Natural Pigments of Anthocyanin and Betalain for Coloring Soy-Based Yogurt Alternative
Article Natural Pigments of Anthocyanin and Betalain for Coloring Soy-Based Yogurt Alternative
Sandra Dias 1, Elisabete M. S. Castanheira 2 , A. Gil Fortes 1 , David M. Pereira 3 and M. Sameiro T. Gonçalves 1,*
1 Centre of Chemistry, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal; [email protected] (S.D.); [email protected] (A.G.F.) 2 Centre of Physics, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal; ecoutinho@fisica.uminho.pt 3 REQUIMTE/LAQV, Laboratory of Pharmacognosy, Department of Chemistry, Faculty of Pharmacy, University of Porto, R. Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal; dpereira@ff.up.pt * Correspondence: [email protected]; Tel.: +351-253-604-372
Received: 1 May 2020; Accepted: 8 June 2020; Published: 11 June 2020
Abstract: The aim of this work was to evaluate the color stability of betalain- and anthocyanin-rich extracts in yogurt-like fermented soy, in order to develop a preliminary understanding of how these pigments behave in this type of food system during storage for 21 days at 4 ◦C. Thus, the extracts of red beetroot, opuntia, hibiscus and red radish were integrated into the yogurt-like fermented soy in two different ways—directly after lyophilization, and encapsulated in nanosystems based in soybean lecithin—as this approach has never been used to further increase the value and potential of the dairy-free alternatives of yogurt-like fermented soy. The results showed that non-encapsulated betalain-rich extracts from red radish are the most promising for coloring yogurt-like fermented soy. However, encapsulated opuntia extracts can also be an alternative to supplement the soy fermented beverages with betalains, without changing significantly the color of the system but giving all its health benefits, due to the protection of the pigments by nanoencapsulation.
Keywords: soy-based yogurt alternative; betalain pigments; anthocyanin pigments; functional food; food dyes
1. Introduction The increased consciousness about the impacts of production and consumption of food on environmental, health and ethical issues has motivated consumers to increase their interest in replacing animal-based foods in their diets. Consequently, the food industry has developed new products from plant-based ingredients, that imitate many of the physicochemical and sensory characteristics associated with animal-derived foods, such as fish, meat, eggs, milk and their derivatives [1–3]. Plant-based yogurt alternatives are among the most important dairy-free options [4], meeting the abovementioned requirements of consumers, specifically those associated with lactose intolerance and other dairy allergies [5]. In recent years, among the plant-based sources used for the production of plant-based yogurt alternatives, the soybean stands out due to the quantity and quality of its protein, as well as its functional properties [6]. Issues related to texture properties and general appearance are the major challenges faced by producers of plant-based yogurt alternatives. It is well-known that plant-derived natural pigments, such as betalains, have become popular for use as natural colorants in the food industry [7,8]. Besides, they present pharmacological properties, such as antioxidant, anti-cancer, anti-lipidemic and antimicrobial activities [9,10]. Beetroot (Beta vulgaris L.) [11–17] and opuntia (Opuntia stricta)[18–27], natural sources of betalains, possess the
Foods 2020, 9, 771; doi:10.3390/foods9060771 www.mdpi.com/journal/foods Foods 2020, 9, 771 2 of 13 betacyanins isobetanine and betanine, along with betaxanthins, such as indicaxanthin, vulgaxanthin I and vulgaxanthin II (beetroot), as well as other derivatives (opuntia). Moreover, anthocyanins, another important class of natural pigments, have been associated with the delay of the start of diseases related to chronic age, owing to their antioxidant activities, and they also restrain the proliferation of cancer cells as a result of their antiproliferative capacities [28–30]. Hibiscus (Roselle sabdariffa L.) [31–34] and radish (Raphanus sativus L.) are two sources of anthocyanins that have also been used as colorants of food and beverages, taking advantage not only of their colorimetric potential, but also of their medicinal properties, and they thus have enormous benefits for health [35,36]. In fact, nowadays, interest in the use of betalains and anthocyanins pigments goes far beyond the color they give to food, which, although very important for the visual aspect, is largely surpassed by the health benefits that they can confer, thus enhancing the global value of the products that consumers increasingly want to be functional foods. However, the instability of extracted betalains and anthocyanins may be a weak point that cannot be forgotten, and encapsulation of these pigments in polysaccharide–proteins, gelatin–maltodextrin, carrageenan and liposomes, has emerged as an interesting possible means of minimizing this weakness [15,16,25–27]. Keeping all this in mind, the aim of the present study was to evaluate the color stability of betalain- and anthocyanin-rich extracts in a soybean dairy-free alternative, which, in addition to conferring a pleasant pink color, can impart health benefits, further enhancing the final value of the product. Thus, red beetroot, opuntia, hibiscus and red radish were used as sources of betalain- and anthocyanin-rich extracts, and yogurt-like fermented soy was chosen as a soy-based dairy-free model. The additions of the extracts were to already-prepared yogurt-like fermented soy, which prevented the pigments from being exposed to inherent issues during the production, such as temperature, which can affect their stability. However, nanoencapsulation studies were performed with the purpose of evaluating the color obtained in yogurt-like fermented soy as well as its stability, in comparison with those qualities obtained with non-encapsulated pigments, during 21 days of storage. The results obtained point to a promising use of red radish for coloring this type of food. However, encapsulated opuntia extracts can be an alternative for supplementing the yogurt-like fermented soy with betalains, without changing significantly the color of the system, but whilst still giving all its health benefits, due to the protection of the pigments by nanoencapsulation.
2. Materials and Methods
2.1. Chemicals and Reagents Ethanol, isopropanol, DMSO, tetrahydrofuran and acetic acid were products from Merck KGaA (Darmstadt, Germany). 3-(4,5-Dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) and Trypan blue were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s Modified Eagle Medium (DMEM), Hank’s balanced salt solution (HBSS), foetal bovine serum (FBS), penicillin–streptomycin solution (penicillin 5000 units/mL and streptomycin 5000 µg/mL) and 0.25% trypsin-EDTA were purchased from GIBCO, Invitrogen™ (Grand Island, NY, USA). The yogurt-like fermented soy (supplier and distributor—Alpro), with Product code (EAN) 5411188104971, contained the following composition according to the label information: water, shelled soybeans (7.9%), sugar, tricalcium citrate, stabilizer (pectin), acidity regulators (sodium citrate, citric acid), aroma, sea salt, antioxidants (extract rich in tocopherols, esters of acids fat from ascorbic acid), vitamins (B12, D2), live yeast (S. thermophilus, L. bulgaricus).
2.2. Plant Material The red beetroot, hibiscus and red radish were obtained from a local grocery store in Portugal as fresh materials (red beetroot and red radish), or in dehydrated form (hibiscus), in October 2019. The fresh opuntia was provided by the Opuntia Farm (Albarrol, Portalegre, Portugal), in May 2019. Foods 2020, 9, 771 3 of 13
2.3. Preparation of Betalain-Rich Extracts The red beetroot and opuntia were hand-peeled and their interiors were cut into small pieces. To these small pieces (5 g) of red beetroot or opuntia, a solution of water/ethanol/acetic acid (66.6:33:0.33, v/v/v) (10 mL) was added, and the mixtures were maintained at room temperature, with irradiation protection for 48 h (red beetroot) or 20 min (opuntia) [37]. Then, the mixtures were filtered and the filtrates subjected to centrifugation in a Z 300 universal centrifuge (Hermle LaborTechnik GmbH, Wehingen, Germany) at 500 rpm for 16 min (in case of opuntia), and solvents were partially evaporated under reduced pressure using the rotary evaporator (Rotavapor® R-210, BÜCHI Labortechnik AG, Flawil, Switzerland) at a temperature of 40 ◦C. At that point, the extracts were subjected to freezing at 80 C and lyophilization (Alpha 1-4 LD Plus—Christ freeze dryer, Martin − ◦ Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany) for five days to produce the corresponding dry extracts which were put in storage in a desiccator until use.
2.4. Preparation of Anthocyanin-Rich Extracts To the hibiscus, in the form it was bought (5 g), a solution of water/ethanol/acetic acid (70:29.7:0.3 v/v/v) (10 mL) was added, and the resulting mixture was maintained with irradiation protection at low temperature (~4 ◦C), for 72 h [38]. Then, the mixture was filtered, and the solvent was subject to partial evaporation under reduced pressure using the rotary evaporator (Rotavapor® R-210, BÜCHI Labortechnik AG, Flawil, Switzerland) at 40 ◦C. At that point, the extract was subjected to freezing at 80 C and lyophilization (Alpha 1-4 LD Plus—Christ freeze dryer, Martin Christ − ◦ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany) over five days, to produce the corresponding dry extract, which was put in storage in a desiccator until use. The red radish was hand-peeled and the peels were cut into small pieces. To these small pieces (25 g), a solution of water/acetic acid (95:5, v/v) (100 mL) was added, and the mixture was kept at low temperature (~4 ◦C) for 18 h. After that, the mixture was filtered, the solvent was partially evaporated, and the resulting extract was subjected to freezing at 80 C followed by lyophilization, according to a − ◦ similar procedure as described above for hibiscus [39].
2.5. Determination of Betalains Content The betalains content of red beetroot extract was previously determined [40]. Ultraviolet –visible (UV-Vis) absorption spectra of opuntia extracts were obtained on a Shimadzu UV-Vis-NIR 3600 Plus spectrophotometer (Shimadzu Corporation, Kyoto, Japan). The experimental spectra were decomposed and fitted to a sum of betacyanins and betaxanthins spectra, to determine the concentration of each type of dye, expressed as mg equivalent betanin/L and mg equivalent indicaxanthin/L, as previously described [40]. Betacyanins were detected at maximum wavelength of 538 nm and betaxanthins at λmax = 480 nm, using Equation (1):
Dye content (mg/L) = (A DF MW 1000)/ε l (1) × × × × where A is the absorbance, DF is the dilution factor, l is the optical path (1 cm), MW is the molecular weight and ε is the molar absorptivity. In Equation (1), values of ε = 6.5 104 M 1 cm 1 and MW = 550 g/mol were used for betacyanin. × − − For betaxanthins, reported values of ε = 4.8 104 M 1 cm 1 and MW = 308 g/mol were utilized [41,42]. × − − 2.6. LRMS and UHPLC-ESI-Q-TOF-MS Analysis of Extracts Low resolution mass spectrometry (LRMS) analyses were carried out on a Thermo Scientific™ LTQ XL™ linear ion trap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Ultra-high performance liquid chromatography with electrospray ionization, coupled to quadrupole-time-of-flight mass spectrometry (UHPLC-ESI-Q-TOF-MS) in positive mode (timsTOF) analyses were performed Foods 2020, 9, 771 4 of 13 at the “Unidad de Espetrometria de Massa e Proteómica, CACTUS”, at University of Santiago de Compostela, Spain. For the UHPLC, a C18 column was used.
2.7. Human Gastric Adenocarcinoma Cell Line Assays The human gastric adenocarcinoma cell line (AGS) was used as a model for gastric tissue. Cells (Sigma-Aldrich, St. Louis, MO, USA) were maintained in DMEM + GlutaMAxTM-1 with 1% penicillin/streptomycin and 10% FBS, at 37 ◦C, in a humidified atmosphere of 5% CO2. For assessing viability, cells were plated at a density of 1.5 104 cells/well, followed by incubation × for 24 h with the samples. After incubation, 0.5 mg/mL MTT solution was added and further incubated for 2 h. The formazan in each well was dissolved in a solution of DMSO/isopropanol (3:1). Lastly, the absorbance at 560 nm was read in a Thermo Scientific™ Multiskan™ GO microplate reader (MA, USA). Viability was expressed as a percentage of the signal of control cells (cell medium).
2.8. Nanoencapsulation Studies The lyophilized extracts of red beetroot, opuntia, hibiscus and red radish were used for nanoencapsulation assays. Ultrapure water Milli-Q grade (MilliporeSigma, St. Louis, MO, USA) was used in all preparation steps. Dilutions in water of the extracts in the range of concentrations 2 10 5 × − –50 10 4 mg/mL were performed to determine the calibration curves and calculate the encapsulation × − efficiency. The calibration curves are linear in this range of extract concentrations (R2 = 0.99). Liposomes were prepared by the ethanolic injection and thin film hydration methods. For the lipid formulation, a commercial lipid mixture used in the food industry was employed, soybean lecithin (Sternchemie, Hamburg, Germany), the composition of which is (% mol/mol) 22% phosphatidylcholine, 20% phosphatidylethanolamine, 20% phosphatidylinositol and 10% phosphatidic acid as main components, with a concentration of 1 10 3 M. In the ethanolic injection method, 2 10 3 g/mL × − × − extract was added to the lipid mixture, and the solvent was evaporated with a stream of ultrapure nitrogen. Then, tetrahydrofuran (0.75 µL) and ethanol (0.75 µL) were added. The mixture was added to Milli-Q grade water (3 mL) under vortexing and the resulting solution was transferred to Amicon tubes and centrifuged (Universal 320 Hettich Zentrifugen, New Delhi, India), for 10 min at 3000 rpm. In the case of the thin film hydration method, soybean lecithin (60 mg) was dissolved in ethanol (3 mL), followed by evaporation under an ultrapure nitrogen stream. Then, 10 10 3 g/mL of the × − aqueous extract solution, previously dissolved in 5 mL of ultrapure water, was added to the lipid mixture. The solution was vortexed for 5 min and then sonicated for the same time. Finally, the resulting mixture was centrifuged in Amicon filter tubes at 3000 rpm for 10 min. The encapsulated and non-encapsulated fractions were both collected, and absorption spectra were measured in a spectrophotometer Shimadzu UV-3600 Plus UV-Vis-NIR. The absorbance of these fractions was determined, allowing us to estimate the concentrations of encapsulated and non-encapsulated pigments, using the calibration curve (absorbance vs. concentration) previously obtained. The encapsulation efficiency, EE(%), was calculated using the relation: EE(%) = (Total amount Amount of nonencapsulated extract)/(Total amount) 100. − × 2.9. Application of Betalain- and Anthocyanin-Rich Extracts to Soy-Based Yogurt Alternative In a transparent Petri dish (5 cm2), at room temperature, to the commercial yogurt-like fermented soy (Alpro) (2 mL), non-encapsulate extracts of red beetroot (4.5 mg), opuntia (4 mg), hibiscus (15.5 mg), red radish (32.5 mg), or encapsulated extracts of the same species (mass indicated in Table S1 in Supplementary Materials) were added by stirring with glass stirring rod. These mixtures were kept in the refrigerator (at a temperature of 4 ◦C) for 21 days, and the color was evaluated visually and using the colorimeter at known time periods (after one day, 7 days, 14 days and 21 days of storage). Foods 2020, 9, 771 5 of 13
2.10. Color Measurement The CIELab parameters, L* (brightness), a* (green versus red coordinate) and b* (blue versus yellow coordinate), were measured on a white calibration block using a Chroma Meter CR-400/410 colorimeter (Konica Minolta, Tokyo, Japan). The yogurt-like fermented soy samples were prepared without or with red beetroot, opuntia, hibiscus or red radish non-encapsulated or encapsulated extracts as mentioned above, and color measurements were made in the transparent Petri dish (5 cm2) where they were prepared. Each sample was analyzed at five distinct points.
2.11. Statistical Analysis For biological assays, results were subjected to the Shapiro–Wilk’s normality test to ensure that it followed a normal distribution. Comparison between the means of controls and each experimental condition was performed using ANOVA. The Grubbs’ test was used to identify outliers. Data was expressed as the mean standard error of the mean of 3 independent experiments, each performed in ± triplicate. GraphPad Prism software was used and values were considered statistically significant with a p 0.05. ≤ 3. Results and Discussion
3.1. Betalain- and Anthocyanin-Rich Extracts In order to obtain betalain- and anthocyanin-rich extracts that can be used in food products, namely dairy-free alternatives such as yogurt-like fermented soy, red beetroot, opuntia, hibiscus and red radish were selected as vegetable sources. After being obtaining, each species was subjected to preliminary studies, in which different experimental parameters were varied, namely the solvent, temperature and time, taking into account the optimization of the pigment extraction process (data not shown). However, for all species only the procedures that resulted in the best results are described, supported by previous published works, which are cited in each case. Thus, starting from red beetroot or opuntia, and using a solution of water/ethanol/acetic acid (66.6:33:0.33, v/v/v) at room temperature, with irradiation protection for 48 h or 20 min, respectively, followed by the usual procedures to separate the filtrates and remove the solvents, it was possible to obtain the corresponding lyophilized betalain-rich extracts [37]. Similarly, starting from hibiscus or red radish, by using water/ethanol/acetic acid (70:29.7:0.3, v/v/v) (hibiscus) [38] or water/acetic acid (95:5, v/v) (red radish) [39], and keeping them at a low temperature (4 ◦C) for 18 h (red radish) or 72 h (hibiscus), the lyophilized anthocyanin-rich extracts were obtained. UV-Vis absorption spectroscopy, LRMS and UHPLC-ESI-Q-TOF-MS were used for characterization of the extracts. Based on UV-Vis absorption spectra of aqueous solutions of the lyophilized extracts, the anthocyanin content was previously estimated for red radish and hibiscus, together with the betalain content for red beetroot [40]. Regarding the new opuntia extract, the UV-Vis spectrum was fitted to a sum of the betacyanins spectrum (maximum at λ = 538 nm) and betaxanthins spectrum (maximum at 480 nm) (Figure1, Table1), as these classes of compounds have characteristic and distinguishable absorption spectra [43]. Using the reported spectra for each of the components [43], the content of each dye was determined by Equation (1), and expressed as mg equivalent betanin/L and mg equivalent indicaxanthin/L, respectively [44,45]. A very low content of betaxanthins in the opuntia extract could be observed, betacyanins being the dominant class of dyes in this plant extract. Foods 2020, 9, 771 6 of 13 FoodsFoods 2020 2020, ,9 9, ,x x FOR FOR PEER PEER REVIEW REVIEW 6 ofof 1414
FigureFigure 1. 1. 1. Absorption AbsorptionAbsorption spectrum spectrum spectrum of of opuntia opuntia of opuntia extract extract extract in in aqueous aque inous aqueous solution solution solution(at (at a a concentration concentration (at a concentration of of 4 4 mg/mL). mg/mL). of Inset:4Inset: mg /Spectral mL).Spectral Inset: decomposition decomposition Spectral decomposition in in the the visible visible inspectral spectr the visibleal region region spectral to to obtain obtain region betacyanins betacyanins to obtain and and betacyanins betaxanthins betaxanthins and concentrations.betaxanthinsconcentrations. concentrations. Table 1. Concentration of pigments in the extracts used determined by UV-Vis absorption. TableTable 1. 1. Concentration Concentration of of pigments pigments in in the the extracts extracts used used determined determined by by UV UV-Vis-Vis absorption. absorption. Extract Betalains (mg/L) ExtractExtract BetalainsBetalains (mg/L) (mg/L) Anthocyanins (g/L) Betacyanins Betaxanthins AnthocyaninsAnthocyanins (g/L) (g/L) BetacyaninsBetacyanins BetaxanthinsBetaxanthins 1 1 RedRed Beetroot BeetrootRed Beetroot 20.1 20.1 1 1 20.1 4.274.27 1 1 - - - Opuntia 4.10 0.13 - OpuntiaOpuntia 4.104.10 0.130.13 - - Hibiscus - - 13.01 1 Hibiscus - - - - 13.01 1 1 Hibiscus Red radish - - 1.27 1 13.01 RedRed radish radish - - - - 1.271.27 1 1 1 Previously determined [40]. 1 1Previously Previously determined determined [40]. [40]. Red beetroot extract, analyzed via LRMS acquired in a positive ionization mode (ESI), displayed a RedRed beetroot beetroot extract extract,, analyzed analyzed via via LRMS LRMS acquired acquired in in a a positive positive ionization ionization mode mode (ESI) (ESI),, displayed displayed peak with m/z 550.78 (base peak), attributed to the betanin pigment, according to the literature [14,40]. aa peak peak with with m/z m/z 550.78 550.78 (base(base peak) peak),, attributed attributed to to the the betanin betanin pigment pigment,, accordingaccording to to the the literature literature In the case of red radish extract, UHPLC ESI-Q-TOF-MS/MS analyses revealed the presence of 16 [14,40].[14,40]. In In the the case case of of red red radish radish extract, extract, UHPLC UHPLC ESI ESI-Q-TOF-MS/MS-Q-TOF-MS/MS analyses analyses revealed revealed the the presence presence compounds, some of which showed molecular mass compatible with pelargonidin derivatives, possibly ofof 16 16 compounds, compounds, some some of of which which show showeded molec molecularular mass mass compatible compatible with with pelargonidin pelargonidin derivatives, derivatives, acylated 3-5-heterosides (Table2). Figure2 (1–3) shows the structure of some pelargonin derivatives, possiblypossibly acylated acylated 3 3-5-heterosides-5-heterosides (Table (Table 2). 2). Figure Figure 2 2 (1 (1–3)–3) shows shows the the structure structure of of some some pelargonin pelargonin with molecular masses compatible with analytical data. derivativesderivatives,, with with molecular molecular mass masseses compatible compatible with with analytical analytical data. data.
. FigureFigure 2. 2. Structures Structures of of pelargonid pelargonidin in derivatives derivatives 1 1––33 putatively putatively present present in in red red radish radishradish extract. extract.extract.
Foods 2020, 9, x FOR PEER REVIEW 7 of 14
Table 2. Mass spectrometry data and putative identification for red radish extract based on the known molecular weight and observed M+. Foods 2020, 9, 771 7 of 13 Entry M+ Compound
1 Table403 2. Mass spectrometry data and putative identification- for red radish extract based on the known 2 molecular271 weight and observed M+. Pelargonidin 3 433 Pelargonidin-3-O-glucoside Entry M+ Compound 4 1181 Pelargonidin-3-O-(caffeoyl, feruloyl)-diglucoside-5-O-(malonyl)-glucoside 5 9891 403Pelargonidin-3-O-(p-coumaroyl)diglucoside - -5-O-(malonyl)-glucoside 2 271 Pelargonidin 6 10053 433Pelargonidin-3 Pelargonidin-3--O-(caffeoyl)diglucosideO-glucoside -5-O-glucoside 7 10194 1181 Pelargonidin-3-PelargonidinO-(ca-ff3eoyl,-O-(feruloyl)sophoroside feruloyl)-diglucoside-5-O-(malonyl)-glucoside-5-O-glucoside 8 11355 989Pelargonidin Pelargonidin-3--(p-coumaroyl,O-(p-coumaroyl)diglucoside-5-p-coumaroyl)-diglucosideO-(malonyl)-glucoside-5-O-(malonyl)-glucoside 6 1005 Pelargonidin-3-O-(caffeoyl)diglucoside-5-O-glucoside 9 11657 1019Pelargonidin-3-O Pelargonidin-3--(feruloyl,p-Ocoumaroyl)-(feruloyl)sophoroside-5--diglucosideO-glucoside-5-O-(malonyl)-glucoside 8 1135 Pelargonidin-(p-coumaroyl,p-coumaroyl)-diglucoside-5-O-(malonyl)-glucoside Given 9 the intended 1165 Pelargonidin-3- applicationO -(feruloyl, in yogurtp-coumaroyl)-diglucoside-5--like fermented soyO, -(malonyl)-glucosideas well as other potential applications in food, the cytotoxicity of the lyophilized extracts in human gastric adenocarcinoma (AGS) cells,Given as thea model intended for application gastric cells, in yogurt-like was evaluated fermented after soy, exposure as well as for other 24potential h. We have applications previously describedin food, that the the cytotoxicity extracts ofof the red lyophilized beetroot, extractshibiscus in and human red gastric radish adenocarcinoma had no significant (AGS) impact cells, as o an cell model for gastric cells, was evaluated after exposure for 24 h. We have previously described that viability in this in vitro model [40]. We now describe that the same is true for the opuntia extract, in the extracts of red beetroot, hibiscus and red radish had no significant impact on cell viability in this which no loss of viability was detected up to 0.5 mg/mL (Figure 3), which points to the lack of toxicity in vitro model [40]. We now describe that the same is true for the opuntia extract, in which no loss of of allviability extracts. was detected up to 0.5 mg/mL (Figure3), which points to the lack of toxicity of all extracts.
Figure 3. Viability of AGS cells exposed to opuntia extract for 24 h, as assessed by the MTT assay. Figure 3. Viability of AGS cells exposed to opuntia extract for 24 h, as assessed by the MTT assay. 3.2. Application of the Extracts to Soy-Based Yogurt Alternative 3.2. Application of the Extracts to Soy-Based Yogurt Alternative In order to evaluate the stability of the color of betalain- and anthocyanin-rich extracts from redIn order beetroot, to evaluate opuntia, hibiscusthe stability and redof the radish, color in of alternative betalain- dairy-free and anthocyanin products,-rich such extracts as soy-based from red beetroot,products, opuntia, a yogurt-like hibiscus fermented and red soy radish was, chosenin alternative as the food dairy model.-free The products, extracts were such applied as soy- inbased products,both non-encapsulated a yogurt-like fermented form and encapsulatedsoy was chosen in liposomes as the food of soybean model. lecithin, The extracts as these were systems applied are in reported as useful in the nanoencapsulation/stabilization of different components, as well as being both non-encapsulated form and encapsulated in liposomes of soybean lecithin, as these systems are additives in the food industry [45]. The ethanolic injection and thin film hydration methods were used, reported as useful in the nanoencapsulation/stabilization of different components, as well as being as these are usually associated with high encapsulation efficiencies, depending on the hydrophilicity additivesor hydrophobicity in the food ofindustry the extracts [45]. [46 The,47]. ethanolic injection and thin film hydration methods were used, as The the extractsse are were usually added associated to already-prepared with high yogurt-like encapsulation fermented efficiencies, soy, preventing depending the pigments on the hydrophilicityfrom being or exposed hydrophobicity to inherent of issues the extracts during the [46,47]. production, such as temperature and pH, which canThe a ff execttracts their stability. were added After addition, to already the- foodprepare systemsd yogurt were kept-like at fermented a low temperature soy, preventing (~4 ◦C) for the pigments21 days from of storage. being exposed to inherent issues during the production, such as temperature and pH, which canThe affect color their obtained stability. in the After yogurt-like addition, fermented the food soy systems samples were was kept evaluated at a low visually temperature and by (~4 °C) formeasuring 21 days colorof storage. indexes in a colorimeter, where L* is the degree of lightness, covering a range from black (0) to white (100), a* is the degree of redness and greenness (from 80 to 0 = green; from 0 The color obtained in the yogurt-like fermented soy samples was − evaluated visually and by measuring color indexes in a colorimeter, where L* is the degree of lightness, covering a range from black (0) to white (100), a* is the degree of redness and greenness (from −80 to 0 = green; from 0 to 100
Foods 2020, 9, x FOR PEER REVIEW 8 of 14 Foods 2020Foods, 9,2020 x FOR, 9, PEER 771 REVIEW 8 of 14 8 of 13 FoodsFoodsFoods 2020 Foods2020 2020, , 9 ,20209 ,9 ,x ,x xFOR FOR, FOR9, x PEER PEER FORPEER PEERREVIEW REVIEW REVIEW REVIEW 88 8of of of 14 14 148 of 14 = red), and b* is the degree of yellowness and blueness (from −100 to 0 = blue; from 0 to +70 = yellow). = =red), red), and and b* b* is is the the degree degree of of yellowness yellowness and and blueness blueness (from (from − 100−100 to to 0 0= =blue; blue; from from 0 0to to +70 +70 = =yellow). yellow). = =red), red),=to red),and 100and b* and =b* isred), is theb* the is degree and thedegree degree b* of isof yellowness the yellowness of degreeyellowness and of and yellowness blueness and blueness blueness (from (from and (from − blueness 100−100 −to 100to 0 0= (fromto =blue; blue;0 = blue; from from100 from0 to 0to to 0 +70 0 =+70 toblue; = +70 =yellow). Tableyellow). = from yellow). S 1 0 in to the Supplementary Material shows the results obtained after the addition of the extracts TableTableTableTable S S 1S 1S 1in 1in in in the the the the Supplementary Supplementary Supplementary Supplementary Material Material Material Material shows shows shows shows the the the the results results results results o o btainedo btainedobtainedbtained after after after after the the the the addition addition −addition addition of of of of the the the the extracts extracts extracts extracts +Table70 = Syellow).1 in the TableSupplementary S1 in the Supplementary Material shows Material the results shows obtained the results after the obtained addition after of the(0the days) additionextracts at regular time periods, namely on the 1st, 7th, 14th and 21st days of storage (Figure 4), in (0(0 days) days) at at regular regular time time periods, periods, namely namely on on the the 1st, 1st, 7th, 7th, 14th 14th and and 21st 21st days days of of storage storage (Figure (Figure 4), 4), in in (0(0 days) days)(0of days) the at at regular extracts regular at regular time (0time days) timeperiods, periods, at periods, regular namely namely namely time on onperiods, the theon 1st, the1st, 7th, namely 1st,7th, 14th7th, 14th on 14thand and the 21stand 1st,21st days21st 7th,days 14thdaysof of storage storage andof storage 21st (Figure (Figure days (Figurecomparison 4), of 4), storagein in4), in with the yogurt-like fermented soy without the addition of any extract. The color of a comparisoncomparisoncomparisoncomparison with with withwith the the thethe yogurt yogurt yogurtyogurt--like-like-likelike fermented fermented fermentedfermented soy soy soysoy without without withoutwithout the the thethe addition addition additionaddition of of ofof any any anyany extract. extract. extract.extract. The The TheThe color color colorcolor of of ofof a a a a comparison(Figure4), in with comparison the yogurt with-like the fermented yogurt-like soy fermented without the soy addition without of the any addition extract. ofThe anycommercial color extract. of a strawberry flavored yogurt was also analyzed (L* = 81.19; a* = 9.66; b* = −0.24), and used commercialcommercial s trawberrystrawberry flavor flavoreded yogurt yogurt was was also also analy analyzedzed (L* (L* = =81.19; 81.19; a* a* = =9.66; 9.66; b* b* = =− 0.24),−0.24), and and used used commercialcommercialcommercialThe color s trawberrystrawberry of strawberry a commercial flavor flavor flavoreded yogurtstrawberry yogurted yogurt was was alsowas flavoredalso analyalso analy analyz yogurtedzed (L* z(L*ed = was =(L*81.19; 81.19; = also 81.19; a* a* analyzed= =9.66; a* 9.66; = 9.66;b* b* = (L* = −b* 0.24),−=0.24), = 81.19;−0.24), and andfor useda*and usedcomparison.= used9.66; It can be observed, by the projection in the color plane (a*,b*),that the most stable forforforfor comparison. comparison. comparison.comparison. It It It It can can cancan be be bebe observed, observed, observed,observed, by by byby the the thethe projection projection projectionprojection in in inin the the thethe color color colorcolor plane plane planeplane (a*,b* (a*,b* (a*,b*(a*,b*),),),that),thatthatthat the the thethe most most mostmost stable stable stablestable forb* = comparison.0.24), and usedIt can for be comparison. observed, by It canthe beprojection observed, in by the the color projection plane in(a*,b* the color),that planethe colors,most (a*,b*),that stable in the whole storage period of 21 days at 4 °C (monitored at 7 days intervals), are the ones colors,colors,colors,colors, in in inin the− the thethe whole whole wholewhole storage storage storagestorage period period periodperiod of of ofof 21 21 2121 days days daysdays at at at at 4 4 4 °4 °C °C°C C (monitore (monitore (monitore(monitoreddd d at at at at 7 7 7 days7 days daysdays intervals), intervals), intervals),intervals), are are areare the the thetheobtained ones ones onesones from hibiscus and from red radish, especially the latter. Further, the yogurt-like fermented colors,the most in stablethe whole colors, storage in the period whole storageof 21 days period at 4 of°C 21 (monitore days at 4d◦ Cat (monitored7 days intervals), at 7 days are intervals), the ones obtainedobtainedobtainedobtained from from from from hibiscus hibiscus hibiscus hibiscus and and and and from from from from red red red red radish, radish, radish, radish, especially especially especially especially the the the the latter. latter. latter. latter. Further Further Further Further, ,the ,the ,the the yogurt yogurt yogurt yogurt--like-like-likelike fermented fermented fermented fermented obtainedare the ones from obtained hibiscus from and hibiscus from red and radish, from especially red radish, the especially latter. Further the latter., the Further, yogurt-like thesoy yogurt-likefermented with the red radish pigment, despite the lower L* value, is the most similar in color to the soysoysoysoy with with with with the the the the red red red red radish radish radish radish pigment, pigment, pigment, pigment, despite despite despite despite the the the the lower lower lower lower L* L* L* L* value, value, value, value, is is is is the the the the most most most most similar similar similar similar in in in in color color colorcolor to to toto the the the the soyfermented with the soy red with radish the red pigment, radish pigment, despite the despite lower the L* lower value, L* value,is the ismost the mostsimilar similar in colorcommercial in color to the to sample (Figure 4). commercialcommercialcommercialcommercial sample sample sample sample (F (F (F (Figureigureigureigure 4). 4). 4). 4). commercialthe commercial sample sample (Figure (Figure 4). 4). The same study was performed with encapsulated extracts in soybean lecithin liposomes, TheTheTheThe same same same same study study study study was was was was performed performed performed performed with with with with encapsulated encapsulated encapsulated encapsulated extracts extracts extracts extracts in in in in soybean soybean soybean soybean lecithin lecithin lecithin lecithin liposomes, liposomes, liposomes, liposomes, The same same study study was was performed performed with with encapsulated encapsulated extracts extracts in soybean in soybean lecithin liposomes, lecithinprepared liposomes, prepared either by ethanolic injection method or thin film hydration (Table S1 and Figures 5 and 6). preparedpreparedpreparedprepared either either either either by by by by ethanolic ethanolic ethanolic ethanolic injection injection injection injection method method method method or or or or thin thin thin thin film film film film hydration hydration hydration hydration (Table (Table (Table (Table S S 1S 1S 1and 1and and and Figures Figures Figures Figures 5 5 5and 5and and and 6). 6). 6). 6). preparedeither by ethanoliceither by injectionethanolic method injection or thinmethod film or hydration thin film (Table hydration S1 and (Table Figures S15 and6 Figures). BothBoth methods5 andmethods 6). have shown very high encapsulation efficiencies for extracts of beetroot, red radish BothBothBothBoth methods methods methodsmethods have have havehave shown shown shownshown very very veryvery high high highhigh encapsulation encapsulation encapsulationencapsulation efficienci efficienci efficienciefficiencieseseses for for forfor extracts extracts extractsextracts of of ofof beetroot, beetroot, beetroot,beetroot, red red redred radish radish radishradish Bothhave methods shown very have high shown encapsulation very high e ffiencapsulationciencies for extractsefficienci ofes beetroot, for extracts red radishof beetroot, and hibiscusand red hibiscus radish [40]. [40]. For the opuntia extract, high encapsulation efficiencies were also obtained (Table andandandand hibiscus hibiscus hibiscushibiscus [40]. [40]. [40].[40]. For For ForFor the the thethe opuntia opuntia opuntiaopuntia extract, extract, extract,extract, high high highhigh encapsulation encapsulation encapsulationencapsulation efficiencies efficiencies efficienciesefficiencies were were werewere also also alsoalso obtained obtained obtainedobtained (Table (Table (Table(Table andFor thehibiscus opuntia [40]. extract, For the high opuntia encapsulation extract, high effi cienciesencapsulation were also efficiencies obtained were (Table also3), obtained although3), although (Table these these were slightly lower for the ethanolic injection method. Nevertheless, it was 3),3),3),3), although although although although these these thesethese were were were were slightly slightly slightlyslightly lower lower lower lower for for for for the the the the ethanolic ethanolic ethanolic ethanolic injection injection injection injection method. method. method. method. Nevertheless, Nevertheless, Nevertheless, Nevertheless, it it it it was was was was 3),were although slightly these lower were for the slightly ethanolic lower injection for the method. ethanolic Nevertheless, injection method. it was observed Nevertheless, thatobserved the it wasthin that the thin film hydration method needs high quantities of the extract to obtain a visually observedobservedobservedobserved that that that that the the the the thin thin thin thin film film film film hydration hydration hydration hydration method method method method needs needs needs needs high high high high quantities quantities quantities quantities of of of of the the the the extract extract extract extract to to to to obtain obtain obtain obtain a a a visually avisually visually visually observedfilm hydration that the method thin film needs hydration high quantities method needs of the high extract quantities to obtain of a the visually extract appealing to obtainappealing pinka visually color, pink color, by comparison with the commercial yogurt. appealingappealingappealingappealing pink pink pink pink color color color color, ,by ,by ,by by comparison comparison comparison comparison with with with with the the the the commercial commercial commercial commercial yogurt. yogurt. yogurt. yogurt. appealingby comparison pink withcolor the, by commercialcomparison yogurt.with the commercial yogurt. Table 3. Encapsulation efficiencies, EE (%) ± SD (%), of the several plant extracts in soybean lecithin TableTableTableTable 3. 3. 3. 3.Encapsulation Encapsulation Encapsulation Encapsulation efficiencies, efficiencies, efficiencies, efficiencies, EE EE EE EE (%) (%) (%) (%) ± ± ± SD ±SD SD SD (%), (%), (%), (%), of of of ofthe the the the several several several several plant plant plant plant extracts extracts extracts extracts in in in insoybean soybean soybean soybean lecithin lecithin lecithin lecithin Table 3. Encapsulation efficiencies, efficiencies, EE (%) ± SD (%), of the several plant extracts in soybean lecithin liposomes , prepared by either by ethanolic injection or thin film hydration (SD: standard deviation). ± liposomesliposomesliposomesliposomesliposomes, ,prepared ,prepared ,prepared prepared, prepared by by by by either either either either by eitherby by by by ethanolic ethanolic ethanolic ethanolic by ethanolic injection injection injection injection injection or or or orthin thin thin thin or film film filmthin film hydration hydration hydrationfilm hydration hydration (SD: (SD: (SD: (SD: standard standard (SD:standard standard standard deviation). deviation). deviation). deviation). deviation). liposomes, prepared by either by ethanolic injection or thin film hydration (SD: standard deviation). Species Ethanolic Injection Thin Film Hydration SpeciesSpeciesSpeciesSpecies EthanolicEthanolicEthanolicEthanolic Injection Injection Injection Injection ThinThinThinThin Film Film Film Film Hydration Hydration Hydration Hydration 1 1 Species SpeciesEthanolic Ethanolic Injection Injection Thin FilmThin Hydration Film Hydration Red Beetroot 98.4 3.1 99.3 3.6 1111 1111 RedRedRedRed Beetroot Beetroot RedBeetroot Beetroot Beetroot 98.498.498.498.4 98.4 3.1 3.1 3.1 3.1 3.1 1 99.399.399.399.3 99.3 3.6 3.6 3.6 3.6 3.6 1 Opuntia 90.9 1.8 92.8 2.0 Red Beetroot 98.4 3.1 1 99.3 3.6 1 OpuntiaOpuntiaOpuntiaOpuntia 90.990.990.990.9 1.8 1.8 1.8 1.8 ± ±92.892.892.892.8 2.0 2.0 2.0 2.0 1 1 Opuntia Opuntia90.9 90.91.8 1.8 92.8 2.0 92.8 2.0 Hibiscus 99.2 4.8 98.1 2.9 1 1 Hibiscus 99.2 4.8 111 ± 98.1± 2.9 111 1 1 HibiscusHibiscusHibiscusHibiscus Hibiscus 99.299.299.2 99.2 4.8 4.8 4.8 99.2 4.8 1 4.8 1 98.1 98.198.12.998.1 1 98.1 2.9 2.9 2.9 2.9 1 Red radish 99.9 6.2 99.6 4.2 1111 ± 1 ± 1 1111 RedRedRedRed radish radish Redradish radish radish Red radish99.999.999.999.9 99.9 6.2 6.2 6.2 6.2 99.9 6.2 1 6.2 99.6 99.699.699.64.299.6 99.6 4.2 4.2 4.2 4.2 4.2 1 1 Previously determined [40]. ± ± 1111 Previously Previously Previously Previously1 Previously1 Previously determined determined determined determined determined [40]. [40]. [40]. [40]. [ 40 [40]. ].
Figure 4. Representation of the colors of the several samples in L*a*b* color scheme, immediately after FigureFigureFigureFigureFigure 4. 4. 4. Representation4. Representation Representation Representation 4. RepresentationRepresentation of of of ofthe the the the colorsof ofcolors colors colorsthe the colorsof colorsof of ofthe the the the severalof ofseveral several severalthe the several several samples samples samples samples samples samples in in in inL*a*b* L*a*b* L*a*b* L*a*b* in in L*a*b* L*a*b*color color color color scheme colorscheme colorscheme scheme scheme scheme,, ,immediately ,immediately ,immediately immediately, immediately immediately after after after after afterafter extract addition (red symbols), and after 1 day (green symbols), 7 days (blue symbols), 14 days (cyan extractextractextractextractextract addition addition addition addition addition (red (red (red (red symbols) symbols) symbols)(red symbols) symbols) symbols),, ,and ,and ,and and after ,after after and after 1 1 after 1 day 1day day day (green1 (green (green day (green (green symbols) symbols) symbols) symbols) symbols) symbols),, ,7 ,7 , 7 days 7days days days, 7 (blue (blue days(blue (blue symbols)(blue symbols) symbols) symbols) symbols) symbols),, ,14 ,14 ,14 14 days days days days, 14 (cyan (cyan days(cyan (cyan (cyan symbols) and 21 days (magenta symbols) of storage at 4 C. Projection in a*,b* plane: open symbols. symbols) and 21 days (magenta symbols) of storage at 4 C. Projection in a*,b* plane: open symbols. symbols)symbols)symbols)symbols) and and and 21 21 and21 days days days 2121 days(magenta days(magenta (magenta (magenta(magenta symbols) symbols) symbols) symbols) symbols) of of of storage storage storage of of storage storage at at at 4 4 at4 C.atC. 4C. ◦4Projection Projection C. ProjectionC. Projection Projection in in in a*,b* ina*,b* a*,b* in a*,b* plane: a*,b*plane: plane: plane: plane: open open open open opensymbols. symbols. symbols. symbols. symbols. : : red beetroot; : opuntia; : hibiscus; : red radish; : commercial strawberry flavor yogurt. : :red :red :red redred beetroot; beetroot; :beetroot; beetroot;red beetroot; beetroot; : :opuntia; ::opuntia; :opuntia; opuntia;: opuntia; : :hibiscus; :hibiscus; : hibiscus;hibiscus; hibiscus;: hibiscus; :: :red : red :red red radish; radish;radish; :radish; radish;red radish; : :commercial : commercialcommercial :commercial commercial : commercial strawberry strawberrystrawberry strawberry strawberry strawberry flavor flavorflavor flavor flavor flavoryogurt. yogurt.yogurt. yogurt. yogurt. yogurt.
FoodsFoodsFoods 2020 20202020, ,9, 99, ,x, xx FOR FORFOR PEER PEERPEER REVIEW REVIEWREVIEW 888 of ofof 14 1414 Foods 2020, 9, x FOR PEER REVIEW 8 of 14 === red), red),red), and andand b* b*b* is isis the thethe degree degreedegree of ofof yellowness yellownessyellowness and andand blueness bluenessblueness (from (from(from − −−100100100 to toto 0 00 = == blue; blue;blue; from fromfrom 0 00 to toto +70 +70+70 = == yellow). yellow).yellow). = red), and b* is the degree of yellowness and blueness (from −100 to 0 = blue; from 0 to +70 = yellow).TableTableTable S SS11 1 in inin the thethe Supplementary SupplementarySupplementary Material MaterialMaterial shows showsshows the thethe results resultsresults o oobtainedbtainedbtained after afterafter the thethe addition additionaddition of ofof the thethe extracts extractsextracts Table S1 in the Supplementary Material shows the results obtained after the addition of the (0extracts(0(0 days) days)days) at atat regular regularregular time timetime periods, periods,periods, namely namelynamely on onon the thethe 1st, 1st,1st, 7th, 7th,7th, 14th 14th14th and andand 21st 21st21st days daysdays of ofof storage storagestorage (Figure (Figure(Figure 4), 4),4), in inin (0 days) at regular time periods, namely on the 1st, 7th, 14th and 21st days of storage (Figurecomparisoncomparisoncomparison 4), in with withwith the thethe yogurt yogurtyogurt--like-likelike fermented fermentedfermented soy soysoy without withoutwithout the thethe addition additionaddition of ofof any anyany extract. extract.extract. The TheThe color colorcolor of ofof a aa comparison with the yogurt-like fermented soy without the addition of any extract. The colorcommercialcommercialcommercial of a s sstrawberrytrawberrytrawberry flavor flavorflavorededed yogurt yogurtyogurt was waswas also alsoalso analy analyanalyzzzededed (L* (L*(L* = == 81.19; 81.19;81.19; a* a*a* = == 9.66; 9.66;9.66; b* b*b* = == − −−0.24),0.24),0.24), and andand used usedused commercial strawberry flavored yogurt was also analyzed (L* = 81.19; a* = 9.66; b* = −0.24), andforforfor used comparison. comparison.comparison. It ItIt can cancan be bebe observed, observed,observed, by byby the thethe projection projectionprojection in inin the thethe color colorcolor plane planeplane (a*,b* (a*,b*(a*,b*),),),thatthatthat the thethe most mostmost stable stablestable for comparison. It can be observed, by the projection in the color plane (a*,b*),that the mostcolors,colors,colors, stable in in in the thethe whole wholewhole storage storagestorage period periodperiod of ofof 21 2121 days daysdays at atat 4 44 ° °C°CC (monitore (monitore(monitoreddd at atat 7 77 days daysdays intervals), intervals),intervals), are areare the thethe ones onesones colors, in the whole storage period of 21 days at 4 °C (monitored at 7 days intervals), are theobtainedobtainedobtained ones from fromfrom hibiscus hibiscushibiscus and andand from fromfrom red redred radish, radish,radish, especially especiallyespecially the thethe latter. latter.latter. Further FurtherFurther, ,, the thethe yogurt yogurtyogurt--like-likelike fermented fermentedfermented obtained from hibiscus and from red radish, especially the latter. Further, the yogurt-like fermentedsoysoysoy with with with the the the red red red radish radish radish pigment, pigment, pigment, despite despite despite the the the lower lower lower L* L* L* value, value, value, is is is the the the most most most similar similar similar in in in colorcolorcolor tototo the the the soy with the red radish pigment, despite the lower L* value, is the most similar in colorcommercialcommercialcommercial to the sample samplesample (F (F(Figureigureigure 4). 4).4). commercial sample (Figure 4). TheTheThe same same same study study study was was was performed performed performed with with with encapsulated encapsulated encapsulated extracts extracts extracts in in in soybean soybean soybean lecithin lecithin lecithin liposomes, liposomes, liposomes, The same study was performed with encapsulated extracts in soybean lecithin liposomes,preparedpreparedprepared either eithereither by byby ethanolic ethanolicethanolic injection injectioninjection method methodmethod or oror thin thinthin film filmfilm hydration hydrationhydration (Table (Table(Table S SS111 and andand Figures FiguresFigures 5 55 and andand 6). 6).6). prepared either by ethanolic injection method or thin film hydration (Table S1 and Figures 5Both BothBothand methods6). methodsmethods have havehave shown shownshown very veryvery high highhigh encapsulation encapsulationencapsulation efficienci efficienciefficiencieseses for forfor extracts extractsextracts of ofof beetroot, beetroot,beetroot, red redred radish radishradish Both methods have shown very high encapsulation efficiencies for extracts of beetroot, redandandand radish hibiscus hibiscushibiscus [40]. [40].[40]. For ForFor the thethe opuntia opuntiaopuntia extract, extract,extract, high highhigh encapsulation encapsulationencapsulation efficiencies efficienciesefficiencies were werewere also alsoalso obtained obtainedobtained (Table (Table(Table and hibiscus [40]. For the opuntia extract, high encapsulation efficiencies were also obtained3),3),3), (Table although although although thesethesethese were were were slightlyslightlyslightly lower lower lower for for for the the the ethanolic ethanolic ethanolic injection injection injection method. method. method. Nevertheless, Nevertheless, Nevertheless, it it it was was was 3), although these were slightly lower for the ethanolic injection method. Nevertheless,observed observedobserved it was that thatthat the thethe thin thinthin film filmfilm hydration hydrationhydration method methodmethod needs needsneeds high highhigh quantities quantitiesquantities of ofof the thethe extract extractextract to toto obtain obtainobtain a aa visually visuallyvisually observed that the thin film hydration method needs high quantities of the extract to obtain a appealingvisuallyappealingappealing pink pinkpink color colorcolor, ,,by byby comparison comparisoncomparison with withwith the thethe commercial commercialcommercial yogurt. yogurt.yogurt. appealing pink color, by comparison with the commercial yogurt. TableTableTable 3. 3.3. Encapsulation EncapsulationEncapsulation efficiencies, efficiencies,efficiencies, EE EEEE (%) (%)(%) ± ±± SD SDSD (%), (%),(%), of ofof the thethe several severalseveral plant plantplant extracts extractsextracts in inin soybean soybeansoybean lecithin lecithinlecithin Table 3. Encapsulation efficiencies, EE (%) ± SD (%), of the several plant extracts in soybean lecithin liposomesliposomesliposomes, ,,prepared preparedprepared by byby either eithereither by byby ethanolic ethanolicethanolic injection injectioninjection or oror thin thinthin film filmfilm hydration hydrationhydration (SD: (SD:(SD: standard standardstandard deviation). deviation).deviation). liposomes, prepared by either by ethanolic injection or thin film hydration (SD: standard deviation). SpeciesSpeciesSpecies EthanolicEthanolicEthanolic Injection InjectionInjection ThinThinThin Film FilmFilm Hydration HydrationHydration Species Ethanolic Injection Thin Film Hydration RedRedRed Beetroot BeetrootBeetroot 98.498.498.4 3.1 3.13.1 1 1 1 99.399.399.3 3.6 3.63.6 1 1 1 Red Beetroot 98.4 3.1 1 99.3 3.6 1 OpuntiaOpuntiaOpuntia 90.990.990.9 1.8 1.81.8 92.892.892.8 2.0 2.02.0 Opuntia 90.9 1.8 92.8 2.0 HibiscusHibiscusHibiscus 99.299.299.2 4.8 4.84.8 1 1 1 98.198.198.1 2.9 2.92.9 1 1 1 Hibiscus 99.2 4.8 1 98.1 2.9 1 RedRedRed radish radishradish 99.999.999.9 6.2 6.26.2 1 1 1 99.699.699.6 4.2 4.24.2 1 1 1 Red radish 99.9 6.2 1 99.6 4.2 1 11 1Previously PreviouslyPreviously determined determineddetermined [40]. [40].[40]. 1 Previously determined [40]. Foods 2020, 9, 771 9 of 13 Foods 2020, 9, x FOR PEER REVIEW 9 of 14 Foods 2020, 9, x FOR PEER REVIEW 9 of 14
Figure 5. Representation of the colors of the several samples in L*a*b*FigureFigureFigure color 4. scheme 4.4. Representation RepresentationRepresentation immediately of ofof the thethe after colors colorscolors of ofof the thethe several severalseveral samples samplessamples in inin L*a*b* L*a*b*L*a*b* color colorcolor scheme schemescheme, ,,immediately immediatelyimmediately after afterafter FigureFigure 5. 5. Representation Representation of of thethe colorscolors of the several samples in L*a*b* colorcolor schemescheme immediately immediately after after Figure 4. Representation of the colorsextract of the addition several (encapsulatedsamples in L*a*b* in soybean color scheme lecithin, immediately liposomes by after ethanolic extractextractextract injection) addition additionaddition (red (red (red(red symbols), symbols) symbols)symbols) and, ,,and andand after afterafter 1 11 day dayday (green (green(green symbols) symbols)symbols), ,,7 77 days daysdays (blue (blue(blue symbols) symbols)symbols), ,,14 1414 days daysdays (cyan (cyan(cyan extractextract addition addition (encapsulated(encapsulated inin soybeansoybean lecithin liposomes byby ethanolicethanolic injection)injection) (red(red symbols) symbols), , extract addition (red symbols), andafter after 11 dayday (green(green symbols),symbols), 7 days days (blue (blue symbols) symbols),, 14 14 days days (cyan (cyan symbols) symbols) and and 21 21 days days (magenta (magenta (magenta symbols) symbols) of of storage storage at at 4 4 C.C. Projection Projection in in a*,b* a*,b* plane: plane: open open symbols. symbols. andand after after 1 1 day day (green (green symbols) symbols),, 77 daysdays (blue symbols), 14 days (cyan(cyan symbols)symbols) and and 21 2121 days daysdays (magenta (magenta (magenta symbols) of storage at 4 C. Projection in a*,b* plane: open symbols. symbols) and 21 days (magenta symbols)symbols) of ofstorage storage at at4 4C.◦ C.Projection Projection in ina*,b* a*,b* plane: plane: open open symbols. symbols. : ::red red red beetroot; beetroot; beetroot; :: :opuntia; opuntia; :: :hibiscus; hibiscus; : :red red radish; radish; : :commercial commercial strawberry strawberry flavor flavor yogurt. yogurt. symbols)symbols) of of storage storage at at 4 4 ° °C.C. ProjectionProjection in a*,b* plane: open symbols. :: redred: red beetroot; beetroot; beetroot; : :opuntia; opuntia;: opuntia; : hibiscus; : red radish; : commercial strawberry flavor yogurt. hibiscus; : red radish. : red beetroot; : opuntia; : hibiscus;: :hibiscus; hibiscus; : red: :red redradish; radish. radish. : commercial strawberry flavor yogurt. It can be concluded that the encapsulation by ethanolic injection method (Figure5) protects the ItIt can can be be concluded concluded thatthat thethe encapsulationencapsulation by ethanolic injectioninjection methodmethod (Figure(Figure 5) 5) protects protects the the dyes, but also changes the color, as the encapsulation systems are yellowish due to the soybean lecithin dyes,dyes, but but also also changes changes the the color color,, asas the the encapsulation systems are are yellowish yellowish due due to to thethe soybeansoybean components.lecithinlecithin components. components. However, However,However, this is not thisthis very isis significant, notnot veryvery significant, as the b* valuesasas thethe b*b* are valuesvalues slightly areare negative,slightly slightly negative, negative, but so are but but the
a*so values.so are are the the These a* a* values. values. results TheseThese suggest resultsresults that suggestsuggest the ethanolic that the injection ethanolic method injectioninjection can methodmethod be an caninterestingcan bebe an an interesting interesting alternative
whenalternativealternative the objective when when is the the to complementobjective objective is is to to the complement complement food system the with food betalain system system or anthocyaninwithwith betalain betalain pigments, or or anthocyanin anthocyanin without causingpigmentspigments a significant, ,without without causing changecausing a ina significantsignificant its color. change in its color. RegardingRegardingRegarding the the the thin thinthin film filmfilm hydrationhydrationhydration method method (Figure (Figure 6)6),,, thethe the colorcolor color waswas was notnot not stablestable stable for for forthe the theperiod period period of of of21 21 days, days, with with with significant significant significant andand and erroneouserroneous erroneous changes changes in color in color parameters parameters andand lightness,lightness, and lightness, indicating indicating indicating a a non non- - a non-protectionprotectionprotection of of the ofthe thedyes dyes dyes or or anan or uncontrolleduncontrolled an uncontrolled (non (non-gradual)-gradual) release release fromfrom from thethe nanocarriers.nanocarriers. the nanocarriers.
Figure 6. Representation of the colors of the several samples in the L*a*b* color scheme immediately FigureFigure 6. 6.Representation Representation of of the the colorscolors ofof thethe severalseveral samples in the L*a*b* color color scheme scheme immediately immediately after extract addition (encapsulated in soybean lecithin liposomes by thin film hydration), (red afterafter extract extract addition addition (encapsulated (encapsulated in soybean in soybean lecithin lecithin liposomes liposomes by thin by film thin hydration), film hydration), (red symbols), (red symbols), and after 1 day (green symbols), 7 days (blue symbols), 14 days (cyan symbols) and 21 days andsymbols) after 1, dayand (greenafter 1 symbols),day (green 7 symbols) days (blue, 7 days symbols), (blue 14symbols) days (cyan, 14 days symbols) (cyan symbols) and 21 days and (magenta 21 days (magenta symbols) of storage at 4 °C. Projection in a*,b* plane: open symbols. : red beetroot; : symbols)(magenta of symbols) storage at of 4 storage◦C. Projection at 4 °C. inProjection a*,b* plane: in a*,b* open plane: symbols. open symbols.: red beetroot; : red beetroot;: opuntia; : : opuntia; : hibiscus; : red radish. hibiscus;opuntia; : red: hibiscus; radish. : red radish.
Foods 2020, 9, 771 10 of 13
In order to obtain a visually satisfactory and interesting pink color comparable with the commercial sample, the yogurt-like fermented soy samples must have positive a* values (which suggest the presence of red pigments), and b* values close to zero, as yellow or blue pigments must not contribute significantly to the resulting color. The data obtained with non-encapsulated extracts show that on day 0, the highest value of a* occurred with red beetroot extract (a* = 7.84), followed by the red radish, opuntia and hibiscus (a* = 3.85) extracts, in that order. Further, on day 0, the highest b* index was displayed by red radish extract (b* = 0.61), followed by hibiscus, red beetroot and opuntia (b* = 5.78). Over the − 21-days storage period, it appears that the values of a* decreased in all samples with non-encapsulated extracts, except for the sample with red radish, which increased (a* = 9.86). As for the b* values, over this period, all samples approached zero, except in the case of the hibiscus extract that increased to a positive value (b* = 5.24), thus registering the contribution of yellow pigments to its color. The visual observation of the samples cohered with the results of the colorimeter, that is, the most stable color was given by the red radish extract. These results may be due to the high amount of pigment added in the case of the radish extract, and its stabilizing properties (copigmentation) [48]. The color obtained with the remaining extracts was lost over time, probably due to the instability of the pigments in the yogurt-like fermented soy at its usual pH (pH value is 4.8) [49–51]. In fact, decreases in pH, a function of the lactic acid bacteria in fermented milk, cause an increase of the hydroxylation of the phenolic rings, which could result in a change of pigments towards blueish tones, as previously observed by other authors for anthocyanin-based extracts [52,53]. Regarding the samples of yogurt-like fermented soy with extracts encapsulated by the ethanol injection method, on day 0, the highest a* value was shown by the sample with red beetroot extract (a* = 0.98), followed by the one with opuntia, red radish and hibiscus (a* = 1.78). On the 21st day, the − − best result appeared in the sample with the red radish extract (a* = 1.19). Considering b* values, on − day 0, the most suitable values were obtained with red beetroot (b* = 1.31) and hibiscus (b* = 1.21) − − extracts (with very close values), and over the 21 days, the extracts in the yogurt-like fermented soy with the best b* values remain unaltered (b* = 1.89, red beetroot; b* = 1.78, hibiscus). The lower values of − − a* and b* in the first day of the experiment are due to the fact that the pigments are encapsulated in soy lecithin. It would be expected that over time these values would improve, as a result of the release of the pigments. However, there is only a slight increase, with the exception of red beetroot, which suggests that red radish, hibiscus and opuntia extracts are protected by nanoencapsulation, and after their release, there is no pigment degradation. As for red beetroot, the decrease in the values of a* and b* may indicate that the encapsulation did not protect the dyes in the yogurt-like fermented soy sample, and the pigments suffered degradation, probably due to the low pH of the yogurt-like fermented soy. In the samples of yogurt-like fermented soy with extracts encapsulated by the thin film hydration method, on day 0, the highest a* values were recorded for opuntia (a* = 9.6), followed by red beetroot, red radish and hibiscus (a* = 1.07). Regarding the b*, the best value was obtained with hibiscus − extract (b* = 1.65), followed by red radish, red beetroot and opuntia (b* = 10.51). Over the 21 days of − − storage, the sample with opuntia extract continued to display the highest a* value (a* = 3.52), followed by hibiscus, red radish and beetroot (a* = 0.48). The best result for b* was recorded in the hibiscus − extract (b* = 0.08). A significant increase in b* over 21 days was registered in the samples with − opuntia, red radish and beetroot extract, but this did not occur with the hibiscus extracts. Further, as mentioned, this method needed a high amount of the extracts in order to obtain a satisfactory color. Overall, considering the results with the encapsulated extracts, it seems that protection of the pigments occurred in the cases of red radish and hibiscus extracts, since there was an increase in both their a* and b* indexes. On the other hand, the application of opuntia and beetroot extracts resulted in a decrease of a* and b* values, which indicates a decrease in the stability of the pigments after their release. Therefore, with regards to application in yogurt-like fermented soy, encapsulation may be considered if there is a need to protect the dyes from degradation, and, for this purpose, a further investigation is needed regarding lipid composition and stability. Foods 2020, 9, 771 11 of 13
4. Conclusions Betalain-rich extracts of red beetroot and opuntia, as well as anthocyanin-rich extracts of hibiscus and red radish, were obtained. Incorporation of the extracts in soybean lecithin nanosystems was performed via ethanolic injection and film hydration, which resulted in moderate to excellent encapsulation efficiencies. Non-encapsulated and encapsulated extracts were added to yogurt-like fermented soy, and color stability was visually and colorimetrically evaluated during storage at ~4 ◦C, for 21 days. The results showed that the non-encapsulated extract of red radish afforded to yogurt-like fermented soy the most appealing pink color, which suggested that this extract may be very promising for future coloring applications in soybean-based food products. On the other hand, nanoencapsulated opuntia extracts can also be used to supplement yogurt-like fermented soy with betalains, imparting their health benefits without changing significantly the color.
Supplementary Materials: The following are available online at http://www.mdpi.com/2304-8158/9/6/771/s1, Table S1: Variations in color index of soy-based yogurt alternative during 21 days of storage. Soy-based yogurt alternative – SY; B – Red beetroot; O – Opuntia; H - Hibicus; R - Red radish; Ei - Ethanolic injection method; Fh - thin film hydration method. Amounts of extracts (mg) are given in brackets. Author Contributions: Conceptualization, S.D. and M.S.T.G.; methodology, S.D., M.S.T.G. and A.G.F.; validation, A.G.F., E.M.S.C. and M.S.T.G.; formal analysis, E.M.S.C. and D.M.P.; investigation, S.D., M.S.T.G., D.M.P. and E.M.S.C.; writing—original draft preparation, M.S.T.G.; writing—review and editing, M.S.T.G., D.M.P., A.G.F. and E.M.S.C.; supervision, E.M.S.C. and M.S.T.G.; project administration, M.S.T.G. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by COMPETE 2020 program, co-financed by the FEDER and the European Union, PTDC/ASP-AGR/30154/2017 (POCI-01-0145-FEDER-030154). Foundation for Science and Technology (FCT, Portugal), and FEDER-COMPETE-QREN-EU funded research centers CQ-UM (UID/QUI/00686/2019), CF-UM-UP (UID/FIS/04650/2019) and REQUIMTE (UIDB/50006/2020). Acknowledgments: Thanks are also due to Joel Borges and Filipe Vaz for the availability of and prompt access to the colorimeter. Conflicts of Interest: The authors declare no conflict of interest.
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