- The objectives of this research were to investigate the optimal conditions for anthocyanins extraction from different anthocyanin rich fruit and to develop microencapsulation formulation for improving stability of anthocyanin extracts. In the extraction optimization study, two extraction methods, "conventional solvent extraction (CE)" and "ultrasound-assisted extraction (UE)" for three different anthocyanin-rich fruit, blueberries, cherries and red pear peels, were investigated. For each extraction method, 3 extraction factors and 3 levels for each factor were evaluated: solvent type (methanol, ethanol and acetone), solvent concentration (60%, 70% and 80%), as well as extraction temperature (50, 60 and 70 °C) for CE or extraction time (20, 40 and 60 min) for UE. A L₉ (3x3) Taguchi design was employed to determine the most significant two factors, and then a completely randomized two factorial (2x2) design was applied to decide the optimal level for each factor (P< 0.05).
The extraction optimization was determined based on the high retention of total phenolic content (TPC), total monomeric anthocyanin (TMA), and radical scavenging capacity (DPPH assay) in extracts. Percent polymeric color (PPC) and individual anthocyanin distribution (HPLC analysis) of the extracts were also monitored to identify possible anthocyanin degradation during extraction. The optimum extraction conditions were identified as: 60% methanol, 50 °C, for 1 hour using CE or 70% methanol, 30 °C, for 20 min using UE for blueberries; 60% ethanol, 70 °C, for 1 hour using CE or 80% ethanol, 30 °C, for 20 min using UE for cherries; 60% methanol, 50 °C, for 1 hour using CE or 60% ethanol, 30 °C, for 60 min using UE for red pear peels. HPLC analysis identified different anthocyanin species from the three fruit extracts. Anthocyanin species, including delphinidin, cyanidin, petunidin, pelargonidin, peonidin, or malvidin with different sugar moiety in fruit extracts were altered by different extraction conditions. Therefore, different conditions for both CE and UE methods should be implemented for specific fruit aiming different anthocyanin compositions.
To prevent the environmental attacks on the stability of anthocyanin extracts during processing or storage, ionic gelation induced microencapsulation was applied to stabilize blueberry anthocyanin extracts (BB ACN) by forming the capsules between a cationic polymer, chitosan (CH), and two different anionic crosslinking agents: 1) sodium tripolyphosphate (TPP), a conventional inorganic agent, and 2) cellulose nanocrystals (CNC) as a newly found organic agent. A 3-step study was implemented. Firstly, the effect of titration direction of different crosslinking agents was evaluated for each formulation group on the yield of microcapsules (YOM),
TMA recovery, and particle characteristics. Secondly, the role of anionic crosslinking agent in encapsulation was investigated, and the encapsulation formulation was optimized to obtain BB ACN microcapsules with higher YOM and TMA recovery. TPC and DPPH for the free phenolic compounds remained in supernatants after collecting anthocyanin microcapsules were also measured, in which the lower TPC and DPPH observed in the supernatants indicated the better encapsulation performance. Thirdly, the effect of the amount of loaded BB ACN (0.41-26.06 cyaniding-3-glucoside mg/mL) on encapsulation efficiency was studied. In addition, ACN distribution in the obtained microcapsules (TMA attached on surface, bound with matrix, or freely existed in core) was also measured for evaluating the stability of formed microcapsules.
Our results showed that the titration direction of the crosslinking agent had no significant effect on TMA recovery as long as the same encapsulation formulation was used. BB-CH-CNC microcapsules exhibited significantly (p<0.05) higher encapsulation efficiency (up to 94%) than BB-CH-TPP. High YOM and TMA recovery was found when the concentration of anionic crosslinking agent (both CNC and TPP) was up to 1.0% (w/v) (mass ratio of chitosan and crosslinking agent = 1:10). Light microscope images clearly showed BB ACN entrapped in microcapsules in use of BB-CH-CNC formulation. ACN distribution in the microcapsules varied depending on the amount of loaded BB ACN. In the BB-CH-TPP microcapsules, 95% of TMA were entrapped in the matrix of wall materials, and the greater the amount of BB ACN loaded, the less the TMA found in the cores. However, BB-CH-CNC microcapsules had more freely available TMA in the cores (up to 48%) with less bound in the matrix and on the surfaces with increasing loading of BB ACN. This study provided new insights on the use of chitosan based microencapsulation technique for stabilizing BB ACN, in which CNC as an anionic crosslinking agent was more effective to produce rigid and stable microcapsules with high encapsulation efficiency, compared to TPP.