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. 2019 Jun 24;56(9):4147–4157. doi: 10.1007/s13197-019-03885-5

Spray-dried microcapsules of anthocyanin-rich extracts from Euterpe edulis M. as an alternative for maintaining color and bioactive compounds in dairy beverages

Emília Maria França Lima 1, Marina Carvalho Martins Madalão 1, Walter Correa dos Santos Jr 1, Patrícia Campos Bernardes 1, Sérgio Henriques Saraiva 1, Pollyanna Ibrahim Silva 1,✉
PMCID: PMC6706512  PMID: 31477986

Abstract

Color is a sensory attribute that influences the acceptance of food and dyes are added into food products to provide them attractiveness. In this context, anthocyanins have merged as an alternative to synthetic dyes. This study aimed to develop food model systems of fermented or unfermented dairy beverages containing added microencapsulated anthocyanin-rich extracts from juçara palm fruit. The stabilities of both pigment and beverage throughout storage in opaque or transparent packaging have been evaluated. Acidity, pH and anthocyanin content in both beverages did not vary during 28 days of storage, and the content of bioactive compounds did not decrease over time. A slight overall color difference that is probably invisible to naked eyes was detected between the beverages analyzed at days 0 and 28. The potential of applying microencapsulated natural pigments into dairy matrices is an effort to increase their nutritional and sensorial values.

Keywords: Antioxidant activity, Phenolic compounds, Natural dyes, Microcapsules, Dairy beverages, Color

Introduction

Color is one of the food sensory characteristics that are readily perceived by consumers, thus playing a decisive role in both acceptance and purchase intention. Therefore, food industry seeks novel attractive products by means of using dyes, either artificial or natural, that stimulate consumer interest (Kaimainen et al. 2015).

Artificial dyes typically provide a wide range of colors as well as high stability, but recent studies have correlated the consumption of some of these dyes with health issues, including allergic processes, possible carcinogenic effects (Polonio and Peres 2009), hyperactivity in children (Kaimainen et al. 2015), and toxicity issues (Osorio et al. 2010). In addition, consumers are becoming increasingly concerned about food quality, which is driving their preference towards natural products. Thus, the replacement of synthetic pigments by their natural counterparts has become a feasible alternative for highlighting the functional properties of natural dyes (e.g., anthocyanins) as well as stimulating studies on their stability and development of new products.

There is a great interest in the use of anthocyanins as natural dyes for food applications (Campos et al. 2017; Oliveira et al. 2015; Sánchez-Bravo et al. 2018). The fruit of juçara (Euterpe edulis Martius)—a palm tree that is native to the Atlantic Forest and commonly used for palm extraction—constitute as a promising source of such pigment. This fruit, which has a dark color due to presence of anthocyanins, mainly cyanidin 3-rutinoside and cyanidin 3-glucoside (Carvalho et al. 2016), can be used as a functional food because of the antioxidant capacity of these phenolic compounds that corresponds to the health-related benefits (Borges et al. 2011; Rigon and Noreña 2015).

The addition of natural antioxidants into food products constitute a possibility of replacing synthetic antioxidants, thus avoiding oxidative reactions that impair the nutritional value and sensory attributes. However, their use is still difficult due to the instability regarding some factors. In order to improve the use of these pigments, microencapsulation appears as a strategy to protect pigments, among other compounds, from adverse processing conditions as well as to provide them greater stability against spoilage factors—such as heat, light, pH, and oxygen—and, as a result, increase their shelf life (Barros and Stringheta 2006; Ferrari et al. 2013). Some polysaccharides have been widely used as encapsulating agents, and maltodextrin—obtained by acid hydrolysis of starches and extensively used in the food industry—is cited by authors as an excellent encapsulating agent (Bicudo et al. 2015; Ferrari et al. 2013). Chong and Wong (2017) concluded that maltodextrin was adequately effective to produce Sapodilla powder with potent antioxidant activity and acceptable color.

There is a great interest in incorporating anthocyanins into dairy products because this practice can turn products, that are already consolidated in the market and widely consumed, more attractive. Among these products, dairy beverages deserve special attention due to the potential of using whey—a by-product generated in cheese production—as means of eliminating its environmental impact as well as providing a nutritious, pleasing, and low-cost product to the consumer (Silva et al. 2017). The overall consumption of dairy beverages has increased, with special emphasis on the fermented ones, which feature sensory characteristics similar to those of yogurt.

Recently, several studies focused on the development of new functional dairy products containing fruit juices, pulp or extracts (Chouchouli et al. 2013; Oliveira et al. 2015; Prudencio et al. 2008; Sánchez-Bravo et al. 2018; Silva et al. 2017), but there is still limited information on the use of such functional compounds in the microencapsulated form and their stability in dairy products.

Therefore, this study aimed to evaluate the incorporation of microencapsulated anthocyanins into model systems of fermented and unfermented dairy beverages as well as to assess the stability of both pigment and product throughout storage in different packaging systems (opaque or transparent).

Materials and methods

Material

Dairy beverages were produced using pasteurized milk (Colagua®, Brazil), whey derived from cheese production, sucrose (Alvinho®, Brazil), corn starch (Maizena®, Brazil), gelatin (Rica Nata®, Brazil), lyophilized cultures for yogurt (Streptococcus salivarius ssp. thermophilus and Lactobacillus delbrueckii ssp bulgaricus) (Rica Nata®, Brazil), and powdered microencapsulated anthocyanins.

Anthocyanin extraction

Frozen juçara pulp was obtained (− 20 °C) directly from producers (Rio Novo do Sul, Espírito Santo State, Brazil). The pulp anthocyanins were extracted with 70% (v/v) ethanol and 70% (w/v) citric acid up to pH of 2.5, in accordance with a methodology proposed by Francis (1982), with modifications. Extraction was performed in the dark and at refrigeration temperature (8 ± 2 °C) for 24 h. The extract was filtered under vacuum using Whatman no 1 paper. Later, it was transferred to a rotary evaporator (Fisatom, model 801, Brazil) for vacuum concentration of 40% of the initial volume at 60 °C. The concentration step of these extracts was defined in preliminary experiments (data not shown).

Anthocyanin microencapsulation

The microcapsules were obtained by drying the concentrated juçara (Euterpe edulis Martius) anthocyanin-rich extracts in a mini spray dryer (Yamato, model ADL 311S, China) after adding maltodextrin 10DE (Cargill®, Brazil) at 30% (w/v) in a ratio of 1:3 (extract:carrier agent, v/v), according to Silva et al. (2013). The following parameters were defined in preliminary experiments and used for drying: outlet air temperature: 50 °C and inlet air temperature 136 °C; maximum compressed air pressure: 0.1 MPa; feed flow rate: 2.0 mL/min; air flow: 0.21 m3/min.

The morphology and size of the microcapsules were analyzed by scanning electron microscopy (SEM). A small amount of sample was fixed on metallic stubs and coated with a thin layer of gold under vacuum using a sputter coater (Balzers, model FDU-010). The samples were analyzed at magnifications of 220 X—10,000 X with an excitation voltage of 20 kV (Silva et al. 2013).

Dairy beverage formulation

The microcapsules and the beverages were produced in laboratorial scale. The fermented dairy beverage comprised 60% of pasteurized milk and 40% of whey, which constituted the milk base. The contents of the other ingredients were based on such milk base, as follows: sugar (12%), gelatin (0.5%), and lyophilized lactic culture (according to the manufacturer’s instructions).

Initially, whey was filtered to remove impurities and heated to 65 °C to inactivate rennet. The filtered milk/sugar/gelatin mixture was incorporated into whey and pasteurized in a thermal bath at 65 °C for 30 min, followed by cooling at 40 °C in an ice bath, with subsequent inoculation of the lactic culture. Fermentation was perfomed at 42 ± 1 °C until pH 4.2 and acidity from 60 to 70 ºDornic were reached. Clots were then broken and microencapsulated anthocyanin—rich extracts from juçara were added under stirring until complete solubilization. The beverage was filled in 100 mL opaque or transparent polyethylene bottles and kept under refrigeration (10 ± 2 °C) prior to the analysis.

Concerning the unfermented dairy beverage, 1.5% of corn starch was further added with other solid ingredients. After pasteurization (65 °C for 30 min), the product was cooled down for approximately 2 h in order to obtain the desirable texture as well as to facilitate microcapsule incorporation and stabilization. The dairy beverages were then stored as described before for the fermented beverage.

Anthocyanin-rich microcapsule incorporation

The mass of powdered microencapsulated anthocyanin to be incorporated into model systems considered the overall color difference (ΔE) between a commercial dairy beverage (used as color standard) and the studied samples. L* (luminosity), a* (intensity of red and green), and b* (intensity of yellow and blue) coordinates were compared using the CIELab color scale (Konica Minolta colorimeter, model CM-5, Japan). 4–8% powdered microencapsulated anthocyanin were added in the dairy beverages in order to obtain ΔE values smaller than 5, which indicate a small color difference between the samples (Obón et al. 2009). The overall color difference was calculated by Eq. 1.

ΔE=ΔL*2+Δa*2+Δb*1/2 1

wherein ΔE = global color difference; ΔL* = variation in L* coordinate; Δa* = variation in a* coordinate; Δb* = variation in b* coordinate.

Stability evaluation

The fermented and unfermented dairy beverages with powdered microencapsulated anthocyanins were stored in transparent and opaque polyethylene bottles at refrigeration temperature (10 ± 2 °C) and submitted to physicochemical analysis after 0, 7, 14, 21, and 28 days, based on the reports of Wallace and Giusti (2008), with modifications.

Total anthocyanin content

Pigments were extracted from dairy beverages in accordance with Prudencio et al. (2008), using a 1.5 N ethanol:HCl solution (85:15 v/v) at a beverage:extractive solution ratio of 1:4 (v/v) in Falcon tubes. After centrifugation (Thermo Scientific Heraeus Megafuge 16R, Germany) for 45 min with a relative centrifugal force of 5000g at 11 °C, the extracts were filtered using Whatman no 1 paper. Anthocyanins were then quantified according with Lees and Francis (1972), with modifications. Briefly, the extract absorbance was measured in a spectrophotometer (BEL Photonics, model SP 2000UV, Brazil) at 510 nm. The results were expressed as mg cyanidin-3-glycoside per 100 g of sample, using the absorption coefficient of 98.2 L/cm g.

Total phenolic content

The total polyphenol content (TPC) was determined with the Folin-Ciocalteau reagent assay by adapting the method proposed by Singleton and Rossi (1965). Initially, the centrifuged extract was diluted in 70% ethanol (v/v). A 0.6 mL aliquot of the extract was mixed with 3.0 mL of Folin-Ciocalteau reagent previously diluted in distilled water (1:10 v/v). The system was allowed to rest for 3 min in the dark, followed by the addition of 2.4 mL of a saturated Na2CO3 solution (7.5% w/v). Absorbance at 760 nm was recorded in a spectrophotometer (BEL Photonics, model SP 2000UV, Brazil) after 1 h of rest in the dark. The TPC was determined using a standard curve of gallic acid (0–150 mg/L) and the results were expressed as mg of gallic acid equivalent per 100 grams of sample (mg GAE/100 g).

Antioxidant activity (ABTS)

The antioxidant activity was measured with the 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) reagent, as described by Re et al. (1999). The radical ABTS·+ was prepared by mixing 10 mL of a 7 mM aqueous ABTS solution and 10 mL of a 2.45 mM potassium persulphate solution. The resulting mixture was then maintained in the dark for 16 h. The absorbance at 734 nm was corrected to 0.700 through the addition of 80% (v/v) ethanol. Later, 3.5 mL of ABTS·+ were allowed to react with 0.5 mL of the extract for 6 min, followed by spectrophotometric measurements. The antioxidant activity was determined using a standard curve of Trolox (0–150 µM), and the results were expressed as Trolox equivalents (µM Trolox g−1).

Antioxidant activity (DPPH)

Antioxidant activity was also evaluated by reacting the antioxidant compounds of the extract with the stable radical 2,2-Diphenyl-1-picrylhydrazyl (DPPH·) following adaptations in the methods proposed by Brand-Willliams et al. (1995) and Espín et al. (2000), and Pukalskas et al. (2002). An aliquot (100 μL) of the extracts was added to a 0.1 mM DPPH methanolic solution (1.0 mL). The mixture was stirred for 15 min in the dark and then the absorbance at 517 nm was determined. Meanwhile, blank absorbance was recorded. The radical scavenging activity of the tested samples, expressed as % RSC (radical scavenger capacity), was calculated by Eq. 2.

%RSC=AB-AA/AB×100 2

wherein: AB = absorbance of the blank; AA = absorbance of the antioxidant-containing sample reacted for 15 min.

Colorimetric analysis

Color was determined through direct measurements of the rectangular coordinate system L*, a*, and b*, using the CIELab color scale, with illuminant D65 and 10° viewing angle, in a Konica Minolta colorimeter (model CM-5, Japan). C* (chromaticity) and h° (hue angle) values were calculated by Eqs. 3 and 4, respectively. The global color difference (ΔE) at times 0 and 28 days was calculated by Eq. 1.

C*=a*2+b*21/2 3
h∘=arctanb*/a* 4

Total titratable acidity and pH

Dairy beverages acidity was determined by titration using a 0.1 mol/L NaOH solution and a potentiometer (Del Lab, model DLA-pH). The results were expressed as g of lactic acid per 100 g of beverage. pH was determined in a previously calibrated digital potentiometer, according to analytical standards of Instituto Adolfo Lutz (2004).

Experimental design and statistical analysis

The experiment was performed in a completely randomized design (CRD) and in a factorial scheme: (i) type of packaging—two levels (opaque and transparent)—and (ii) time—five levels (0, 7, 14, 21, and 28 days)—and three repetitions for each type of dairy beverage (i.e., fermented and unfermented). Analysis were conducted in triplicate and data were submitted to analysis of variance (ANOVA) and regression, as needed, using Statistica®, version 10 (StatSoft Inc., Tulsa, USA).

Results and discussion

Anthocyanin-rich microcapsule incorporation into dairy beverages

The increase in concentration of microcapsules caused an L* decrease resulting in a darker dairy beverage, and, at the same time, an increase of coordinate a*, indicating a greater red color intensity, being the most interesting parameter. Dairy beverages with 6% added powdered microencapsulated anthocyanin presented the lowest ΔE value (5.07), which was the ideal concentration, while highest value (ΔE = 8.96) was observed with the addition of 4% of microencapsulated anthocyanin. The pigment percentage to be added into product was then determined in such a way to obtain attractive color similar to that of commercial dairy beverage, composed by synthetic red dyes.

Microcapsules characterization

By SEM (Fig. 1), microcapsules presented a similar morphology, predominantly smooth and spherical, and an average size smaller than 10 μm, fitting into the microscale range due the formation of capsules with a size between 0.2 and 5000 µm (Barros and Stringheta 2006). The addition of large particles to food matrices can affect the texture of food products; therefore, particles with a diameter smaller than 100 µm are the most suitable (Annan et al. 2008). Smoother microcapsules are desirable for the stability of encapsulated pigments and have showed greater anthocyanin retention, which may be associated with a better accommodation of the pigment in the microparticle (Carvalho et al. 2016; Osorio et al. 2010).

Fig. 1.

Fig. 1

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Photomicrographs by SEM of spray-dried microcapsules of anthocyanin-rich extract from juçara’s pulp with maltodextrin. Magnifications of 500 and 2500 X

Physicochemical analysis on dairy beverages

Table 1 presents the ANOVA results for each of the studied response variables in fermented and unfermented dairy beverages. As storage time did not contribute to the decrease in anthocyanin and phenolic contents, the kinetics of anthocyanin and phenolic compounds degradation in these model systems was not calculated.

Table 1.

ANOVA (F and p values) of anthocyanins content, antioxidant activity, total phenolic content, pH and total acidity: effect of time, packaging and time*packaging interactions for fermented and unfermented dairy beverages

Response variables Source Fermented Unfermented
F p F p
Total anthocyanins content (mg/100 g) Time 2.209ns 0.10468 1.044ns 0.40936
Packaging 0.308ns 0.58488 0.001ns 0.97638
Time*Pack. 0.159ns 0.95648 0.035ns 0.99740
Antioxidant activity (ABTS) (µM Trolox g−1) Time 3.581* 0.02335 1.903ns 0.14928
Packaging 27.094* 0.00004 185.816* 0.00000
Time*Pack. 4.526* 0.00912 0.060ns 0.99287
Antioxidant activity (DPPH) (% RSC) Time 5.540* 0.00360 0.720ns 0.58553
Packaging 0.580ns 0.45673 0.010ns 0.94291
Time*Pack. 0.660ns 0.62864 1.710ns 0.18673
Total phenolic content (mg GAE/100 g) Time 2.230ns 0.10218 0.743ns 0.57407
Packaging 2.756ns 0.11249 71.349* 0.00000
Time*Pack. 0.466ns 0.76022 1.770ns 0.17444
pH Time 1.350ns 0.28773 1.310ns 0.29949
Packaging 0.000ns 0.99157 0.200 ns 0.65790
Time*Pack. 0.010ns 0.99990 0.020ns 0.99936
Total titratable acidity (g/100 g) Time 1.431ns 0.26033 0.105ns 0.97957
Packaging 0.001ns 0.97079 0.006ns 0.93854
Time*Pack. 0.177ns 0.94771 0.009ns 0.99980
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*Significant (p < 0.05);nsNot significant (p > 0.05). % RSC radical scavenger capacity, GAE gallic acid equivalent

Anthocyanin content

An analysis of anthocyanins in concentrated extract (prior to spray drying microencapsulation) was made, and an average value of 740.08 mg/100 g was found. Juçara can be considered a promising source of anthocyanins. Novello et al. (2015) and Borges et al. (2011) optimized the extraction of juçara’s pulp anthocyanins with different solvents and acids, and they found maximum values of 124.9 mg/100 g and 259.6 mg/100 g, respectively. Additionally, they have also demonstrated its potential use as a dye in food products.

Fermented milks are perceived as healthy foods, and the development of new products containing fruits and their bioactive compounds may provide a food valued by health consumers. In this sense, it is necessary to evaluate the effect of fermentation on some characteristics, like antioxidant properties, anthocyanin content and color, and the microbial counts of lactic acid bacteria (Sánchez-Bravo et al. 2018).

In fermented dairy beverage, anthocyanin content remained stable throughout the investigated storage period in both packaging types (p > 0.05) (Table 1). The average values are shown in Table 2. Slightly acid pH, refrigeration and mainly microencapsulation are factors that contributed to the maintenance of pigment stability (Bernardes et al. 2019). Similar result was reported by Prudencio et al. (2008), that evaluated the addition of anthocyanins and betalains into petit-suisse cheese and observed that the dyes were stable during 40 days of storage, supporting the application of both natural dyes as well as assigning functional properties to the product. However, Silva et al. (2017) observed a decrease (p < 0.05) in total anthocyanin content in fermented dairy beverage with addition of blueberry, and suggested that during storage time chemical reactions lead to destabilization of pigment. In the present study, the microencapsulation of juçara extract contributed to the maintenance of anthocyanins in the medium, despite chemical reactions and interactions between pigment and milk components that can occur and reduce the pigment availability (Oliveira et al. 2015; Silva et al. 2017).

Table 2.

Mean values of anthocyanin content, total polyphenol content, total titratable acidity and overall color difference of fermented and unfermented dairy beverages model systems

Beverage/Packaging types Fermented Unfermented
Opaque Transparent Opaque Transparent
Anthocyanin (mg/100 g) 3.22 ± 0.63a 3.23 ± 0.50a 3.22 ± 0.63a 3.23 ± 0.50a
TPC (mg GAE/100 g) 78.69 ± 9.11a 72.12 ± 4.53a 98.28 ± 8.72a 67.84 ± 11.53b
pH 3.86 ± 0.07a 3.86 ± 0.08a 4.29 ± 0.18a 4.26 ± 0.19a
Total titratable acidity (g/100 g) 1.07 ± 0.07a 1.08 ± 0.05a 0.65 ± 0.12a 0.64 ± 0.12a
ΔE 3.10 ± 0.11a 2.17 ± 0.11a 3.56 ± 1.42a 2.10 ± 0.59a
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Data shown as mean values ± SD of three repetitions. Means followed by the same letter on rows for each packaging type within each dairy beverage did not differ statistically (p < 0.05) by Tukey test at 5% significance. TPC Total polyphenol content; ΔE overall color difference (measured in times 0 and 28 days)

In unfermented dairy beverage, similar results were observed (Table 1), where the pigment remained stable during 28 days for both beverages and both packaging types (p > 0.05) (mean values are presented in Table 2). Although incidence of light is a factor that contributes to anthocyanins degradation (Bernardes et al. 2019), in the present study, as anthocyanin content remained stable, the use of opaque packaging is not necessary, if the objective is protect the pigment against light. Kaimainen et al. (2015) commented on the importance of the addition of natural pigments in the sensory acceptance of model systems of juices, and noticed that the more intense color of anthocyanin-rich juices may result in an increased product acceptance. The addition of natural pigments in beverages is facilitated when fruit extracts are microencapsulated. In the present study, microencapsulation was shown to protect the bioactive compounds from the adverse environmental conditions, such as exposure to light and oxygen, which can cause damages such as oxidation and colorless (Osorio et al. 2010), as well as interactions with other components of the dairy beverage.

Antioxidant activity–ABTS

In the antioxidant activity determination by ABTS cation radical assay, storage time and packaging type exerted a cooperative influence (p < 0.05) on the antioxidant activity of the fermented dairy beverages (Table 1). The effect of time within each packaging type was analyzed by regression, but none of the tested models (linear and second-degree polynomial) was significant. The effect of packaging condition over time was then investigated, and the different packaging systems differed only at the seventh day (Fig. 2). However, the overall values indicates a constant trend: 306.67 (time 0) to 295.56 (time 28) µM Trolox g−1 and 300.00 to 373.33 µM Trolox g−1 for opaque and transparent packaging, respectively.

Fig. 2.

Fig. 2

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Antioxidant activity values of fermented dairy beverages throughout storage time, as determined by the ABTS (μM Trolox g−1). Means followed by the same letter within the same time do not differ by Tukey test (p > 0.05)

In unfermented dairy beverages, antioxidant activity (ABTS) was only influenced (p < 0.05) by packaging type and remained unchanged during the investigated storage time (Table 1). The average antioxidant activity found for unfermented beverage stored in opaque packaging was 564.00 ± 55.59 μM Trolox g−1, versus 373.00 ± 43.91 μM Trolox g−1 for transparent packaging. This difference was observed since the beginning of the storage time interval and remained proportional up to the 28th day of storage. Oliveira et al. (2015), when studying the addition of strawberry pulp in yogurt, observed a reduction (18%) in the antioxidant capacity after 28 days of storage under refrigeration, while in the fruit preparation (control), the ABTS assay indicated a decrease of only 4%.

Antioxidant activity can be modified by the encapsulation process and/or interactions with the wall material. Aguiar et al. (2017) evaluated microencapsulation by spray drying of natural antioxidants and showed higher values after the process, proving that microencapsulation does not compromise the antioxidant capacity. Here, both the antioxidant activity and the anthocyanin content can be considered constant, which corroborates the efficiency and importance of pigment microencapsulation. In addition, this observation strengthens the relationship between antioxidant activity and anthocyanin content in food products, as demonstrated by Kardum et al. (2014) when observed that anthocyanins contribute to strong antioxidant activity of fresh berries and commercial juice.

Antioxidant activity–DPPH

In fermented dairy beverage, antioxidant activity measured by the stable DPPH· radical scavenging was influenced exclusively by storage time (p < 0.05) (Table 1), but no regression model was significant. High scavenging rates were observed for both packaging: from 80.68 to 86.40% at days 0 and 28, respectively, and this increase over time may indicate a controlled release of the microencapsulated compounds that display antioxidant activity, e.g. anthocyanins and phenolic compounds, in a complex food matrix (fermented dairy beverage), as also demonstrated by Campos et al. (2017) with açaí fermented milk. The authors observed that antioxidant activity (DPPH) of product showed excellent correlation with anthocyanins, and values decreased with reduced concentration of these components.

In unfermented dairy beverages, antioxidant activity (DPPH) remained unchanged (p > 0.05) throughout the investigated storage time interval (Table 1), being 81.70 ± 2.34% for beverage stored in opaque packaging and 81.63 ± 2.55% for transparent packaging.

Bernstein and Noreña (2015) evaluated antioxidant activity (DPPH) in microcapsules of anthocyanins, indicating that microencapsulation was efficient in protecting the pigment, the main component responsible for the antioxidant activity. The stabilization of the antioxidant activity observed here is a promising result for the use of anthocyanin-rich microcapsules in model systems of dairy beverages, given the importance of the consumption of foods featuring good antioxidant capacity as well as of the reduction of synthetic antioxidants in foods.

Both methods (i.e., ABTS and DPPH) demonstrated the stability of the antioxidant activity in dairy beverages throughout the 28 days of storage. This result is positive and fulfils the initial expectations of this study by promoting the stabilization of the bioactive compounds of juçara pulp through microencapsulation by spray drying. The methods used to determine the antioxidant activity of food products are based on different mechanisms and use different sources of radicals or oxidant compounds. As a consequence, the obtained results are expressed in different units, making the comparison between them difficult.

Total phenolic content (TPC)

Total phenolic content in the fermented dairy beverages remained constant (p > 0.05) throughout the investigated storage time, regardless of packaging type (Table 1). The average values are shown in Table 2. The similar behavior for anthocyanins and total phenolic compounds could be related to the fact that anthocyanins constitute a class of phenolic compounds called flavonoids, that are sources of bioactive substances with antioxidant capacity (Carvalho et al. 2016).

Stability of phenolic compounds of strawberries was also observed by Oliveira et al. (2015) in yogurt matrix, since concentrations of catechin, epicatechin and kaempferol decreased only after 28 days of storage of the preparations. Silva et al. (2017) evaluated the TPC concentration in fermented dairy beverage with blueberry juice and have found values ranging from 15.35 (minimum juice concentration) to 24.28 mg GAE/100 mL (maximum extract concentration), values lower than those found in the beverages of the present study. This can be explained by the fact that fermentation process and storage time can influence TPC values, since the phenolic content in fermented milk is affected by pH, initial content of the fruit and bacterial strain (Sánchez-Bravo et al. 2018).

In unfermented dairy beverage, similarly to the antioxidant activity by ABTS method, packaging type influenced total phenolic compounds in the beverage (p < 0.05) (Table 1). There are reports on the numerous applications of phenolic compounds as functional ingredients in dairy products. This functionality is based on the capacity of polyphenols to interact with milk proteins—to which the former has good affinity—and form soluble complexes. Some models suggest that these complexes are formed by multiple weak interactions between amino acid side chains and polyphenol aromatic rings, indicating that this association is a surface phenomenon that is influenced by factors such as nature of protein residues and nature of polyphenol, temperature, and presence of other components (e.g., sugars). However, the quantitative impact of specific interactions should be further studied in order to define future recommendations for product development (O’Connell and Fox 2001; Oliveira et al. 2015; Sánchez-Bravo et al. 2018).

The content of anthocyanins and phenolic compounds remained the same over the investigated time, probably due to the protection provided to bioactive compounds by microencapsulation. Thus, the degradation kinetics of phenolic compounds was not evaluated.

pH and acidity

Both pH and acidity of fermented beverages remained constant (p > 0.05) throughout storage time, regardless of the packaging type (Table 1). This result was expected, provided that these variables are correlated. The difference between the values of these variables in differently packaged beverages was small (Table 2). Similar observation was made by Chouchouli et al. (2013), Silva et al. (2017) and Sánchez-Bravo et al. (2018). These authors reported that the addition of fruit extracts into dairy products have not altered neither their pH nor their acidity throughout storage. The high titratable acidity found in our product is attributed to the fruit natural acidity, milk base and, mainly, the acidification step for the preparation of ethanolic juçara extracts.

As observed for fermented beverage, pH and acidity of unfermented beverage remained constant throughout storage (p > 0.05), regardless of packaging type (Table 1). Commercial unfermented dairy beverages have pH close to neutrality, which is a result of their main components, namely milk and whey. The low pH values found (Table 2) are due to the high acidity of the extracts, provided that anthocyanin extraction is performed by acidification of the pulp to pH 2.5, as described in Sect. Anthocyanin extraction. Thus, the use of acidulants in the production of unfermented dairy beverage is not necessary. pH is one of the factors that exerts the greatest influence in anthocyanin color maintenance in food products. Acid medium favor the stability and predominance of cation flavylium, that presents red color, while neutral to alkaline pH make anthocyanins unstable and convert them to the colorless carbinol and chalcone pseudo bases (Osorio et al. 2010; Zaidel et al. 2014). Titratable acidity was lower in unfermented than in fermented dairy beverages due to fermentation process, characterized by production of mainly lactic acid by Lactobacillus, Streptococcus and other lactic acid bacteria. The type of starting culture should be considered during the study because there is a diversity of metabolic properties associated to preservation, nutrients available and taste of product (Campos et al. 2017; Sánchez-Bravo et al. 2018).

Acidic products (e.g., such as yogurt and other fermented dairy beverages) become promising for the incorporation of anthocyanins due to their intrinsic characteristics as well as the need to store at refrigeration temperature, which also influences pigment stability (O’Connell and Fox 2001; Sánchez-Bravo et al. 2018). Additionally, these pH and temperature conditions favor the microbiological stability and, as a consequence, product conservation.

Colorimetric analysis

The average values of coordinates L*, a*, b* and parameters C* and hº were similar for both beverage types (Fig. 3). They presented high luminosity (L), which is assigned to its main ingredients—milk and whey, which have a white color as well as an L* value close to 100.00. The positive value of the coordinate a* indicates a tendency to originate the red color, being this the coordinate of greatest interest because of anthocyanin coloration. The coordinate b* indicates the blue-to-yellow intensity, with its positive value an indication of a tendency towards yellow. Chroma (C*) is a quantitative color attribute that evaluates the degree of difference between two shades having the same luminosity. In the present study, C* followed the trend of the coordinate a*, indicating that the red color was the most expressive in determining the color of the product. The chromatic hue (hº) was positioned in the first quadrant in the color solid, indicating that color of the dairy beverage is between red and yellow, tending to red, since 0 and 360º angles are attributed to red, while 90º, 180º, and 270º angles represent yellow, green, and blue shades, respectively. The lower the hº, the closer the beverage is to a* axis. The higher the angle, the closer the beverage is to the b* axis (Fig. 3).

Fig. 3.

Fig. 3

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Colorimetric coordinates of fermented and unfermented dairy beverages model systems in both packaging types. Mean values ± SD of three repetitions

Color perception by means of colorimetric coordinates in an isolated method that present difficult interpretation. Thus, one of the best parameters to describe color variation is global color difference (ΔE), a combination of the coordinates L*, a*, and b*. ΔE was measured to indicate the total distance between color of two samples (namely those stored for 0 and 28 days) in the three-dimensional CIELab color space (Pathare et al. 2012). In the present study, the ΔE found for both beverages stored in opaque and transparent packaging was lower than 4.0 (Table 2). This may be considered good because values ranging from 1.5 to 5.0 suggest small color variation, whereas values higher than 5 indicate an evident color variation (Obón et al. 2009).

Wallace and Giusti (2008) evaluated the feasibility of using powdered anthocyanins as dyes in yogurt and observed high color stability in the product, suggesting that natural dyes could be used in dairy matrices. They found results similar to those obtained in our study (L* = 65, a* = 12, b* = − 6).

Microencapsulation of juçara extracts contributed to the maintenance of color of dairy beverages, in opaque and transparent packages, probably due to the protection that this process give to anthocyanins. In the microencapsulation by spray dryer a carrier agent (maltodextrin) exert protection to a core material (bioactive compounds of juçara extract), which allows an extended release of anthocyanins and polyphenols, and protection of these components against adverse surrounding conditions. Thus, encapsulated natural bioactives have better heat, light and pH stability and this impacts directly on the color of the products (Zaidel et al. 2014).

Conclusion

Powdered microencapsulated anthocyanin-rich extracts provided both fermented and unfermented dairy beverages (model systems) with colors similar to a commercial product as well as stable pigments, pH, and acidity. There was no reduction in the contents of bioactive compounds at the end of storage time, indicating the potential use of microencapsulated natural pigments in dairy matrices. In addition, it has been demonstrated that the use of opaque packaging in this type of product is dispensable. This is an advantage, since it enables the use of various packages (opaque and transparent), according to preference, design and cost, maintaining the anthocyanin stability.

It is suggested that the synthetic red dyes used in dairy beverages can be replaced by anthocyanin-rich extracts microencapsulated with maltodextrin by spray dryer, since it was obtained a product featuring good color stability. In addition, the natural pigments increased the nutritional value of the product, with juçara pulp a potential source of anthocyanins.

It should be taken into account that a model system of dairy beverages was developed here. It is necessary further work on the development of new products as well as on the study of the sensory characteristics, for incorporation in the market and availability to consumers, the main motivators of the development of this type of research.

Acknowledgements

Authors would like to thank Brazilian Federal Agency for the Support and Evaluation of Graduate Education (CAPES) for scholarship to E.M.F. Lima and National Council for Scientific and Technological Development (CNPq) for the financial support (Grant Number: 478246/2013-7). They also thank professor Renê Silva (Laboratory of Microscopy–Department of Physics) and Nucleus of Microscopy and Microanalysis (Federal University of Viçosa, MG, Brazil), for SEM analysis.

Footnotes

Publisher's Note

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Contributor Information

Emília Maria França Lima, Email: emiliamflima@gmail.com.

Marina Carvalho Martins Madalão, Email: marina_cmartins@yahoo.com.br.

Walter Correa dos Santos, Jr., Email: w.c.junior@live.com

Patrícia Campos Bernardes, Email: paticbernardes@gmail.com.

Sérgio Henriques Saraiva, Email: sergiohsaraiva@gmail.com.

Pollyanna Ibrahim Silva, Email: pollyannaibrahim@gmail.com.

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