Optimization of the Microencapsulation of Anthocyanins from Haskap Berries (Lonicera caerulea L.) in Ca-Alginate Beads for Oral Delivery

Giovana B. Celli, Amyl Ghanem, and Marianne S. Brooks

Dalhousie University, Halifax, NS, B3H 4R2, Canada

giovanacelli@dal.ca

ABSTRACT SUMMARY The aim of this study was to optimize the

encapsulation of anthocyanins from haskap berries (Lonicera caerulea L) in Ca-alginate beads using Box-Behnken (BB) design. Three variables were evaluated: sodium alginate (SA) and calcium chloride (CC) concentrations, and time (T) that the beads remained in the crosslinking solution. Encapsulation efficiency ranged from 17.97 to 63.12%. The mathematical model showed a high coefficient of determination (R2 = 97.98%) and the optimum conditions for the encapsulation were as follows: 9% (w/w) alginate, 2% CaCl2 (w/v) and 10 min in the crosslinking solution.

INTRODUCTION

Studies have shown that the consumption of a variety of berries can be associated with a number of positive health-related effects, such as reduction of cancer [1]. Haskap berries have high levels of anthocyanins and are recognized as the elixir of life by Japanese aborigines [2]. These fruits have a pleasant tart taste that is not often appealing to all consumers, which could limit the consumption of fresh fruits. In addition, isolated anthocyanins are very susceptible to degradation by pH, temperature, light, etc. Encapsulation is an effective technique to increase the stability of these compounds and for the elaboration of value-added products and nutraceuticals.

This work aimed to optimize the microencapsulation of anthocyanins extracted from haskap berries in Ca-alginate beads. The optimized encapsulate will be later evaluated by its thermal stability in comparison to the extract. This data prepares the way to determine the best applications of these encapsulates in food processes. EXPERIMENTAL METHODS

Freeze dried haskap berries (var. Indigo gem) were extracted with 80% ethanol (v/v) (0.5% formic acid, v/v) in a ratio of 1:25 (g/mL) under stirring at 600 rpm for 10 min at room temperature. The extract was filtered and evaporated at 40 ºC under vacuum.

BB design was used to optimize the encapsulation of this extract in Ca-alginate beads using the following variables and levels: low viscosity sodium alginate (9, 9.5, 10%, w/w) and calcium

chloride concentrations (2, 2.5, 3%, w/v, acidified with 2% (v/v) acetic acid glacial), and time in the crosslinking solution (10, 20, 30 min) (Table 1). The pH of the extract was adjusted to >4.5 before addition to the alginate solution (extract:alginate solution ratio 1:4 by volume) and extruded into the crosslinking solution through a 23G needle using a peristaltic pump (average flow rate of 0.3 mL/min). The encapsulation efficiencies (EE, %) were indirectly assessed by measuring the total anthocyanin content in the recovered CaCl2 solution (amount non-entrapped) by a colorimetric method [3].

RESULTS AND DISCUSSION An example of beads with encapsulated extract is shown in Figure 1.

Figure 1 – Beads produced with 10% (w/w) alginate after adjusting the pH and extruded to an acidified 3% (w/v) CaCl2 solution (ratio extract:alginate solution 1:19 by volume) (left – fresh beads; right – after drying overnight at room temperature, in the dark) The EE% results are shown in Table 1. Table 1 – BB design matrix used in this study N. Alginate

(%) CaCl2 (%)

Time (min)

EE (%)

1 9.0 2.0 20 42.25 2 10.0 2.0 20 46.20 3 9.0 3.0 20 17.97 4 10.0 3.0 20 50.66 5 9.0 2.5 10 57.84 6 10.0 2.5 10 63.12 7 9.0 2.5 30 39.39 8 10.0 2.5 30 62.27 9 9.5 2.0 10 51.85 10 9.5 3.0 10 46.50 11 9.5 2.0 30 36.79 12 9.5 3.0 30 32.32

13 9.5 2.5 20 30.57 14 9.5 2.5 20 27.90 15 9.5 2.5 20 29.33

The contour plots obtained from Minitab® 17.1.0

show that all variables assessed affected the encapsulation efficiency (Figure 2). Five anthocyanins were identified by HPLC in the evaporated extract prepared: 1) cyanidin 3,5-di-glucoside; 2) cyanidin 3-glucoside; 3) cyanidin 3-rutinoside; 4) pelargonidin 3-glucoside; and 5) peonidin 3-glucoside. These compounds are hydrophilic and the shorter the beads remained the crosslinking solution, the higher was their retention in the beads. Depending on the concentration of alginate and CaCl2 used, the loss of water while the gel shrinks could contribute to the leakage of anthocyanin from the beads. In addition, anthocyanins can present different resonant structures depending on the pH. It could be hypothesized that higher concentration of CaCl2 could lead to an electrostatic repulsion and attraction with calcium and chlorine, respectively, which could contribute to the leakage of anthocyanins from the beads.

The optimized model can be described by the

following equation:

EE (%) = 5162.8 + 48.2A2 + 0.1T2 – 988.5A – 281.3CC – 14.9T + 28.7ACC + 0.9AT + 0.04CCT

The optimal conditions would be 9% (w/w) of sodium alginate, 2% (w/v) of CaCl2 and 10 min in the

crosslinking solution, with predicted EE of approximately 70%.

It should be noted that the pH of the evaporated haskap extract was very low (approximately 2.9) and its adjustment was essential to prevent immediate gelation of the alginate solution and enable extrusion. However anthocyanin stability is also dependent on the pH and this should be considered when determining the experimental pH range.

CONCLUSION Results obtained from the BB design indicated that the optimal conditions for the encapsulation of anthocyanins from haskap berries were: 9% sodium alginate, 2% CaCl2 and 10 min, with predicted EE of 70%. The model obtained fits the experimental data well and has a high R2 (97.98%). We demonstrated that pH is an important factor to consider during the preparation of anthocyanins beads, as it can affect the stability of both the anthocyanin and the alginate. REFERENCES 1. Gordillo, G. et al. Antioxid. Redox Signaling

2009, 11, 47-58. 2. Bors, B. et al. Haskap breeding and production -

Final report. Saskatchewan Agriculture, 2012. 3. Giusti, M.; Wrolstad, R.E. Current Protocols in

Food Analytical Chemistry 2001, 1-13. New York: John Wiley & Sons.

ACKNOWLEDGMENTS

The authors acknowledge the National Council for Research and Development (CNPq – Brazil) and the Natural Sciences and Engineering Research Council (NSERC) of Canada for financial support.

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Figure 2 – Contour plots show that the highest efficiency is achieved with the lowest concentration of alginate and CaCl2, and in the shortest crosslinking time.