*3.2. Phycobiliprotein Purification*

PBP purification was conducted according to the method of Ranjithak and Kaushik [63] with modifications. Briefly, cyanobacterial cell suspensions were digested overnight with lysozymes at a final concentration of 1.0 mg/mL in a 50 mM phosphate bu ffer of pH 7.2 at 37 ◦C. Next, the cell suspensions were sonicated in a water–ice bath for 2 min at 15-s intervals (Microson Ultrasonic Cell Disruptor XL, Farmingdale, NY, USA). One volume of the sonicated extract was diluted with one volume of phosphate bu ffer and centrifuged at 10,000 rpm for 15 min (RC 5B Plus Sorvall SS-34, Newtown, USA). The PBP-rich supernatants were recovered, and 500 μL of aliquots were used to obtain the corresponding 280–700 nm UV/VIS absorption spectrum. The selective precipitation of proteins from the remaining supernatants was conducted by adding ammonium sulfate, with continuous stirring at 4 ◦C to reach a 0%–35% saturation and a 35%–60% saturation to enrich precipitates with PE and PC, respectively. After 24 h, each protein precipitate was resuspended in 1.0 mL of phosphate bu ffer and dialyzed in 12,000 Da cut-o ff cellulose dialysis tubing (Sigma, Steimheim, Germany) against distilled water at 4 ◦C. Aliquots of the dialyzed PBP solutions were used to obtain the corresponding 280–700 nm absorption spectra. The absorption maxima 620 nm (PE), 565 nm (PC) and 650 nm (APC) were used to compute their concentration according to the following equations:

[PC] (mg/mL) = ([(A615 − A730) - 0.476 (A652 − A730)] × 1/5.34) [APC] (mg/mL) = ([(A652 − A730) - 0.208 (A615 − A730)] × 1/5.09) [PE]] (mg/mL) = ([((A562 − A730) - 0.241 [PC] − 0.849 [APC]] × 1/9.62)

PBPs were further purified by ion exchange chromatography by using a DEAE (Diethylaminoethyl-cellulose) column (7.0 by 1.5 cm). Two or three milliliters of a PBP-dialyzed solution was loaded in the column and eluted with a 0.03–1.0 M NaCl gradient at room temperature. One-milliliter fractions were collected, and the colored fractions were pooled and scanned to obtain the UV (Ultraviolet)–Vis (Visible) absorption spectra. Concentration and purity were inferred by using the corresponding equations. The collected purified PBPs were lyophilized and stored at −20 ◦C until used.

## *3.3. PBPs Stability*

Triplicates of each purified PBP solution were incubated in a 50 mM phosphate buffer of pH 7.2 at temperatures from 0 to 80 ◦C over 72 h. Protein stability was evaluated in triplicate assays at room temperature in a 50 mM sodium phosphate buffer adjusted to pH 1 to 14. Changes in PBP concentration were obtained from the corresponding UV–Vis absorption spectra every 24 h. In addition, purified PE from the LLC-10 strain and PC from the LLA-10 strain were subjected to incubations at 138 ◦C for 4 s; visible absorption spectra were obtained before and after the heat treatment to evaluate changes in stability.

#### *3.4. PBPs Antioxidant Capabilities*

The biomass from cyanobacterial strains *Nostoc* sp. Llayta (LLC-10) and *Nostoc* sp. Caquena (CAQ-15) at the exponential growth phase was recovered by centrifugation (4000 rpm, 10 min; rotor Sorvall SS-34) and washed twice with a 0.9% NaCl solution. The washed cell pellets were extracted with 4.0 mL of 70% methanol and vortexed at maximal speed for 2 min in a Genic2 multitube holder (Daigger Sci. Ind., model G560E, Bohemia, NY, USA). The methanol extracts were sonicated on a water–ice bath (Microson Ultrasonic Cell Disruptor XL, Farmingdale, NY, USA) for 3 min at 10-s intervals and clarified by centrifugation (4000 rpm, 10 min), and then the supernatants were saved. The methanol extracts were filtered through 0.2 μm SFCA (Surfactant-Free Cellulose Acetate) syringe filters (Ultra Cruz) and stored at −20 ◦C. The antioxidant capacity of the methanol extracts and the purified PBP was performed by ABTS and FRAP assays according to Re et al. (1999) and Benzie and Strain (1996), respectively [64,65]. Results were expressed as μmoles of Trolox equivalents per 100 g of fresh mass or milligram of pigment.

## *3.5. Toxicity Assays*

Wild-type *C. elegans* var. Bristol-N2 and the *Escherichia coli* strain OP50 were obtained from the *Caenorhabditis* Genetics Center (St. Paul, MN, USA). The nematode was plate-propagated as previously described [66]. *C. elegans* var. Bristol-N2 was cultured on nematode growth agar that was seeded with either *E. coli* OP50 or *E. coli* strain B, following the procedure reported by Brenner [67]. Gravid worms were gently shaken at room temperature in 10 volumes of a fresh 1% NaClO/0.5 M NaOH solution. Carcasses and other debris were dissolved after 5–10 min, and resistant eggs (50% to 100% viable eggs) were collected and washed several times in an M-9 buffer [46]. The M-9 buffer was prepared with 1.5 g of KH2PO4, 3.0 g of Na2HPO4, 2.5 g of NaCl, 0.5 mL of 1 M MgSO4, and sterile distilled water to a final volume of 500 mL [68]. Eggs were deposit in agar plates for 48 h, and young worms (larval stage 4 and 5) were harvested and resuspended in the M-9 buffer.

#### *3.6. Preparation and Sensory Analyses of Dairy Prototypes*

Ten mL of skimmed milk were mixed with purified PBP pigments at a final concentration of 1.2 mg PC/mL and 0.3 or 1.4 mg PE/mL. The skim milk that was used in this work was a commercial liquid product prepared by Colun (Cooperativa Agrícola y Lechera de la Unión Ltd., La Unión, Chile) with a content of 3.3% of total protein, 0.05% total fat, 4.7% carbohydrates, 32 mg% sodium, 115 mg% calcium and 90 mg% phosphorus. According with the manufactures, this skim milk was processed by UHT technology (Ultra High Temperature) at 138 ◦C for 4 s. The acceptability of PBP-containing dairy prototypes was evaluated by sensory preferences. The study used skimmed milk as a common matrix, and three dairy prototypes were designed: Prototype N◦ 1 (PC from the LLC-10 strain), Prototype N◦ 2 (PE from the CAQ-15 strain) and Prototype N◦ 3 (PE from the LLC-10 strain). A five-point hedonic scale test was used to measure appearance, smell, texture and flavor. The tests involved ten university students as impartial judges who did not have previous training in sensory-type analyses. Consumer acceptability was measured with five-point hedonic scale (1—dislike extremely; 2—dislike slightly; 3—neither like nor dislike; 4—like slightly; 5—like extremely). Data were subjected to variance analysis (ANOVA) with a significance level of *p* < 0.05.
