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Microencapsulation by spray-drying of polyphenols extracted from red chicory and red cabbage: Effects on stability and color properties
Journal Pre-proofs Microencapsulation by spray-drying of polyphenols extracted from red chicory and red cabbage: effects on stability and color properties Francesca Zanoni, Martina Primiterra, Nicola Angeli, Gianni Zoccatelli PII: DOI: Reference:
S0308-8146(19)31654-1 https://doi.org/10.1016/j.foodchem.2019.125535 FOCH 125535
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
14 May 2019 5 September 2019 13 September 2019
Please cite this article as: Zanoni, F., Primiterra, M., Angeli, N., Zoccatelli, G., Microencapsulation by spray-drying of polyphenols extracted from red chicory and red cabbage: effects on stability and color properties, Food Chemistry (2019), doi: https://doi.org/10.1016/j.foodchem.2019.125535
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Microencapsulation by spray-drying of polyphenols extracted from red chicory and red cabbage: effects on stability and color properties
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Francesca Zanoni , Martina Primiterra , Nicola Angeli , Gianni Zoccatelli
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University of Verona, Department of Biotechnology, Verona, Italy
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Sphera Encapsulation Srl, Verona, Italy
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MUSE, Limnology and Phycology Section, Trento, Italy
*Corresponding Author: Gianni Zoccatelli, PhD Dipartimento di Biotecnologie Università degli Studi di Verona Strada Le Grazie, 15 - CV1 I-37134 Verona, Italy Tel: +39 045 8027952 Fax: +39 045 8027929 e-mail: [email protected]
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Abstract The research of antioxidants and natural pigments to replace synthetic molecules is increasingly considering wastes from plant food supply chains. Red chicory (RCH) and red cabbage (RCA) are rich sources of polyphenols (PP), especially anthocyanins, well know natural pigments possessing strong antioxidant capacity and beneficial health effects. The aim of this work was to compare different solvents for PP extraction and to evaluate the effect of spraydrying encapsulation using modified starch on PP, antioxidant capacity (AOC) and color properties. Methanol:water (70:30) showed the best extraction capacity, while ethanol:water (70:30) extracts displayed the highest thermal stability. Ethanol:water extracts were spray-dried with a yield of 95-99% for both crops, while the efficiency of PP encapsulation was 79% (RCA) and 88% (RCH). Encapsulation improved retention of PP and AOC upon thermal treatment (RCH: 20-30%, RCA: 44-55%) without altering color properties. This process can be employed for the development of functional foods and supplements.
Keywords Red Chicory (Chicorium intibus L.), Red cabbage (Brassica oleracea L. var capitata f.rubra), polyphenols, microencapsulation; antioxidant capacity (AOC); spray-drying. Abbreviations: RCH: red Chicory; RCHE: red Chicory extract; RCA: red cabbage; RCAE: red cabbage extract; EtOH: ethanol, MetOH: methanol; aw: water activity; GA: gallic acid; GAE: gallic acid equivalents; TE: Trolox equivalents; PP: Polyphenols; TP: total polyphenols; SP: superficial polyphenols; EE: Encapsulation efficiency; YE: yield of encapsulation; Abs: absorbance; f.w.: fresh weight; OSA: octenyl succinic anhydride; ABTS: 2,2'-azino-bis (3ethylbenzothiazoline-6-sulphonic acid.
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1.
Introduction
The research for natural antioxidants and the growing importance in the management and requalification of industrial food wastes is increasingly considering plant food by-products as source of valuable molecules, e.g. polyphenols, carotenoids and fibers (Lante, Nardi, Zocca, Giacomini, & Corich, 2011). The exploitation of this type of resources gives the possibility to lower the amount of food wastes, hence diminishing the negative impact on the environment due to the phytotoxic effect of the high organic matter content (Lavelli, Harsha, Laureati, & Pagliarini, 2017). In particular, among the different classes of molecules with health promoting effects, during the last 10 years, researchers and food industries have been focusing their attention on polyphenols. The reasons rely on the fact that they are present in great abundance in our diet, and are involved in the prevention of various chronic-degenerative pathologies such as cardiovascular diseases (Scalbert, Manach, Morand, Remesy, & Jimenez, 2005), diabetes (Matsui, Ogunwande, Abesundara, & Matsumoto, 2006), neurodegenerative diseases (Levites, Amit, Youdim, & Mandel, 2002) and osteoporosis (Dudaric, Fuzinac-Smojver, Muhvic, & Giacometti, 2015). Red chicory (Chicorium intibus L.) and red cabbage (Brassica oleracea L. var capitata f.rubra) are both important sources of polyphenols. Moreover, the wastes produced during their processing, consisting mainly of leaves and steams, can reach up to 40-50% of the total harvested material, that for the great part is still nutritionally and hygienically valid especially in the case of the fourth-range production where edible material is eliminated also to improve the aspect of the food and increase the acceptability of the consumer ; Lante, Nardi, Zocca, Giacomini, & Corich, 2011; Llorach, Tomas-Barberan, & Ferreres, 2004). RCH is a typical winter vegetable indigenous to Europe, North and Western America that has gained attention for its content of phytochemicals with potential health benefits, such as phenolic acids, flavonoids and anthocyanins (Bais & Ravishankar, 2001). The high content of anthocyanins, that account for the 50-55% of the total phenolic compounds, is responsible for the characteristic deep red color of the leaves. Among all the leafy vegetables that are consumed raw, red chicory has been recognized to have the highest content of polyphenols (Gazzani, Daglia, Papetti, & Gregotti, 2000;). RCA, in the past indigenous of the Mediterranean zone, is now one of the most important vegetables grown around the world (Wiczkowski, Szawara-Nowak, & Topolska, 2013). The abundance and the relative low cost of production, if compared to other vegetables rich in polyphenols (e.g. black carrot and purple potatoes), make it an attractive source
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of natural red pigments (Bridle & Timberlake, 1997; ). Its use has been reported in the treatment of inflammatory bowel disease (Zielińska, Lewandowska, Podsedek, Cygankiewicz, Jacenik, Sałaga, et al., 2015), and in the attenuation of cardiac and hepatic oxidative stress (Sankhari, Thounaojam, Jadeja, Devkar, & Ramachandran, 2012). It is worth considering that the production and harvest of these vegetables are seasonal activities and the by-products obtained from their processing need to be handled in a limited time frame, since once extracted, polyphenols are highly susceptible to degradation (Patras, Brunton, O'Donnell, & Tiwari, 2010). Spray-drying has been widely used to dry a number of foods and pharmaceutical preparations, among which many containing heat-sensitive molecules such as polyphenols (Mahdavi, Jafari, Ghorbani, & Assadpoor, 2014; Shishir & Chen, 2017). It is the most common technique used to turn liquids with high solid content into powders. In addition, it is recognized as an economic process by which it is possible to produce high quality powders. To our knowledge, no work about the stabilization of polyphenols from red chicory through spray-drying has been addressed, and only two works about the stabilization of polyphenols from red cabbage have been described (Bernstein & Noreña, 2015; Hongmei & Meng, 2015). In these studies only the production and characterization of the powders were tackled without evaluating their performance in a simulating medium. The aim of this work was to assess the quality and the thermal stability of phenolic extracts from red chicory and cabbage leaves obtained by different solvents (water, and hydro-alcoholic mixtures of ethanol and methanol) and to improve the stability of polyphenols and antioxidant capacity by spray-drying encapsulation without altering the color properties of the pigments.
2. Materials and methods: Materials Fresh Cichorium intybus L. (red chicory var. Chioggia) and Brassica oleracea L. (red cabbage var. Capitata f. rubra) were purchased from a local market. Modified starch CAPSUL® was provided by Ingredion (Illinois, US). Folin-Ciocalteau reagent, sodium carbonate, formic acid, methanol, ABTS, potassium persulfate, 37% chloridric acid, ethyl acetate, Trolox®, gallic acid and ethanol were obtained from Sigma Aldrich (St. Louis, MO, US).
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2.1. Extraction of polyphenols Polyphenols were extracted from RCH and RCA leaves combining different protocols with some modifications (Cefola, Carbone, Minasi, & Pace, 2016; Heimler, Isolani, Vignolini, & Romani, 2009; Innocenti, Gallori, Giaccherini, Ieri, Vincieri, & Mulinacci, 2005; McDougall, Fyffe, Dobson, & Stewart, 2007; Pereira, de Arruda, & Stefani, 2015). Briefly, 500 g of RCH fresh leaves were ground with a SAMA K45 cutter (Dito SAMA, Italy). The obtained sample was extracted with the solvent for 4 h in continuous agitation (ratio sample: solvent 1:1). Formic acid was used to adjust the pH of the sample to 3.3-3.5. The extract was filtered through Whatman filter paper in a Buchner funnel. The obtained solution was kept at 4°C in the dark until use. Three different extraction solvents were tested: water, 30% water:70% EtOH and 30% water:70% MetOH. The same operations were conducted with RCA. Methanol and ethanol were then removed by evaporation with a rotary evaporator (Büchi, Switzerland) prior use/storage.
2.2. Polyphenols quantification The Folin-Ciocalteau assay was performed following a method previously proposed (Singleton, Orthofer, & LamuelaRaventos, 1999) with some modifications. Briefly, the reaction was conducted in a 96 well micro-plate (Sarstedt, Germany). An amount of 5 ul of each sample was incubated for 3 minutes with 150 ul of Folin-Ciocalteau reagent (previously diluted at 1:15 with water). Finally, 40 ul of 20% sodium carbonate solution was added and left to react for 30 minutes in the dark. The absorbance was measured using a micro-plate reader (BioTek, Milan, Italy) at 750 nm. Water was used as blank. The calibration curve was carried out using GA as standard. The results were expressed as milligrams GAE per gram of f.w. All the experiments were performed in triplicate.
2.3. Antioxidant capacity The procedure proposed by Thaipong and co-workers was followed (Thaipong, Boonprakob, Crosby, Cisneros-Zevallos, & Byrne, 2006) with some modifications. The stock solutions consisting of 7.4 mM ABTS and 2.6 mM potassium persulfate were prepared. The working solution was prepared by mixing the two stock solutions in equal volumes and allowing them to react for 12 h at room temperature in the dark. The solution was then diluted opportunely with phosphate-buffered saline (10 mM phosphate buffer, 150 mM NaCl, pH 7.4) to obtain an absorbance of 0.75 units at 734 nm. Fresh ABTS solution was prepared for each assay. Samples (20 μl) were placed in a 96-well plate and left to
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react with 180 μl of the ABTS solution for 2 h in the dark. The absorbance was measured at 734 nm using a micro-plate reader. Trolox served as standard. The results were expressed in μmols TE per gram f.w. All the experiments were performed in triplicate.
2.4. Thermal stability The stability of RCHE and RCAE before and after encapsulation was studied at 100°C. The analyses were conducted placing samples of the extracts or of the powders (100 mg resuspended in 1 mL of water, final pH 3.5-3.6) in a thermostatic water bath for 3 hours. Samples were collected every hour to quantify PP and AOC. In the case of powders 5 ml of ethanol was added to the samples that were centrifuged at 4500 g for 10 minutes to remove insoluble material. PP and AOC were measured on the supernatants.
2.5. Microparticles production and characterization 2.5.1. Microencapsulation by spray-drying Among the different extracts those carried out in ethanol were chosen to be microencapsulated. CAPSUL® modified starch was rehydrated directly in 100 ml of the extracts for 1 hour prior its use at a final concentration of 15%. The atomization process was performed with the use of a Mini-Spray dryer B-290 (Büchi). The conditions used were as 3
follows: drying air flow rate 40 m /h; inlet air temperature 140±3; outlet air temperature 70±3 and a feed flow rate of 5 mL/min. The formed microparticles were collected at the bottom of the cyclone separator. The RCHE and RCAE powders were kept in aluminum sealed bags and stored at -20°C until use.
2.5.2. Yield of encapsulation The YE was calculated as percentage of the ratio between the powder collected at the end of the process and the amount of solid used to initially feed the spray-drying system. The YE was expressed as:
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2.5.3. Water activity The Aw of the spray-dry powders was measured in triplicate using a Rototronic (Bassersdorf, Switzerland) device filling the 4.5 cm diameter disposable plates with the powder and incubating at 20°C.
2.5.4. Encapsulation efficiency TP and SP of the powders were evaluated following a protocol previously proposed (Mandavi, Jafari, Assadpour, & Ghorbani, 2016) with some modifications. In order to obtain the SP, 100 mg of powder was incubated with 10 ml of ethanol. The solution was left in agitation for 1 minute in a rotator mixer, and the sample was centrifuged at 4500 g for 10 minutes. The supernatant was recovered and filtered at 0.45 um. For the evaluation of the TP, 100 mg of powder was resuspended in 1 ml of water. The sample was placed in a sonication bath for 30 minutes at room temperature to allow the rupture of the microparticles in solution. Ethanol (10 ml) was added and the solution was left in agitation for 30 minutes in a rotator mixer. The sample was centrifuged at 4500 g for 10 minutes and then filtered at 0.45 um. The quantification of the polyphenols content was performed by Folin-Ciocalteau as described above. The EE was calculated using the subsequent formula:
(
2.5.5. Optical microscopy Spray-dried powders were resuspended in mineral oil. The oil was dispersed on a glass slide and images were taken using an EVOS optical microscope (Thermo Fisher Scientific, US). Microparticles diameters have been measured by analyzing digital images using the software ImageJ (http://imagej.nih.gov/ij). One hundred microparticles have been measured for each batch.
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2.5.6. Scanning electron microscopy The observation of microparticles surface morphology was carried out using a scanning electron microscopeZeiss XVP (Carl Zeiss SMT Ltd., Cambridge, UK). The samples were attached to a graphite tape and then covered with a thin film of gold at high vacuum (Agar Sputter Coater B7340, Agar Scientific Ltd., Stansted, Essex, UK).
2.6. UV-vis spectra analysis UV-vis spectra of RCHE and RCAE before and after encapsulation were analyzed using a Evolution 201 UV-vis spectrophotometer (Thermo Scientific). The samples were diluted 1:40 in 10 different media consisting of water in which the pH was modified opportunely with HCl and NaOH to represent a pH range from 1 to 10. The spectra were recorded between 300 and 750 nm.
2.7. Statistical analysis All measurements, if not differently stated, were performed in triplicates. The statistically significant differences among the obtained data were analyzed using t-test. The differences were considered significant with p<0.05. The Pearson coefficient was computed to test the correlation between polyphenol content of the extracts and antioxidant capacity. Data are reported as the average ± the standard deviation.
3. Results and discussion 3.1 Polyphenol extraction Three different solvents were used to extract polyphenols from the edible parts of RCH and RCA: water, and hydroalcoholic mixtures of ethanol and methanol. In general methanol exhibited the best extracting capacity leading to PP content in RCHE of about 1.70 mg GAE/g f.w. (Fig.1 A) not far from the values observed in other works dealing with different red chicory varieties (D'Evoli, Morroni, Lombardi-Boccia, Lucarini, Hrelia, Cantelli-Forti, et al., 2013; Heimler,
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Isolani, Vignolini, Tombelli, & Romani, 2007; Innocenti, Gallori, Giaccherini, Ieri, Vincieri, & Mulinacci, 2005; Lavelli, 2008). In the case of RCAE the amount of extracted PP was higher, on average 2.20 mg GAE/g f.w. (Fig.1 A), confirming previous data that indicated a higher PP content of RCA leaves in comparison to RCH (Innocenti, Gallori, Giaccherini, Ieri, Vincieri, & Mulinacci, 2005; Kaulmann, Jonville, Schneider, Hoffmann, & Bohn, 2014). A significant difference was found between the RCAE EtOH and RCAE MetOH, with the latter presenting a PP content about 25% higher. The AOC investigated by ABTS assay showed a good correlation with the PP content in the case of RCHEs (R=0.85) and a strong correlation (R=0.99) in the case of RCAEs (Fig.1 B).
3.2 Stability assessment of the extracts To evaluate the stability of the extracted PP, RCHE and RCAE were treated at 100°C for 3 hours to simulate the boiling process and the amount of retained PP and AOC was measured. Fig. 2 shows the percent retainment of polyphenol and AOC as a function of the time of boiling. For both the samples a significant loss of PP was observed after 3 hours of treatment. In the case of RCHE, the water extract showed the lower stability, with a loss of PP of 90% after 3 hours (Fig.2 A) and a decrease of the AOC of 80% (Fig. 2 C). The trend displayed by this sample was quite interesting since within 2 hours of treatment it showed the higher PP retainment but after 3 hours this value dramatically decreased. Differently from the water extract, RCHE MetOH and RCHE EtOH showed higher PP and AOC % retainment (Fig 2A, Fig. 2C). In particular, RCHE EtOH displayed 65% retainment of AOC, even though the AOC of the crude extract was definitely lower than that of RCHE MetOH (Fig. 1B). In the case of RCA the time-dependent thermal treatments of the two hydro-alcoholic extracts showed clearly different patterns, being PP and AOC % retainment of the RCAE EtOH 25-30% higher than that of RCAE MetOH (Fig. 2B, Fig. 2D). In particular, the PP % retainment of RCAE MetOH was very close to that of RCAE H2O (Fig. 2B) while, in the case of the AOC, the stability of the MetOH extract was definitely the lowest (Fig. 2D). It is noteworthy that the PP and AOC % retainment of RCAE EtOH (42% and 40% respectively, Fig. 2B, Fig. 2D) were lower if compared to the PP and AOC % retainment of RCHE EtOH (64% and 70% respectively, Fig. 2A, Fig. 2C)
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indicating that somehow the polyphenols extracted from red cabbage are less stable that those extracted from red chicory. This result was unexpected since anthocyanins from red cabbage, which represent a consistent amount of polyphenols, when extracted in similar conditions were shown to be very stable due to their acylation (Wiczkowski, Szawara-Nowak, & Topolska, 2013). The reason of these apparently contradictory results is probably the different ratio of anthocyanins to other polyphenols (mainly hydroxycinnamic acids). Indeed, while in RCH (var. Chioggia) this value is around 0.20, so with hydroxycinnamic acids being 5 times higher than anthocyanins (Tardugno, Pozzebon, Beggio, Del Turco, & Pojana, 2018), in RCA this ratio is close to 4.0, so with anthocyanins definitely more concentrated than hydroxycinnamic acids (Mizgier, Kucharska, Sokol-Letowska, Kolniak-Ostek, Kidon, & Fecka, 2016), and being anthocyanins sensibly more labile to degradation than other polyphenol classes (Brownmiller, Howard, & Prior, 2008), the heating process produced a more marked degradation of RCA polyphenols despite the protective effect of acylation. Due to the higher stability of the EtOH extracts, and to the fact that methanol could raise some safety concerns, RCHE EtOH and RCAE EtOH were chosen for the subsequent microencapsulation.
3.3 RCHE and RCAE PP encapsulation For this process CAPSUL® was selected as encapsulant matrix. This is a native starch modified with OSA. Due to the amphiphilic character of the grafted functional groups, OSA-starch presents excellent film forming properties (Sweedman, Tizzotti, Schafer, & Gilbert, 2013). The obtained powders were characterized by a pink-purple color in the case of RCHE (Fig. 3A) and of an intense violet color in the case of RCAE powder (Fig. 3B). Optical microscope observation (Fig. 3C, Fig. 3D) showed the presence of particles with different dimensions, ranging from 1 μm to 30 μm. The particles seem to be present as agglomerated structures composed by encapsulates, visible in both the powders and represented by dark purple spots, and by small transparent empty particles (similar to inclusion bodies) disposed around the encapsulates containing the PP. Scanning electron microscopy analysis of RCAE powder (Fig. 3F) confirmed what observed by optical microcopy: indeed, irregular and spherical particles with different dimensions were visible. In both the powders (Fig. 3E, Fig. 3F) the strong presence of dented collapsed structures was observable, with smaller particles that tended to aggregate among them or with bigger particles. These structures were more visible in the case of RCHE powder. This peculiar morphology could be due to the low feed rate applied during the drying step that, coupled to the low amount of
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solids, renders the drying process too fast leading to the production of small and collapsed particles (; Garcia-Tejeda, Salinas-Moreno, Hernandez-Martinez, & Martinez-Bustos, 2016; Osorio, Acevedo, Hillebrand, Carriazo, Winterhalter, & Morales, 2010). The combination with other materials, i.e. whey proteins, may improve the surface smoothness and decreased the surface indentation (Sheu & Rosenberg, 1998). Indeed Bernstein et Noreña (2015) obtained similar dented particles when using gum arabic as a carrier, while the same process based on polydestrose gave globular and smooth particles.
The YE was 95.2 ± 2.0% in the case of RCHE and 99.1 ± 0.5% in the case of RCAE. These data were difficult to compare for the scarce literature dealing with the encapsulation of these plant extracts. The water activity of the powders was 0.144 ± 0.026 in the case of RCHE powder and 0.162 ± 0.031 in the case of RCAE. These values indicate that the obtained powders should be microbiologically stable. The SP was 6.1 ± 0.5% in the case of RCHE and 5.7 ± 0.7% in the case of RCAE. This information is of great importance because the superficial fraction of polyphenols, that is not embedded in the encapsulates, is more prone to oxidation. These values can be probably reduced adjusting spray-drying operational parameters in order to promote a better particles formation (e.g. inlet temperature and feed rate). Also, the increase of solids content (i.e. starch), and the inclusion of further polymers, like maltodextrin and gum arabic, could contribute in reducing the SP. TP and SP allowed to calculate EE of 79.1 ± 3.0% for RCHE and 88.2 ± 1.3% for RCAE. These data are not in accordance with the results described in Fig. 2 since, despite showing lower stability, red cabbage PP exhibited better spray-drying retention. This higher resistance can be a consequence of the protection exerted by OSA-starch. The capacity of PP to interact with starch is in facts well known. In particular, it was highlighted that the structure of PP can influence the degree and the way of interaction with the polysaccharide (Zhu, 2015). This could be the rationale of the different protecting behavior exerted by starch towards the two extracts since acylated PP present in RCAE might differently interact with the polysaccharide.
3.4 Powder stability evaluation To understand whether the encapsulation process might have improved the stability of the polyphenols, the spray-dry powders were resuspended in water and subjected to thermal treatment at 100°C as described for the extracts. The results are showed in Fig. 4. The RCHE powder showed on average 90% retainment of both PP and AOC compared to the original extract, meaning an improved thermal stability of 27% for PP and 20% for AOC. Similar results were
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obtained for the RCAE powder, with PP and AOC % retainment of 86% and 94% respectively. These values mean a thermal stability increment of 40% for PP and 54% for AOC. The data clearly show that despite the original extracts are characterized by strong differences with respect to stability as a consequence of the different polyphenols present (anthocyanins Vs hydroxycinnamic acids), the encapsulation process based on OSA-starch matrix provided a protective effect leading to a substantial similar stability. The results are of great importance since very frequently the stability of the encapsulated active molecules is analyzed only in terms of shelf life during storage at specific conditions of temperature and humidity, leaving an important issue such as the stability of the active during processing not addressed. This information is very important when an industrial heating process has to be deigned or tuned. In the present case the resuspension of the powders in water simulated a boiling process, but it would be interesting to evaluate their performance in relation to other kinds of processes.
3.5. Color properties of RCHE and RCAE powders Anthocyanins-rich extracts are frequently used as natural pigments for food and supplements applications. The encapsulation process can influence the color properties of these pigments due to their possible interaction with the solids added, i.e. OSA-starch, and considering the heat treatment that the molecules undergo after atomization. Hence, it is crucial to evaluate the color properties of encapsulated extracts. For this reason the extracts and the resuspended powders have been buffered at different pH value, from 1 to 10 in order to highlight possible differences. In the range of pH between 1 and 3 the two extracts showed a purple-red color peculiar of the flavylium cation structure (Fig. 5) with a maximum of absorption at 520 nm (Fig. 6A, Fig. 6C). The absorbance at this wavelength normally decreases in the pH range between 4 to 6, due to the flavylium cation hydration that leads to the uncolored chalcone and carbinol pseudobase forms (Francis, 1989). This was particularly visible in the case of RCHE extract (Fig. 5), whereas for RCAE the presence of purplish chinoidal forms was probably the reason of the color observed in this pH range. In basic conditions (pH 7-10), a shift in the absorbance was observed to higher wavelengths, around 580 nm for RCHE and 620 nm for RCAE (Fig. 6A and 6C respectively). This bathochromic shift is a consequence of the conversion of anthocyanins to unstable chinoidal structures characterized by an intense blue color in the case of RCAE (Cevallos-Casals & Cisneros-Zevallos, 2004). Differently, the exposure of RCHE anthocyanins to basic pH values led to a simultaneous increase in the absorbance at 380-420 nm (Fig. 6A) due to presence of chalcones and trans-chalcones characterized by a yellow-green color (Fig. 5, line 1). This phenomenon is visible also for RCAE a pH 10, where the blue
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color observed at pH 9 shifted to yellow (Fig. 5, line 2). This was confirmed by the absorption spectrum obtained at this pH showing a maximum at 380 nm (Fig. 6C). In the case of RCHE powder, the absorption spectra registered at different pH values were very similar to those of the starting extract (Fig. 6A, Fig. 6B) indicating that the encapsulation process did not influence the light-absorption properties of the anthocyanins in the visible range. In the case of RCAE powder (Fig. 6D) a small (5 nm) hypsochromic shift (so towards smaller wavelengths) of the maxima was observed in comparison to the starting extract along all the pHs tested. Additionally, the color at pH 6 appeared pinkish instead of blue (Fig. 5, line 3 vs line 4). This correlates with the spectra registered at pH 5 and 6, that in the case of RCAE powder are perfectly overlapping (Fig. 6D) while for RCAE they differ in the 500-700 nm range. These phenomena can be explained considering the presence of starch that might have affected the optical properties of the anthocyanins by interacting with their structure, even though we cannot exclude the contribute of the high operating temperature of spray-drying. On the whole, the color properties of the anthocyanins were almost unaffected by the encapsulation process. This results gives the possibility to consider these powders for further applications i.e. as sensors for food spoilage detection during storage (Pourjavaher, Almasi, Meshkini, Pirsa, & Parandi, 2017). The release of specific molecules during microbial growth, e.g. organic acids, ammonia, etc., could change the color of the anthocyanins, possibly grafted to edible biofilms, indicating the progress of the deterioration. Encapsulation might increase the performance of these sensors since the anthocyanins are more stable, with the possibility to work at different conditions. In additions, it gives the possibility to confine the off-flavors/tastes deriving from the vegetable source, like in the case of cabbage. This would reduce the impact on the organoleptic level increasing the acceptability of the product by the consumer (Lavelli, Harsha, Laureati, & Pagliarini, 2017).
4. Conclusions In the present work an extraction procedure from the edible parts of red chicory and red cabbage was established using ethanol 70% as solvent. The extracts were found instable if exposed to high temperature, especially in the case of red cabbage. The encapsulation process led to an increase of the thermal stability of polyphenols and AOC of 2030% for red chicory and of 44-55% for red cabbage. Upon microencapsulation, the pH-dependent light-absorption properties of the pigments were almost entirely preserved. These characteristics make the microencapsulated powders here developed promising functional ingredients and innovative pH sensors.
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Acknowledgments: This work was supported by the Grant of the University of Verona. We thank Dr. Giulia Donà for her valuable technical assistance.
Figure captions
Fig. 1. PP content (panel A) and AOC (panel B) of RCHE and RCAE extracts. Statistically significant differences (p<0.05) within each extract are indicated by different letters.
Fig. 2. Time-dependent percentage retainment of PP (A and B) and AOC (C and D) of RCH (A and C) and RCA (B and D) extracts upon heat treatment at 100°C for 3 hours.
Fig. 3. Appearance (A and B), optical microscopy images (C and D) and scanning electron microscopy images (E and F) of RCHE powder (left side) and RCAE powders (right side).
Fig. 4. Time-dependent percentage retainment of PP (A) and AOC (B) of RCHE, RCHE powder, RACE and RCAE powder upon heat treatment at 100°C for 3 hours. The powders were resuspended in water before the treatment. The pH of the samples was 3.4-3.6.
Fig. 5. Color appearance of extracts and solubilized powders at different pH values (pH 1-10). 1: RCHE, 2: RCHE powder, 3: RCAE, 4: RCAE powder. The powders were resuspended in water before the treatment. The pH of the samples was adjusted with HCl and NaOH.
Fig. 6. Visible light absorption spectra of extracts and solubilized powders at different pH values (pH 1-10). The samples are the same presented in Fig. 5.
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Declaration of interests
☐ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
☒The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
Gianni Zoccatelli and Francesca Zanoni are shareholders of Sphera Encapsulation SRL
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Highlights 1. 2. 3. 4. 5.
Polyphenols of red chicory extracts are more stable than those of cabbage Encapsulation improves the thermal stability of polyphenols Encapsulation by spray-drying does not affect the color properties of the pigments Red cabbage polyphenols were more efficiently encapsulated than those of chicory Spray-dry powders can be used to develop functional foods and supplements
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S0308-8146(19)31654-1 https://doi.org/10.1016/j.foodchem.2019.125535 FOCH 125535
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
14 May 2019 5 September 2019 13 September 2019
Please cite this article as: Zanoni, F., Primiterra, M., Angeli, N., Zoccatelli, G., Microencapsulation by spray-drying of polyphenols extracted from red chicory and red cabbage: effects on stability and color properties, Food Chemistry (2019), doi: https://doi.org/10.1016/j.foodchem.2019.125535
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© 2019 Elsevier Ltd. All rights reserved.
Microencapsulation by spray-drying of polyphenols extracted from red chicory and red cabbage: effects on stability and color properties
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Francesca Zanoni , Martina Primiterra , Nicola Angeli , Gianni Zoccatelli
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University of Verona, Department of Biotechnology, Verona, Italy
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Sphera Encapsulation Srl, Verona, Italy
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MUSE, Limnology and Phycology Section, Trento, Italy
*Corresponding Author: Gianni Zoccatelli, PhD Dipartimento di Biotecnologie Università degli Studi di Verona Strada Le Grazie, 15 - CV1 I-37134 Verona, Italy Tel: +39 045 8027952 Fax: +39 045 8027929 e-mail: [email protected]
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Abstract The research of antioxidants and natural pigments to replace synthetic molecules is increasingly considering wastes from plant food supply chains. Red chicory (RCH) and red cabbage (RCA) are rich sources of polyphenols (PP), especially anthocyanins, well know natural pigments possessing strong antioxidant capacity and beneficial health effects. The aim of this work was to compare different solvents for PP extraction and to evaluate the effect of spraydrying encapsulation using modified starch on PP, antioxidant capacity (AOC) and color properties. Methanol:water (70:30) showed the best extraction capacity, while ethanol:water (70:30) extracts displayed the highest thermal stability. Ethanol:water extracts were spray-dried with a yield of 95-99% for both crops, while the efficiency of PP encapsulation was 79% (RCA) and 88% (RCH). Encapsulation improved retention of PP and AOC upon thermal treatment (RCH: 20-30%, RCA: 44-55%) without altering color properties. This process can be employed for the development of functional foods and supplements.
Keywords Red Chicory (Chicorium intibus L.), Red cabbage (Brassica oleracea L. var capitata f.rubra), polyphenols, microencapsulation; antioxidant capacity (AOC); spray-drying. Abbreviations: RCH: red Chicory; RCHE: red Chicory extract; RCA: red cabbage; RCAE: red cabbage extract; EtOH: ethanol, MetOH: methanol; aw: water activity; GA: gallic acid; GAE: gallic acid equivalents; TE: Trolox equivalents; PP: Polyphenols; TP: total polyphenols; SP: superficial polyphenols; EE: Encapsulation efficiency; YE: yield of encapsulation; Abs: absorbance; f.w.: fresh weight; OSA: octenyl succinic anhydride; ABTS: 2,2'-azino-bis (3ethylbenzothiazoline-6-sulphonic acid.
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1.
Introduction
The research for natural antioxidants and the growing importance in the management and requalification of industrial food wastes is increasingly considering plant food by-products as source of valuable molecules, e.g. polyphenols, carotenoids and fibers (Lante, Nardi, Zocca, Giacomini, & Corich, 2011). The exploitation of this type of resources gives the possibility to lower the amount of food wastes, hence diminishing the negative impact on the environment due to the phytotoxic effect of the high organic matter content (Lavelli, Harsha, Laureati, & Pagliarini, 2017). In particular, among the different classes of molecules with health promoting effects, during the last 10 years, researchers and food industries have been focusing their attention on polyphenols. The reasons rely on the fact that they are present in great abundance in our diet, and are involved in the prevention of various chronic-degenerative pathologies such as cardiovascular diseases (Scalbert, Manach, Morand, Remesy, & Jimenez, 2005), diabetes (Matsui, Ogunwande, Abesundara, & Matsumoto, 2006), neurodegenerative diseases (Levites, Amit, Youdim, & Mandel, 2002) and osteoporosis (Dudaric, Fuzinac-Smojver, Muhvic, & Giacometti, 2015). Red chicory (Chicorium intibus L.) and red cabbage (Brassica oleracea L. var capitata f.rubra) are both important sources of polyphenols. Moreover, the wastes produced during their processing, consisting mainly of leaves and steams, can reach up to 40-50% of the total harvested material, that for the great part is still nutritionally and hygienically valid especially in the case of the fourth-range production where edible material is eliminated also to improve the aspect of the food and increase the acceptability of the consumer ; Lante, Nardi, Zocca, Giacomini, & Corich, 2011; Llorach, Tomas-Barberan, & Ferreres, 2004). RCH is a typical winter vegetable indigenous to Europe, North and Western America that has gained attention for its content of phytochemicals with potential health benefits, such as phenolic acids, flavonoids and anthocyanins (Bais & Ravishankar, 2001). The high content of anthocyanins, that account for the 50-55% of the total phenolic compounds, is responsible for the characteristic deep red color of the leaves. Among all the leafy vegetables that are consumed raw, red chicory has been recognized to have the highest content of polyphenols (Gazzani, Daglia, Papetti, & Gregotti, 2000;). RCA, in the past indigenous of the Mediterranean zone, is now one of the most important vegetables grown around the world (Wiczkowski, Szawara-Nowak, & Topolska, 2013). The abundance and the relative low cost of production, if compared to other vegetables rich in polyphenols (e.g. black carrot and purple potatoes), make it an attractive source
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of natural red pigments (Bridle & Timberlake, 1997; ). Its use has been reported in the treatment of inflammatory bowel disease (Zielińska, Lewandowska, Podsedek, Cygankiewicz, Jacenik, Sałaga, et al., 2015), and in the attenuation of cardiac and hepatic oxidative stress (Sankhari, Thounaojam, Jadeja, Devkar, & Ramachandran, 2012). It is worth considering that the production and harvest of these vegetables are seasonal activities and the by-products obtained from their processing need to be handled in a limited time frame, since once extracted, polyphenols are highly susceptible to degradation (Patras, Brunton, O'Donnell, & Tiwari, 2010). Spray-drying has been widely used to dry a number of foods and pharmaceutical preparations, among which many containing heat-sensitive molecules such as polyphenols (Mahdavi, Jafari, Ghorbani, & Assadpoor, 2014; Shishir & Chen, 2017). It is the most common technique used to turn liquids with high solid content into powders. In addition, it is recognized as an economic process by which it is possible to produce high quality powders. To our knowledge, no work about the stabilization of polyphenols from red chicory through spray-drying has been addressed, and only two works about the stabilization of polyphenols from red cabbage have been described (Bernstein & Noreña, 2015; Hongmei & Meng, 2015). In these studies only the production and characterization of the powders were tackled without evaluating their performance in a simulating medium. The aim of this work was to assess the quality and the thermal stability of phenolic extracts from red chicory and cabbage leaves obtained by different solvents (water, and hydro-alcoholic mixtures of ethanol and methanol) and to improve the stability of polyphenols and antioxidant capacity by spray-drying encapsulation without altering the color properties of the pigments.
2. Materials and methods: Materials Fresh Cichorium intybus L. (red chicory var. Chioggia) and Brassica oleracea L. (red cabbage var. Capitata f. rubra) were purchased from a local market. Modified starch CAPSUL® was provided by Ingredion (Illinois, US). Folin-Ciocalteau reagent, sodium carbonate, formic acid, methanol, ABTS, potassium persulfate, 37% chloridric acid, ethyl acetate, Trolox®, gallic acid and ethanol were obtained from Sigma Aldrich (St. Louis, MO, US).
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2.1. Extraction of polyphenols Polyphenols were extracted from RCH and RCA leaves combining different protocols with some modifications (Cefola, Carbone, Minasi, & Pace, 2016; Heimler, Isolani, Vignolini, & Romani, 2009; Innocenti, Gallori, Giaccherini, Ieri, Vincieri, & Mulinacci, 2005; McDougall, Fyffe, Dobson, & Stewart, 2007; Pereira, de Arruda, & Stefani, 2015). Briefly, 500 g of RCH fresh leaves were ground with a SAMA K45 cutter (Dito SAMA, Italy). The obtained sample was extracted with the solvent for 4 h in continuous agitation (ratio sample: solvent 1:1). Formic acid was used to adjust the pH of the sample to 3.3-3.5. The extract was filtered through Whatman filter paper in a Buchner funnel. The obtained solution was kept at 4°C in the dark until use. Three different extraction solvents were tested: water, 30% water:70% EtOH and 30% water:70% MetOH. The same operations were conducted with RCA. Methanol and ethanol were then removed by evaporation with a rotary evaporator (Büchi, Switzerland) prior use/storage.
2.2. Polyphenols quantification The Folin-Ciocalteau assay was performed following a method previously proposed (Singleton, Orthofer, & LamuelaRaventos, 1999) with some modifications. Briefly, the reaction was conducted in a 96 well micro-plate (Sarstedt, Germany). An amount of 5 ul of each sample was incubated for 3 minutes with 150 ul of Folin-Ciocalteau reagent (previously diluted at 1:15 with water). Finally, 40 ul of 20% sodium carbonate solution was added and left to react for 30 minutes in the dark. The absorbance was measured using a micro-plate reader (BioTek, Milan, Italy) at 750 nm. Water was used as blank. The calibration curve was carried out using GA as standard. The results were expressed as milligrams GAE per gram of f.w. All the experiments were performed in triplicate.
2.3. Antioxidant capacity The procedure proposed by Thaipong and co-workers was followed (Thaipong, Boonprakob, Crosby, Cisneros-Zevallos, & Byrne, 2006) with some modifications. The stock solutions consisting of 7.4 mM ABTS and 2.6 mM potassium persulfate were prepared. The working solution was prepared by mixing the two stock solutions in equal volumes and allowing them to react for 12 h at room temperature in the dark. The solution was then diluted opportunely with phosphate-buffered saline (10 mM phosphate buffer, 150 mM NaCl, pH 7.4) to obtain an absorbance of 0.75 units at 734 nm. Fresh ABTS solution was prepared for each assay. Samples (20 μl) were placed in a 96-well plate and left to
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react with 180 μl of the ABTS solution for 2 h in the dark. The absorbance was measured at 734 nm using a micro-plate reader. Trolox served as standard. The results were expressed in μmols TE per gram f.w. All the experiments were performed in triplicate.
2.4. Thermal stability The stability of RCHE and RCAE before and after encapsulation was studied at 100°C. The analyses were conducted placing samples of the extracts or of the powders (100 mg resuspended in 1 mL of water, final pH 3.5-3.6) in a thermostatic water bath for 3 hours. Samples were collected every hour to quantify PP and AOC. In the case of powders 5 ml of ethanol was added to the samples that were centrifuged at 4500 g for 10 minutes to remove insoluble material. PP and AOC were measured on the supernatants.
2.5. Microparticles production and characterization 2.5.1. Microencapsulation by spray-drying Among the different extracts those carried out in ethanol were chosen to be microencapsulated. CAPSUL® modified starch was rehydrated directly in 100 ml of the extracts for 1 hour prior its use at a final concentration of 15%. The atomization process was performed with the use of a Mini-Spray dryer B-290 (Büchi). The conditions used were as 3
follows: drying air flow rate 40 m /h; inlet air temperature 140±3; outlet air temperature 70±3 and a feed flow rate of 5 mL/min. The formed microparticles were collected at the bottom of the cyclone separator. The RCHE and RCAE powders were kept in aluminum sealed bags and stored at -20°C until use.
2.5.2. Yield of encapsulation The YE was calculated as percentage of the ratio between the powder collected at the end of the process and the amount of solid used to initially feed the spray-drying system. The YE was expressed as:
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2.5.3. Water activity The Aw of the spray-dry powders was measured in triplicate using a Rototronic (Bassersdorf, Switzerland) device filling the 4.5 cm diameter disposable plates with the powder and incubating at 20°C.
2.5.4. Encapsulation efficiency TP and SP of the powders were evaluated following a protocol previously proposed (Mandavi, Jafari, Assadpour, & Ghorbani, 2016) with some modifications. In order to obtain the SP, 100 mg of powder was incubated with 10 ml of ethanol. The solution was left in agitation for 1 minute in a rotator mixer, and the sample was centrifuged at 4500 g for 10 minutes. The supernatant was recovered and filtered at 0.45 um. For the evaluation of the TP, 100 mg of powder was resuspended in 1 ml of water. The sample was placed in a sonication bath for 30 minutes at room temperature to allow the rupture of the microparticles in solution. Ethanol (10 ml) was added and the solution was left in agitation for 30 minutes in a rotator mixer. The sample was centrifuged at 4500 g for 10 minutes and then filtered at 0.45 um. The quantification of the polyphenols content was performed by Folin-Ciocalteau as described above. The EE was calculated using the subsequent formula:
(
2.5.5. Optical microscopy Spray-dried powders were resuspended in mineral oil. The oil was dispersed on a glass slide and images were taken using an EVOS optical microscope (Thermo Fisher Scientific, US). Microparticles diameters have been measured by analyzing digital images using the software ImageJ (http://imagej.nih.gov/ij). One hundred microparticles have been measured for each batch.
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2.5.6. Scanning electron microscopy The observation of microparticles surface morphology was carried out using a scanning electron microscopeZeiss XVP (Carl Zeiss SMT Ltd., Cambridge, UK). The samples were attached to a graphite tape and then covered with a thin film of gold at high vacuum (Agar Sputter Coater B7340, Agar Scientific Ltd., Stansted, Essex, UK).
2.6. UV-vis spectra analysis UV-vis spectra of RCHE and RCAE before and after encapsulation were analyzed using a Evolution 201 UV-vis spectrophotometer (Thermo Scientific). The samples were diluted 1:40 in 10 different media consisting of water in which the pH was modified opportunely with HCl and NaOH to represent a pH range from 1 to 10. The spectra were recorded between 300 and 750 nm.
2.7. Statistical analysis All measurements, if not differently stated, were performed in triplicates. The statistically significant differences among the obtained data were analyzed using t-test. The differences were considered significant with p<0.05. The Pearson coefficient was computed to test the correlation between polyphenol content of the extracts and antioxidant capacity. Data are reported as the average ± the standard deviation.
3. Results and discussion 3.1 Polyphenol extraction Three different solvents were used to extract polyphenols from the edible parts of RCH and RCA: water, and hydroalcoholic mixtures of ethanol and methanol. In general methanol exhibited the best extracting capacity leading to PP content in RCHE of about 1.70 mg GAE/g f.w. (Fig.1 A) not far from the values observed in other works dealing with different red chicory varieties (D'Evoli, Morroni, Lombardi-Boccia, Lucarini, Hrelia, Cantelli-Forti, et al., 2013; Heimler,
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Isolani, Vignolini, Tombelli, & Romani, 2007; Innocenti, Gallori, Giaccherini, Ieri, Vincieri, & Mulinacci, 2005; Lavelli, 2008). In the case of RCAE the amount of extracted PP was higher, on average 2.20 mg GAE/g f.w. (Fig.1 A), confirming previous data that indicated a higher PP content of RCA leaves in comparison to RCH (Innocenti, Gallori, Giaccherini, Ieri, Vincieri, & Mulinacci, 2005; Kaulmann, Jonville, Schneider, Hoffmann, & Bohn, 2014). A significant difference was found between the RCAE EtOH and RCAE MetOH, with the latter presenting a PP content about 25% higher. The AOC investigated by ABTS assay showed a good correlation with the PP content in the case of RCHEs (R=0.85) and a strong correlation (R=0.99) in the case of RCAEs (Fig.1 B).
3.2 Stability assessment of the extracts To evaluate the stability of the extracted PP, RCHE and RCAE were treated at 100°C for 3 hours to simulate the boiling process and the amount of retained PP and AOC was measured. Fig. 2 shows the percent retainment of polyphenol and AOC as a function of the time of boiling. For both the samples a significant loss of PP was observed after 3 hours of treatment. In the case of RCHE, the water extract showed the lower stability, with a loss of PP of 90% after 3 hours (Fig.2 A) and a decrease of the AOC of 80% (Fig. 2 C). The trend displayed by this sample was quite interesting since within 2 hours of treatment it showed the higher PP retainment but after 3 hours this value dramatically decreased. Differently from the water extract, RCHE MetOH and RCHE EtOH showed higher PP and AOC % retainment (Fig 2A, Fig. 2C). In particular, RCHE EtOH displayed 65% retainment of AOC, even though the AOC of the crude extract was definitely lower than that of RCHE MetOH (Fig. 1B). In the case of RCA the time-dependent thermal treatments of the two hydro-alcoholic extracts showed clearly different patterns, being PP and AOC % retainment of the RCAE EtOH 25-30% higher than that of RCAE MetOH (Fig. 2B, Fig. 2D). In particular, the PP % retainment of RCAE MetOH was very close to that of RCAE H2O (Fig. 2B) while, in the case of the AOC, the stability of the MetOH extract was definitely the lowest (Fig. 2D). It is noteworthy that the PP and AOC % retainment of RCAE EtOH (42% and 40% respectively, Fig. 2B, Fig. 2D) were lower if compared to the PP and AOC % retainment of RCHE EtOH (64% and 70% respectively, Fig. 2A, Fig. 2C)
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indicating that somehow the polyphenols extracted from red cabbage are less stable that those extracted from red chicory. This result was unexpected since anthocyanins from red cabbage, which represent a consistent amount of polyphenols, when extracted in similar conditions were shown to be very stable due to their acylation (Wiczkowski, Szawara-Nowak, & Topolska, 2013). The reason of these apparently contradictory results is probably the different ratio of anthocyanins to other polyphenols (mainly hydroxycinnamic acids). Indeed, while in RCH (var. Chioggia) this value is around 0.20, so with hydroxycinnamic acids being 5 times higher than anthocyanins (Tardugno, Pozzebon, Beggio, Del Turco, & Pojana, 2018), in RCA this ratio is close to 4.0, so with anthocyanins definitely more concentrated than hydroxycinnamic acids (Mizgier, Kucharska, Sokol-Letowska, Kolniak-Ostek, Kidon, & Fecka, 2016), and being anthocyanins sensibly more labile to degradation than other polyphenol classes (Brownmiller, Howard, & Prior, 2008), the heating process produced a more marked degradation of RCA polyphenols despite the protective effect of acylation. Due to the higher stability of the EtOH extracts, and to the fact that methanol could raise some safety concerns, RCHE EtOH and RCAE EtOH were chosen for the subsequent microencapsulation.
3.3 RCHE and RCAE PP encapsulation For this process CAPSUL® was selected as encapsulant matrix. This is a native starch modified with OSA. Due to the amphiphilic character of the grafted functional groups, OSA-starch presents excellent film forming properties (Sweedman, Tizzotti, Schafer, & Gilbert, 2013). The obtained powders were characterized by a pink-purple color in the case of RCHE (Fig. 3A) and of an intense violet color in the case of RCAE powder (Fig. 3B). Optical microscope observation (Fig. 3C, Fig. 3D) showed the presence of particles with different dimensions, ranging from 1 μm to 30 μm. The particles seem to be present as agglomerated structures composed by encapsulates, visible in both the powders and represented by dark purple spots, and by small transparent empty particles (similar to inclusion bodies) disposed around the encapsulates containing the PP. Scanning electron microscopy analysis of RCAE powder (Fig. 3F) confirmed what observed by optical microcopy: indeed, irregular and spherical particles with different dimensions were visible. In both the powders (Fig. 3E, Fig. 3F) the strong presence of dented collapsed structures was observable, with smaller particles that tended to aggregate among them or with bigger particles. These structures were more visible in the case of RCHE powder. This peculiar morphology could be due to the low feed rate applied during the drying step that, coupled to the low amount of
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solids, renders the drying process too fast leading to the production of small and collapsed particles (; Garcia-Tejeda, Salinas-Moreno, Hernandez-Martinez, & Martinez-Bustos, 2016; Osorio, Acevedo, Hillebrand, Carriazo, Winterhalter, & Morales, 2010). The combination with other materials, i.e. whey proteins, may improve the surface smoothness and decreased the surface indentation (Sheu & Rosenberg, 1998). Indeed Bernstein et Noreña (2015) obtained similar dented particles when using gum arabic as a carrier, while the same process based on polydestrose gave globular and smooth particles.
The YE was 95.2 ± 2.0% in the case of RCHE and 99.1 ± 0.5% in the case of RCAE. These data were difficult to compare for the scarce literature dealing with the encapsulation of these plant extracts. The water activity of the powders was 0.144 ± 0.026 in the case of RCHE powder and 0.162 ± 0.031 in the case of RCAE. These values indicate that the obtained powders should be microbiologically stable. The SP was 6.1 ± 0.5% in the case of RCHE and 5.7 ± 0.7% in the case of RCAE. This information is of great importance because the superficial fraction of polyphenols, that is not embedded in the encapsulates, is more prone to oxidation. These values can be probably reduced adjusting spray-drying operational parameters in order to promote a better particles formation (e.g. inlet temperature and feed rate). Also, the increase of solids content (i.e. starch), and the inclusion of further polymers, like maltodextrin and gum arabic, could contribute in reducing the SP. TP and SP allowed to calculate EE of 79.1 ± 3.0% for RCHE and 88.2 ± 1.3% for RCAE. These data are not in accordance with the results described in Fig. 2 since, despite showing lower stability, red cabbage PP exhibited better spray-drying retention. This higher resistance can be a consequence of the protection exerted by OSA-starch. The capacity of PP to interact with starch is in facts well known. In particular, it was highlighted that the structure of PP can influence the degree and the way of interaction with the polysaccharide (Zhu, 2015). This could be the rationale of the different protecting behavior exerted by starch towards the two extracts since acylated PP present in RCAE might differently interact with the polysaccharide.
3.4 Powder stability evaluation To understand whether the encapsulation process might have improved the stability of the polyphenols, the spray-dry powders were resuspended in water and subjected to thermal treatment at 100°C as described for the extracts. The results are showed in Fig. 4. The RCHE powder showed on average 90% retainment of both PP and AOC compared to the original extract, meaning an improved thermal stability of 27% for PP and 20% for AOC. Similar results were
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obtained for the RCAE powder, with PP and AOC % retainment of 86% and 94% respectively. These values mean a thermal stability increment of 40% for PP and 54% for AOC. The data clearly show that despite the original extracts are characterized by strong differences with respect to stability as a consequence of the different polyphenols present (anthocyanins Vs hydroxycinnamic acids), the encapsulation process based on OSA-starch matrix provided a protective effect leading to a substantial similar stability. The results are of great importance since very frequently the stability of the encapsulated active molecules is analyzed only in terms of shelf life during storage at specific conditions of temperature and humidity, leaving an important issue such as the stability of the active during processing not addressed. This information is very important when an industrial heating process has to be deigned or tuned. In the present case the resuspension of the powders in water simulated a boiling process, but it would be interesting to evaluate their performance in relation to other kinds of processes.
3.5. Color properties of RCHE and RCAE powders Anthocyanins-rich extracts are frequently used as natural pigments for food and supplements applications. The encapsulation process can influence the color properties of these pigments due to their possible interaction with the solids added, i.e. OSA-starch, and considering the heat treatment that the molecules undergo after atomization. Hence, it is crucial to evaluate the color properties of encapsulated extracts. For this reason the extracts and the resuspended powders have been buffered at different pH value, from 1 to 10 in order to highlight possible differences. In the range of pH between 1 and 3 the two extracts showed a purple-red color peculiar of the flavylium cation structure (Fig. 5) with a maximum of absorption at 520 nm (Fig. 6A, Fig. 6C). The absorbance at this wavelength normally decreases in the pH range between 4 to 6, due to the flavylium cation hydration that leads to the uncolored chalcone and carbinol pseudobase forms (Francis, 1989). This was particularly visible in the case of RCHE extract (Fig. 5), whereas for RCAE the presence of purplish chinoidal forms was probably the reason of the color observed in this pH range. In basic conditions (pH 7-10), a shift in the absorbance was observed to higher wavelengths, around 580 nm for RCHE and 620 nm for RCAE (Fig. 6A and 6C respectively). This bathochromic shift is a consequence of the conversion of anthocyanins to unstable chinoidal structures characterized by an intense blue color in the case of RCAE (Cevallos-Casals & Cisneros-Zevallos, 2004). Differently, the exposure of RCHE anthocyanins to basic pH values led to a simultaneous increase in the absorbance at 380-420 nm (Fig. 6A) due to presence of chalcones and trans-chalcones characterized by a yellow-green color (Fig. 5, line 1). This phenomenon is visible also for RCAE a pH 10, where the blue
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color observed at pH 9 shifted to yellow (Fig. 5, line 2). This was confirmed by the absorption spectrum obtained at this pH showing a maximum at 380 nm (Fig. 6C). In the case of RCHE powder, the absorption spectra registered at different pH values were very similar to those of the starting extract (Fig. 6A, Fig. 6B) indicating that the encapsulation process did not influence the light-absorption properties of the anthocyanins in the visible range. In the case of RCAE powder (Fig. 6D) a small (5 nm) hypsochromic shift (so towards smaller wavelengths) of the maxima was observed in comparison to the starting extract along all the pHs tested. Additionally, the color at pH 6 appeared pinkish instead of blue (Fig. 5, line 3 vs line 4). This correlates with the spectra registered at pH 5 and 6, that in the case of RCAE powder are perfectly overlapping (Fig. 6D) while for RCAE they differ in the 500-700 nm range. These phenomena can be explained considering the presence of starch that might have affected the optical properties of the anthocyanins by interacting with their structure, even though we cannot exclude the contribute of the high operating temperature of spray-drying. On the whole, the color properties of the anthocyanins were almost unaffected by the encapsulation process. This results gives the possibility to consider these powders for further applications i.e. as sensors for food spoilage detection during storage (Pourjavaher, Almasi, Meshkini, Pirsa, & Parandi, 2017). The release of specific molecules during microbial growth, e.g. organic acids, ammonia, etc., could change the color of the anthocyanins, possibly grafted to edible biofilms, indicating the progress of the deterioration. Encapsulation might increase the performance of these sensors since the anthocyanins are more stable, with the possibility to work at different conditions. In additions, it gives the possibility to confine the off-flavors/tastes deriving from the vegetable source, like in the case of cabbage. This would reduce the impact on the organoleptic level increasing the acceptability of the product by the consumer (Lavelli, Harsha, Laureati, & Pagliarini, 2017).
4. Conclusions In the present work an extraction procedure from the edible parts of red chicory and red cabbage was established using ethanol 70% as solvent. The extracts were found instable if exposed to high temperature, especially in the case of red cabbage. The encapsulation process led to an increase of the thermal stability of polyphenols and AOC of 2030% for red chicory and of 44-55% for red cabbage. Upon microencapsulation, the pH-dependent light-absorption properties of the pigments were almost entirely preserved. These characteristics make the microencapsulated powders here developed promising functional ingredients and innovative pH sensors.
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Acknowledgments: This work was supported by the Grant of the University of Verona. We thank Dr. Giulia Donà for her valuable technical assistance.
Figure captions
Fig. 1. PP content (panel A) and AOC (panel B) of RCHE and RCAE extracts. Statistically significant differences (p<0.05) within each extract are indicated by different letters.
Fig. 2. Time-dependent percentage retainment of PP (A and B) and AOC (C and D) of RCH (A and C) and RCA (B and D) extracts upon heat treatment at 100°C for 3 hours.
Fig. 3. Appearance (A and B), optical microscopy images (C and D) and scanning electron microscopy images (E and F) of RCHE powder (left side) and RCAE powders (right side).
Fig. 4. Time-dependent percentage retainment of PP (A) and AOC (B) of RCHE, RCHE powder, RACE and RCAE powder upon heat treatment at 100°C for 3 hours. The powders were resuspended in water before the treatment. The pH of the samples was 3.4-3.6.
Fig. 5. Color appearance of extracts and solubilized powders at different pH values (pH 1-10). 1: RCHE, 2: RCHE powder, 3: RCAE, 4: RCAE powder. The powders were resuspended in water before the treatment. The pH of the samples was adjusted with HCl and NaOH.
Fig. 6. Visible light absorption spectra of extracts and solubilized powders at different pH values (pH 1-10). The samples are the same presented in Fig. 5.
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Declaration of interests
☐ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
☒The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
Gianni Zoccatelli and Francesca Zanoni are shareholders of Sphera Encapsulation SRL
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Highlights 1. 2. 3. 4. 5.
Polyphenols of red chicory extracts are more stable than those of cabbage Encapsulation improves the thermal stability of polyphenols Encapsulation by spray-drying does not affect the color properties of the pigments Red cabbage polyphenols were more efficiently encapsulated than those of chicory Spray-dry powders can be used to develop functional foods and supplements
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