Project Number: 8030-31000-005-011-A
Project Type: Cooperative Agreement
Start Date: Feb 15, 2020
End Date: Jun 30, 2021
The purpose of this agreement is to evaluate the stability of astaxanthin in microalgal cells (McaAst) and to determine if the astaxanthin can potentially be used by Atlantic salmon. Encapsulating the microalgal cells will increase stability of the astaxanthin.
(1) Encapsulation: Microalgae containing astaxanthin (McaAst). McaAst will either be encapsulated or freeze dried and stored in a -80 ºC freezer until the encapsulation process has been completed. McaAst will be encapsulated in a food-grade hydrocolloid, sodium alginate (NaAlg) by ionotropic gelation techniques. About 1 - 5% NaAlg will be dispersed in milliQ water at room temperature under vigorous magnetic stirring. McaAst at 1 - 3% will be added to the dispersed NaAlg and further mixed. The blended dispersion of NaAlg and McaAst will be introduced in 20 mL plastic syringes and pumped into 0.1 M calcium chloride solution under low magnetic stirring. The microcapsulated McaAst will be washed and dried at room temperature. Encapsulation efficiency (EE) of McaAst in NaAlg will be calculated. Scanning electron microscopy (SEM), confocal laser scanning microscopy (Fig. 1) and particle size analysis will be used to study the morphology and size of the microcapsules. In vitro release study of encapsulated McaAst will be determined per the method described by Llorens et al. (2015) with slight modification. In brief, McaAst microcapsules will be incubated in PBS (0.01 M, pH 7.4) (release media) at 37oC and under shaking at 100 rpm. Aliquots of the release media will be withdrawn at 15, 30, 60, 90 and 120 min and absorbance read at 455 nm. (2) Stability Test: Triplicate freeze-dried McaAst samples will be stored in a cool room used to store fish feed for 8 weeks. Samples will be freeze dried McaAst, (reference), 1% NaAlg + McaAst, 2% NaAlg + McaAst, and 3% NaAlg + McaAst. Samples will be taken every week and analyzed in duplicate using high pressure liquid chromatography (HPLC) to determine the stability of the astaxanthin. Currently HPLC analysis is the best way to evaluate the amount of astaxanthin in salmon muscle tissue and algal cells. (3) In vitro Digestibility study: In vitro digestibility study will be used to predict accessibilty encapsulated McaAst. The two treatments that show the greatest stability in the previous test will be used in this study along with the reference McaAst. The method described by Rungruangsak-Torrissen et al. (2002) with slight modification will be used, that is instead of determining the amino acid reactive groups, astaxanthin content will be measured. The samples after digestion will be treated as described by Zhou et al. (2018). (4) HPLC analysis: Astaxanthin content of salmon fillet will be determined using HPLC per the method described by Ljungqvist et al. (2012). Standards will be purchased from Chromadex (Irvine, CA). (5) In vivo study: A digestibility study will be conducted using 500 g or greater Atlantic salmon at the USDA ARS National Cold Water Marine Aquaculture Center in Franklin, ME. About 180 g will be stocked in nine 1.0 m3 tanks (20 fish per tank depending on size) and supplied with 4-L min-1 of oxygen saturated water at a temperature of 13°C to14°C from a recirculating biological filtration system (32 ppt salinity).