Orientadora: Profa. Dra. Grasiele Scaramal Madrona

Data da Defesa: 17/02/2017



The blackberry (Rubus fruticosus) is a fruit belonging to the family Rosaceae, genus Rubus, with 400 to 500 species, known as berries. The berries are small fruits, with sweet taste and rounded format, characterized by their high antioxidant potential due to the contents of total phenolic compounds and flavonoids. In addition, they have high content of anthocyanins, mainly anthocyanin cyanidin 3-glucoside. In fruit processing industries, about 20% of pomace is produced, basically composed of seeds and barks that still contain a large amount of phenolic compounds, such as anthocyanins. The limiting factor for the use of anthocyanins is their lower stability, being affected by several parameters such as pH, copigmentation, light, temperature and oxygen. In orther to minimize these adverse effects, microencapsulation can assist with many useful properties, the encapsulation provides a degree of stabilization for active compounds, avoiding deteriorating reactions.
The aim of the present study was to microencapsulate and characterize the extracts (aqueous and hydroalcoholic) of the blackberry pomace by applying spray drying process, evaluating the stability against light, temperature and pH variations.
On the blackberry pomace was realized analyzes of pH, titratable acidity, moisture, ashes, color using CIEL*a*b* system an hue (H) and chromaticity (C) angles were calculated. Two extracts of the blackberry pomace were prepared: aqueous extract and hydroalcoholic extract. Response surface methodology was used to evaluate the effect of the temperature and extraction time in the aqueous extraction of anthocyanins. The aqueous extract was obtained from the dilution of the blackberry pomace in distilled water (500 mg/mL) and mechanically shaken at 60°C for 45 minutes. The hydroalcoholic extract was obtained by diluting the pomace (500 mg/mL) in ethyl alcohol 80% (v/v), under mechanical stirring for 48 hours, filtration and rotation at 65°C until complete evaporation of the solvent. Afterwards, the extracts were filtered and submitted to drying in spray dryer. Maltodextrin (DE 10) was used as an encapsulating agent in the microencapsulation process. The carrier maltodextrin was added directly to the filtrates, in a ratio of 1:1 (w/w), by mechanical agitation. The aqueous and hydroalcoholic extracts mixed with maltodextrin were dried in mini spray dryer placed in plastic containers and stored under freezing (-18ºC). The determination of total phenolic compounds, total monomeric anthocyanins content, antioxidant activity by the radical sequestration method DPPH and Iron Reduction Method (FRAP), were determined by colorimetrical methods. Encapsulation yield was calculated, chromatographic analyzes by high performance liquid chromatography in a Shimadzu HPLC were performed and cyanidin-3-glucoside, galic acid, ellagic acid and quercetin concentrations determined, the particle morphology was performed by scanning electron microscopy. Microcapsules were evaluated during 36 days for temperatures of 4 and 25 °C, light and no Light using two fluorescent lamps of 20W and a dark chamber. Color, total phenolic compounds and total monomeric anthocyanins were evaluated. Samples (extracts and microcapsules) were evaluated in different Mcllvaine buffer (pH solutions 2.0, 3.5, 5.0 and 6.5), analyzes of color, total phenolic compounds, total monomeric anthocyanins and antioxidant activity by DPPH method. Color difference values were calculated to study color changes, the first-order reaction rate constants (k) and half-lives (t1/2), i.e. the times needed for 50% degradation of anthocyanins, were calculated. All analyzes were performed in triplicate and submitted to analysis of variance and Tukey's test (p <0.05) for the minimum significant difference between the means using the statistical program STATISTICA version 7.0.
The pomace has higher phenolic compounds contents and lower anthocyanins contents than the pulp. It is noted that dry aqueous extract are lighter than dry hydroalcoholic extract. When comparing a*, b* and H° parameters of microcapsules and dry extracts, the values are very close, this results demonstrate that encapsulating agent protected the color compounds of samples during the drying process. The phenolic compounds gallic acid followed by the ellagic were the major compounds in chromatographic analyzes, and the anthocyanins cyanidin was observed in higher concentration in all samples, followed by quercetin. Still analyzing the anthocyanins, hydroalcohol extraction was more efficient (1.5 times) in terms of encapsulation than aqueous extraction. However, for extraction of phenolic compounds the highest efficiency was when aqueous solution was used (1.2 times). In relation to the morphology, it was observed that the non-encapsulated extracts presented a more amorphous and irregular form than the microcapsules, indicating that the material that was encapsulated is actually protected. About temperature and light stability, it was observed that the use of temperature 4 °C kept the a* values constant, indicating greater red intensity in the samples and better stability in this condition. There was an increase for phenolic compounds in aqueous extraction microcapsule at 4 °C, whereas was observed at 25 °C, with light and without light, no significant difference during 36 days of storage. It was also observed that phenolic compounds were not influenced in hydroalcoholic extraction microcapsule under the different light and temperature conditions. Anthocyanins did not show degradation in aqueous extraction microcapsule at 4 °C and 25 °C during 36 days of storage, whereas in hydroalcoholic extraction the microcapsule manteined the anthocyanins content at 4 °C and at 25 °C low losses. Where observed light influenced the degradation of anthocyanins, with a loss in aqueous extraction microcapsule with light, in no light there was no losses. In the hydroalcoholic extraction microcapsule, the sample with light presented greater degradation as compared to no light. About pH stability, extracts presented higher color variations (ΔΕ) in pH 2.0, 3.5 and 5.0 as compared to the microcapsules, indicating that microencapsulation provided a higher color stability. When evaluating microcapsules as pH variations, the lower color variation was observed at pH 2.0, showing more anthocyanins stability at low pH. Regarding phenolic compounds, most samples presented an increse, while hydroalcoholic extract was degradated in pHs 3.5, 5.0 and 6.5 for 7 storage days. In anthocyanins it was observed that low pH kept all samples stable, the degradation increases with a pH increasement, the same relation was observed in antioxidant activity. About extractions, aqueous extraction has lower loss percentage during storage days in pHs (2.0, 3.5 and 5.0), thus use water as a solvent presented more advantageous than hydroalcoholic extraction. The degradation of the anthocyanins follow the first order reaction rate constants, and the half-lives of microcapsules were larger (2 to 7 times) than the extracts, and the largest half-lives were at low pH. Half-lives decreased and the degradation constants increased with pH increased.
The use of blackberry pomace is promising, since it has important antioxidant compounds, which when encapsulated may have technological applications by the food industry. Microencapsulation by spray dryer and with maltodextrin was efficient for the protection of phenolic compounds and anthocyanins during the studied storage period and the different conditions proposed to light, temperature and pH. Aqueous extraction was the only one that did not present anthocyanin loss in the temperature of 4 ºC, indicating its potential since this type of aqueous extraction is low cost and ecofriendly (does not use organic solvents). In general, higher stability was observed in samples stored at 4 °C and without light. At pH stability, the degradation of anthocyanins in the microcapsule was lower than the extracts, and the higher half-lives were in low pHs, until about 14 days.
Keywords: anthocyanins, phenolic compounds, antioxidant activity, HPLC, microencapsulation, spray dryer, maltodextrin

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