Document Type : Research Article


Gorgan University of Agricultural Sciences and Natural Resources


Introduction: Controlling and targeting release of bioactive compounds have a key role in improving their functional properties such as antioxidant and anti-disease activities. Encapsulation is one of the best methods for protection and controlling release of bioactive ingredients. Indeed, in this process, protection and controlling release of ingredients as core materials are performed by surrounding of them via variety of wall materials. Emulsions are most popular encapsulation systems that are classified in variety types such as single layer emulsion, multi-layer emulsion, doubleemulsion, and etc. Hydrophilic bioactive compounds can be loaded in inner aqueous phase of water in oil in water (W/O/W) double-emulsions. The stability of doubleemulsions is low due to presence of two interfaces in them.Applying a thermodynamically stable W/O emulsion (e.g., micro-emulsion) as a primary emulsion and using of complex biopolymers as emulsifier and stabilizer in outer phase of doubleemulsions can improve their stability (Dickinson, 2011; Boyer et al, 2012).
Saffron bioactive compounds include crocin, picrocrocin, and saffranal are widely used for a variety of functional and healthy goals in food and pharmaceutical industries. These compounds have many different functions, including anti-carcinogenic, anti-oxidant, anti-depressant, anti-apoptotic, anti-tussive, anti-nociceptive, anti-inflammatory and anti-thrombotic properties (Moraga et al, 2004).
In the present study, our main goal was kinetically evaluated release of crocin, picrocrocin and saffranal from inner phase to outer phase of doubleemulsion during 22 days storage by Zero order, Fist order, Higuchi, and Hixson-Crowell.
Materials and method: Saffron was provided from Torbatheydariyeh farms, Khorasan-e-razavi, Iran. Sunflower oil and sodium azide were purchased from FRICO (Sirjan, Iran) and Sigma-Aldrich (St. Louis, USA), respectively. Whey protein concentrate (80% protein) and sorbitanmonooleate (span 80) were obtained from Sapoto cheese (USA) and Merck (Germany), respectively. Maltodextrin was obtained from Qinhuangdao starch Co. (DE 16-20, China) and citrus pectin with a degree of methyl esterification of 71.1% and galacturonic acid >65% was purchased from MP biomedical (Netherland). All other chemicals used in this study were of analytical grade.
For extraction of crocin, picrocrocin and saffranal, a total of 10 grams of saffron sample was macerated in 150 mL of water in a glass bottle, covered with aluminum foil (to prevent direct exposure to light), and was placed in an incubator shaker (Kavooshmega, Iran) for 24 hours at 30oC. Then, this solution was homogenized (10000 rpm for 10 minutes, HeidolphSilentcrusher, Germany) for maximum extraction of saffron compounds. Finally, the extract was filtered under vacuum by using a Whatman No. 1 (11 mm) filter paper, and kept in the freezer at -18oC prior to any examination. ISO/TS 3632 procedure (2003) was used for the measurement of saffron compounds.
The doubleemulsions were prepared in two-step:
(a) Frist, primary W/O micro-emulsions were produced by two formulations: 60:30:10% and 62:33:5% of sunflower oil, span 80, and saffron extract, respectively.
(b) Then, the W/O micro-emulsions was gradually added into the outer aqueous phase contains why protein concentrate (WPC)/maltodextrin or WPC/pectin/maltodextrin while blending by a homogenizer (12000 rpm for 5 minutes at 10oC, HeidolphSilentcrusher, Germany) and then these coarse emulsions were further emulsified using mentioned homogenizer (15000 rpm for 8 minutes at 10oC). All doubleemulsions were composed of 25% primary emulsion and 75% outer aqueous phase
Droplet size of doubleemulsions after one day and 22 days storage weremeasured using Zetasizer (Malvern Instruments, Worcestershire, UK).
The released components in the outer aqueous phase were measured by evaluation of encapsulation efficiencyof the ratio of crocin, picrocrocin, and saffranalat a specific time:
E (%) = 100- (C2×100/C1) (1)
Where C2 is the percentage of crocin, picrocrocin and saffranal in outer aqueous phase and C1 equals to the percentage of compounds in inner aqueous phase.
C2 is a released into outer aqueous phase relative to the total amount present in the outer aqueous phase if all compounds were released (M ∞).
The viscosity of emulsions was measured using a programmable viscometer (model LVDV -II + Pro, Brookfield Engineering Laboratories, USA) and by a ULA spindle.
The released are kinetly evaluated by Zero order, Fist order, Higuchi, and Hixson-Crowell.
The experiments were all carried out in triplicate. The collected data were analyzed by one-way ANOVA; the means were compared by the Duncan's multiple range tests at the 5% level through SPSS version 21 (IBM, USA).
Results and Discussion: As shown in fig. 1, the droplet size of produced W/O micro-emulsions were lower than 200 nm. In fact, these droplets are water droplets containing bioactive compounds of saffron dispersed within oil phase that surrounded with Span 80 (Fig. 2).Also, it was found that by increase of saffron extract (from 5% to 10%) as dispersed phase in W/O micro-emulsions, droplet size and poly-dispersityindex (PDI) weresignificantly (P< 0.05) affected (Table. 3).
As shown in table. 4, crocin, picrocrocin, and saffranal had a same release trend, but the release rate of crocin was lower than saffranal and picrocrocin. As regard to R2, SSE, and RMSE from kinetic modeling in table. 5, the firstorder was a best model for release of crocin, and zero order was a best model for release of picrocrocin and saffranal. Also, kinetic date of release showed that the high release of crocin, saffranal, and picrocrocin was observed by increasing the dispersed phase content of primary W/O micro-emulsion and also it was found that WPC/pectindelayed the release of encapsulated ingredients more than single WPC (Table. 5). Indeed, the using of complex biopolymers as the external binary film of doubleemulsions causes a resistance to release for inner compounds (Dickinson, 2011).
As shown in fig. 3, the viscosity of doubleemulsions stability with WPC/pectin complex was higher than doubleemulsions stabilized by only WPC. This can confirm the higher stability of stabilized doubleemulsions with complex biopolymers (Olivieri et al, 2003).