by Kuswandi and Nurfawaidi (2017) showed the application of methyl red and bromocresol purple used separately as pH sensors for the detection of red meat spoilage [ 11 ]. In the case of milk, there is a decrease in pH due to the presence of lactic acid, a by-product of bacterial growth [ 13 ]. A study conducted with apples has shown the correlation between apple ripeness, alteration of texture, and flavor and the pH measured as a consequence of aldehyde emission. In this research, the application of a sensor based on methyl red for the detection of aldehyde was demonstrated [ 14 ]. Hence, the use of an on-package colorimetric and pH-responsive sensor is a sensitive, simple, user-friendly, and cost-effective alternative to monitor food freshness. For the present study, the focus was to develop a solution to monitor microbiological activity as well as food degradation using functionalized inks with pH indicators directly printed on cardboard, for further application on the inside of food packages. For this a dual-sensor approach was tested, using a combination of two pH indicators with sensitivity for different areas of the pH scale. The use of two different indicators combined in the same solution allows a higher accuracy of the results, avoiding false positives [ 11 ]. The pH indicators used in this work were bromothymol blue (BB), methyl red (MR), methyl red sodium (MRs), methyl orange (MO), and bromocresol purple (BP). The cardboards functionalized with these pH indicators were able to change their color, depending on whether they came into contact with acidic or alkaline solutions. Furthermore, the cardboard printed with a double sensor exhibited the capacity to detect the spoilage of three different foods, fish, meat, and fruit. Thus, this study demonstrated the potential of pH-sensitive inks on cardboard packages as a tool for signaling food deterioration and/or microorganism proliferation. 2. Materials and Methods 2.1. Materials Methyl red (C 15 H 15 N 3 O 2 ), Methyl red sodium salt (C 15 H 15 N 3 O 2 Na), Methyl orange (C 14 H 14 N 3 NaO 3 S), bromocresol purple (C 21 H 16 Br 2 O 5 S), and bromothymol blue (C 27 H 28 Br 2 O 5 S) were purchased from Sigma-Aldrich. The polymeric base for ink preparation was provided by JoséNeves & Cia., Lda (Ponte, Portugal), as well as the cardboard. The filter paper, used as substrate at the beginning of this work, was purchased from Normax. 2.2. Preparation of pH-Sensitive Aqueous Solutions and Application on Paper Substrates The pH-sensitive solutions were formulated exclusively thinking about their potential for food monitorization. The selection of bromothymol blue (BB), methyl red (MR), methyl red sodium (MRs), methyl orange (MO), and bromocresol purple (BP) indicators were based on previous research works [ 11 , 14 , 15 ]. Three different combinations of these indicators were tested to prepare the final indicator solutions. The first, where the indicators MR:MRs were combined in a ratio of 1:1; the second, combined MO:BP with the same ratio (1:1); and the last, which was a combination of MR:BB in a ratio of 3:2. The ratios used were also based on the previous research cited above. The preparation of the solution requires distilled water and magnetic stirring (300 rpm). The concentration of the indicators in water was 0.5, 1, and 2% (w/v). The 1 and 2% (w/v) concentrations were used to study the influence of concentration on the time-response of substrates. After 1 h of mixing, the Materials 2022,15, 6431 3 of 15 solution was passed to a Petri dish where the filter paper was immersed for another hour. Following that, the filter paper was removed and dried at room temperature. 2.3. Preparation of pH-Sensitive Inks and Application on Cardboard Substrates The conditions used for ink preparation were similar to those given above for aqueous solutions. At this stage, the aqueous base has been replaced by the polymer base. The polymer used was liquid with a varnish base and had a viscosity between 200 and 300 mPa/s, required to carry out the printing technique. Furthermore, this polymeric solution was selected in order to meet JoséNeves’ requirements for further industrial application by flexography. The combinations of the above-referred indicators were dissolved in the polymer maintaining vigorous agitation for 1 h. The inks were then ready to print on the cardboard substrates using stamping or flexography techniques. For each sample, one layer of ink was used, and all samples were dried at room temperature. The cardboard samples obtained can be seen in Figure 1. Figure 1. Cardboard samples after printing with pH-sensitive inks functionalized with ( a ) MR:MRs 1% (w/v), (b) MR:BB 1% (w/v), (c) MO:BP 1% (w/v), and (d) MO:BP 2% (w/v). 2.4. Characterization of pH-Sensitive Aqueous Solutions and Paper Substrates for Color Color characterization was carried out in aqueous solutions and filter paper substrates. Initially, a direct contact assay was performed. To cover the required pH range from 3 to 12, two different solutions were used. To achieve an acidic pH, a solution of 1% (w/v) of citric acid in water was made and added dropwise until reaching the desired pH. On the other hand, 1% (w/v) monosodium phosphate (NH 2 PO 4 ) aqueous solution was used to obtain an alkaline pH. After achieving the required pH, the color of all samples was evaluated using the RGB color model. The influence of concentration on color and time-response of the samples was also evaluated. As mentioned in Section 2.2 filter paper substrates were functionalized with pH-sensitive solutions in concentrations 0.5, 1, and 2% (w/v). The samples were then placed on top of Petri dishes filled with 3 mL of pure ammonia for 300 s, without direct contact. After that, RGB color coordinates were measured. ImageJ software was used to determine the RGB (red, green, and blue) values from the images. The RGB model is a combination of the different amounts of red, green, and blue primary lights to produce a variety of colors. Each of the primary colors corresponds to a value between 0 and 255. When all coordinates correspond to 0 the color black is obtained, and if they correspond to 255, the color white is achieved [ 16 ]. Image acquisition was performed in environmental conditions with a digital camera Canon PowerShot SX530 HS, with 16 megapixels resolution and 4.3 mm focal length. 2.5. Adhesion and Abrasion Properties of Cardboard Substrates The determination of the abrasion resistance of printed materials consisted of using a circular cardboard sample from the area where the ink was applied with a diameter of 127 mm. The Martindale equipment was used in this article to simulate the constant Materials 2022,15, 6431 4 of 15 abrasion that the cardboard would undergo in transportation, storage, and other normal processes. In this specific case of packaging, a printing universal paper sheet with approximately 80 g/m 2 was used as a base pattern on which 100 abrasion cycles were performed. A cycle refers to one full rotation with a pressure of 9 kPa and a rotational frequency of 44.5 ±2.4 min−1. This method was adapted from the standard ASTM D5264 [17]. The adhesion method covers the procedures for evaluating coating adhesion to substrates. The approach used was an adaptation of methodology B mentioned in standard ASTM D3359 [ 18 ]. In this sense, 2.5 cm × 2.5 cm squares were drawn in a test area equal to 6.25 cm 2 , resulting in a grid of 25 squares. It is important to bear that the test must be carried out in an area where the coating is as uniform as possible. Afterwards, the test area was lightly pressed with a cutting element. After the test area was well delineated, a section of tape was placed diagonally over the grid and left there for 90 s. Then, the tape was removed at an angle of 180º through a single movement. The analysis grid was evaluated before and after in ImageJ software to calculate the percentage of coating removed from the substrate. 2.6. Evaluation of Cardboard Substrates’ Sensitivity to Gaseous Atmospheres To assess the sensitivity of the prepared cardboard samples to the presence of gases (acidic and alkaline), a set-up was prepared in which each sample was left for 5 min to guarantee the formation of a saturated environment. The set-up constructed consists of a combination of a flask containing 2 mL of acid or base liquid and a 250 mL glass beaker covered with parafilm (Figure 2). Since the base (ammonia) and/or acid (HCl) used are volatile, the gaseous environment is easily created. After 5 min, the color response was recorded. To understand the change in colors the RGB coordinates were measured using ImageJ software, before and after exposure. Image acquisition was performed as described in Section 2.4. Figure 2. Developed set-up to evaluate the gas sensitivity of the samples. 2.7. Cardboard Detection of Food Spoilage After exposing the sensors to environments saturated with acidic and alkaline chemical agents, a test was run to simulate real-life conditions. The indicator methyl orange + bromocresol purple (MO:BP) was used in this test at concentrations of 1% (w/v) and 2% (w/v). These functionalized cardboard samples were then placed in an isolated environment with three different foods: 75 g of fresh salmon, 75 g of beef steak, and strawberry. The assay was performed until the pH sensor changed color or the food showed visible signs of degradation (change in smell and color). A control condition, in which the functionalized cardboard is subjected to a closed environment in the absence of food and under the same temperature and humidity conditions, was also introduced. The experiment was carried out at room temperature (approximately 20 ◦ C). The color of the samples was evaluated on the first and last days of the trial, and RGB coordinates were obtained using ImageJ software. Image acquisition was performed as described in Section 2.4. Materials 2022,15, 6431 5 of 15 2.8. Statistical Analysis Statistical analysis was performed to compare the color coordinates in the assays using cardboard samples. The results are presented as the average of three replicates performed, and the respective standard deviation. The results were analyzed using a one-way ANOVA test and multiple comparisons were performed using Šídák’s test. Statistical analysis was achieved using GraphPad Prism 6 Software. A critical value for significance of p< 0.05 was used throughout the study. 3. Results and Discussion 3.1. Measurement of RGB Coordinates of pH-Sensitive Aqueous Solutions Considering the functional property of the mentioned indicators, after the preparation of the solutions with a concentration of 0.5% (w/v), the color change induced by changing the pH was tested. A study of the RGB color coordinates obtained for each of the prepared indicator mixture solutions was carried out, which is presented below in Figure 3. Figure 3. Graphical correlation of the aqueous solution color changes in response to pH. Results obtained for ( a ) MR:MRs 0.5% (w/v), ( b ) MR:BB 0.5% (w/v), and ( c ) MO:BP 0.5% (w/v) are expressed in RGB coordinates and the color obtained for every pH value is represented. It is possible to verify that with the variation of the pH, the RGB coordinates also change. However, it is noticeable that, for all sensors, the colors presented at some pH values are very similar, with close RGB values. This means the indicators have a similar response to those pH values, and, as seen by image representation, the color difference is not visible to the naked eye. For MR:MRs (Figure 3a), the highest variation in the RGB coordinates occurs between the values of 5 and 6, within the working range of these pH indicators [ 19 ]. The color changes observed were as expected and are due to a shift in the maximum wavelength absorbed by the compounds. In acid conditions methyl red strongly absorbs at a wavelength of approximately 515 nm, and when subjected to a basic pH, due to deprotonation, this compound has a peak at approximately 431 nm. For the curve correspondent to MR:BB solutions the same was observed. However, in this case, a higher slope was visible from pH 5 to 6, showing a change in the color spectrum, Materials 2022,15, 6431 6 of 15 from orange to green. This result is explained by the reference work zone of the used indicators, which are integrated into the pH range of 4.4 to 6.0 for MR and pH 6.0 to 7.6 for BB [ 20 ]. In this case, the color changes are due to methyl red’s absorption profile described above, and to bromothymol blue, which absorbs at 433 nm when in contact with an acidic solution and at 615.5 nm at an alkaline pH [21]. For MO:BP solutions, the highest variation of RGB coordinates is seen between pH 4 and 7. For a pH of 7 and 8, the colors obtained are quite identical, and for the lower end of the pH scale, the same was verified. This was to be expected since the turning point of methyl orange is from 3.1 to 4.4 [ 22 ], and the pH range where bromocresol purple changes its color is between 5.2 and 6.8 [ 23 ]. MO changes its color due to a hypsochromic shift in maximum absorption wavelength from 508 nm to 466 nm, in acid and basic conditions, respectively [ 24 ]. On the other hand, bromocresol purple strongly absorbs at a wavelength of approximately 430 nm in acidic conditions and demonstrates a shift to 590 nm in basic solutions [23]. 3.2. Measurement of RGB Coordinates of pH-Sensitive Paper After the preparation of the solutions, they were applied on filter paper to measure the absorption efficiency of the indicators. These samples were also subjected to an analysis of RGB components. Following the application of the solutions on filter paper, the study of the correlation between pH and color obtained by the paper samples was carried out. As can be seen in Figure 4, it was immediately noticeable for all sensors a change in the RGB coordinates after application of the acid and base solutions in the filter paper. Thus, it can be stated that the color of the substrate used will have a direct influence on the obtained final color. Although aqueous solutions have exhibited lower RGB coordinates, the variation remains similar to those obtained for paper substrates. This difference is probably due to the lower concentration of the solutions, which makes them slightly more translucent. When applied to paper, the color is much more compact and coherent, and also lighter due to the introduction of the white of the substrate. This is proved by the increase in the number of RGB coordinates, corresponding to a lighter color. Figure 4. Graphical correlation of RGB color coordinates and the pH of filter paper samples functionalized with aqueous solutions of ( a ) MR:MRs, ( b ) MR:BB, and ( c ) MO:BP at a concentration of 0.5% (w/v), and (d) corresponding color palette. Materials 2022,15, 6431 7 of 15 As shown in Figure 4d, it was possible to obtain a color palette with a coherent correlation between pH and color. As can be seen, by varying the pH of the solution, the color presented by the substrate also changed. This result allowed assessing the response of the pH indicators to changes in their surrounding environment. For example, looking at the paper substrate with MO:BP at pH 7 it is possible to detect a brown color, but when the sample was placed in contact with an acidic environment its color tended to change to orange. Considering these results, and since food spoilage is characterized by a change in its atmospheric pH, the relation obtained can be easily extrapolated to a label that could inform consumers about the deterioration and/or microorganisms’ presence in food. To corroborate this sentence and to optimize the sensor concentration, it was necessary to make other assays where the time and response of the substrates were measured. 3.3. Influence of Concentration on Color and Time-Response of the Samples To evaluate the influence of concentration in the time-response of the substrates to gaseous environment detection tests were carried out. For this, three Petri dishes were prepared in which 3 mL of pure NH 4 were placed. Subsequently, the paper samples were placed on the plates, without direct contact with the solution, spacing about 1 cm (Figure 5). Figure 5. Illustration of the set-up used for detection of ammonia gases by substrates functionalized with pH indicators MR:MRs for 0.5, 1, and 2% (from left to right). The RGB color model was once again applied to measure the color difference between the samples over 300 s. Thus, it was possible to confirm the indicator concentration influence on the RGB value presented by each sample. As can be seen in Figure 6, with the increase in the concentration, the RGB coordinates decrease. Since the value of the RGB coordinates decreases towards 0, corresponding to black, it is understood that the higher the solution concentration the darker the sample will become. As shown in Figure 6, it was also found that the response time of different concentration samples is similar, since the change in their color happens at equal times. However, to the naked eye, this change is not perceptible, and because of that, it gives the illusion that the response time was different. For MR:MRs (Figure 6a), if monitoring the 2% (w/v) sample, the color changes from yellow to red appears to be faster than for a lower concentration, such as 0.5 and/or 1%. This perspective is entirely because the variation in number of the color coordinate is higher, making the detection more perceptive to the naked eye. The same was verified for the remaining combinations of indicators, MR:BB and MO:BP. Materials 2022,15, 6431 8 of 15 Figure 6. Influence of the concentration of indicators on the color change of ( a ) MR:MR-functionalized substrates, ( b ) MR:BB-functionalized substrates, and ( c ) MO:BP-functionalized substrates. Results are expressed in RGB color coordinates. It is concluded that the concentration used has a direct influence on the color hue presented by the samples, being darker as more concentrated. However, the time-response is the same, with variation in RGB coordinates at similar times. The detection by the naked eye is the more visible the higher the concentration. 3.4. Adhesive and Abrasive Properties Adhesive and abrasive properties were analyzed after cardboard functionalization with the sensors MR:MRs, MR:BB, and MO:BP. The abrasion property has a great influence on cardboard substrates, especially if they are used for packaging. When the product has high abrasion resistance and does not appear to wear out after several cycles, it is considered more robust and usually lasts longer [ 25 ]. Additionally, when it comes to packaging the inks need to have high abrasion resistance and adhesive properties to maintain the information visible to the consumer or distributor throughout its life cycle and, at the same time, preserve the aesthetic appeal of the package. The results obtained for abrasion resistance can be evaluated by visual analysis of the wear surface and/or by the mass difference before and after abrasive cycles (Figure 7A). Looking at the images presented in Figure 7A, it can be seen that the wear resistance of the cardboard samples is quite similar at 0 and 100 cycles. For the three sensors, it is not possible to visualize any changes in color or uniformity of the surface of the samples. In addition, watching the graph representation of the sample’s weight before and after cycles is shown that the achieved differences were not significant. Thus, it was concluded that the samples have high wear resistance for 100 cycles. Materials 2022,15, 6431 9 of 15 Figure 7. Results obtained for the ( A ) abrasion resistance of the samples ( 1 ) MR:MRs 1% (w/v), ( 2 ) MO:BP 1% (w/v), ( 3 ) MR:BB 1% (w/v), and ( 4 ) weight of samples before and after 100 abrasive cycles. Results obtained for ( B ) adhesion test of the samples ( 1 ) MR:MRs 1% (w/v), ( 2 ) MO:BP 1% (w/v), and ( 3 ) MR:BB 1% (w/v). The ( B4 ) percentage of the adhesion was calculated by the difference between total area and removed area. Adhesion is another important property in packaging products. It evaluates the resistance to a small peeling force of a coating on a cardboard surface. If the coating is weakly bonded to the substrate, then it can be easily pulled off and the adhesion is considered weak [ 25 ]. The adhesion test used follows an adaptation of the ASTM D3359 standard. In the results presented in Figure 7B), it is observed that the adhesion of the pH-sensitive ink was very similar between samples, before and after pulling off the tape. The analysis of the adhesion percentage is calculated by the difference between the removed area after the test and the total area (before the test), using ImageJ software. Therefore, it is clear that the sample with the lowest adhesion was the one functionalized with the mixture of MR:MR indicators, with a value of 91.1%. Thus, all samples present a good adhesion resistance with no significant differences in surface color and uniformity, and a good level of adhesion greater than 90%.