The phycobiliproteins are water-soluble proteins and pigments found in the cytoplasm or the stroma of the chloroplast (Glazer et al., 1976; Pagels et al., 2019). Red micro and macroalgae, Cyanobacteria, and Cryptophytes are the only sources of reddish-red pigment, R-phycoerythrin (R-PE), and their primary function is to trap light energy between 495 and 650 nm wavelengths and transfer it to chlorophyll a of the photosynthetic reaction center of Photosystem II through other biliproteins as phycocyanin and allophycocyanin (Sekar and Chandramohan, 2008; Castro-Varela et al., 2021; Roy and Pabbi, 2022). R-PE is an oligomeric protein of 240 kDa with three subunits α (about 16 kDa), β (about 21 kDa), and γ (about 39 kDa), and they are bound to specific cysteines by thioether bonds (Li et al., 2019). The phycobiliproteins study mainly focuses on therapeutic applications (as bioactive), i.e., anti-inflammatory, antiviral, hepatoprotective, and anticarcinogenic capacities of R-PE have been reported (Thangam et al., 2015; Senthilkumar et al., 2018; Ulagesan et al., 2021).

The red algae often contain high levels of proteins (Hasan R. and Fao, 2009; Manivannan et al., 2009; Gómez-Ordóñez et al., 2010), in contrast to the brown algae with lower content (Angell et al., 2016). In this sense, the nutritional and the biological properties based on the protein coming from the R-PE from the red algae could be used as functional ingredients in several phases of food fortification, such as Kappaphycus alverezii (fish cutlet), Gelidium amansii (hamburger patties), Porphyra umbilicalis (restructured meats) (Mamatha et al., 2007; Moreira et al., 2010; Jeon and Choi, 2012; Le Guillard et al., 2015; Angell et al., 2016; Ngamnikom et al., 2017). The red macroalga Sarcopeltis skottsbergii (formerly Gigartina skottsbergii), endemic to the southern region of South America (Buschmann et al., 2008), presents a high content of the R-phycoerythrin, sulfated polysaccharide and mycosporine-like amino acids (MAAs) (Roleda et al., 2008).

R-PE is located within the phycobilisomes connected to Photosystem II in the chloroplasts; thus, cell disruption is required for the efficient release during the extraction process. There are several available methods for extraction of phycobiliproteins such as osmotic shock (Kawsar et al., 2011), maceration in the presence of liquid nitrogen in phosphate buffer (Munier et al., 2014), freeze grinding (Galland-Irmouli et al., 2000), freezing and thawing (Senthilkumar et al., 2013), ultrasonication (US) (Le Guillard et al., 2015) and homogenization (Pereira et al., 2020). Due to the complexity of structures and properties of bioactive compounds and the structure of the extracted materials, there is no available universal extraction protocol (Ciko et al., 2018). For this reason, it is essential to carry out extraction studies that allow us to obtain from the biological material high yield and bioactivity (Michalak and Chojnacka, 2014) by applying advanced extraction techniques (Ciko et al., 2018).

Therefore, exploring cost-effective extraction and separation methods for R-PE is necessary. Disruption of the rigid cell wall is a critical step required to increase the availability of algal proteins for extraction (Barba et al., 2015). Recently, extraction technologies, especially those considered green processes such as methods involving extraction with water, have gained attention to exploring marine resources for obtaining functional ingredients.

Ultrasound-Assisted Extraction (UAE) has attracted the attention of researchers for its application in food and allied industries (Michalak and Chojnacka, 2014; Chemat et al., 2017) and acts by creating compression and decompression through sound waves at the frequency of 20 kHz. Thus, several mechanisms for UAE action have been identified, including fragmentation, erosion, sonocapillary effect, sonoporation, local shear stress, and destruction-detexturation of plant cell wall matrix (Le Guillard et al., 2015; Chemat et al., 2017). An overall effect of these mechanisms results in disruption of cell wall. However, a given mechanism’s relative extent of contribution varies with the type of vegetal biomass and process parameters such as amplitude wave or time (Alexandre et al., 2017; Chemat et al., 2017). Another modern non-conventional alternative method to recover intracellular components is high-pressure homogenization (HPH), considered one of the greenest technologies, which showed a considerably shorter extraction time and higher yield than other conventional techniques (Ciko et al., 2018). This method is typically performed by forcing a liquid through a narrow nozzle at high pressure and establishing high shear stress. The use of mild temperatures makes this process especially attractive in extracting thermosensitive bioactive compounds (Alexandre et al., 2017). The membrane technologies are well suited to use with seaweed as part of a biorefinery process to maximize the valorization of all components within algae and avoiding the presence of heavy metals in the final product (Yaich et al., 2011). Some researchers have suggested that the combination of extraction technologies and the incorporation of membrane technologies could be used to isolate algal proteins using the same principles of molecular weight cut-offs used in the dairy industry. Ultrafiltration (UF) membranes constitute a physical barrier that retains all compounds bigger than the membrane molecular weight cut-off. UF could then be used to isolate proteins and other macromolecules between 1 and 200 kDa, as it has been validated on an industrial scale (food and feed industry) to generate enriched fractions less than 10, 5, 3 and 1 kDa. UF was used to isolate R-PE protein from macroalgae Grateloupia turuturu following cell homogenization, retaining about 100% of the protein without denaturation (Denis et al., 2009).

In this regard, determining a behavior pattern between R-phycoerythrin and the extraction method, we could consider a new attribute that reinforces the use of green methods for sustainability and development of a bioprocess. In fact, considering that there is no universal extraction protocol that ensures the quality of the structures and properties of the pigments (Liu et al., 2019). Therefore, it is believed that a specific extraction process must be established for each biological material extracted to obtain extracts with desirable bioactivity and high yield.

Response Surface Methodology (RSM) is a statistical tool used to determine and optimize the optimal experimental conditions to achieve maximum yields with minimum time and resource consumption (Wani et al., 2017). In this way, this study aimed to optimize the extraction process to obtain crude extracts with a higher yield of phycoerythrin from S. skottsbergii using RSM. Two green methods, UAE, and HPH, including the variables for UAE (time, amplitude, and solvent) and HPH (Pressure, number of passes, and solvent), were investigated for their influence on the yield of the extracts. UF technology, antioxidant activity, soluble protein content, and photosynthetic pigments were tested to evaluate the concentration and purity of R-PE in the aqueous extracts.

Red seaweeds, such as Porphyra sp. (Nori), have relatively high protein content (Mouritsen et al., 2018; Abdala-Díaz et al., 2019). However, in our study, the measured content of protein for S. skottsbergii (5.46%DW) was lowest than that of Porphyra sp. (Rhodophyta) (15.6%DW), but high respect to the brown algae Laminaria ochroleuca (Phaeophyceae) (Alginate source) (4.8% DW) reported by Abdala-Díaz et al. (2019). Protein levels may vary in different species, geographical areas, seasons, and extractions methodologies (Pereira et al., 2020). Seaweeds, especially red seaweeds, appear to be an essential source of proteins and biliproteins (Mouritsen et al., 2018; Pereira et al., 2020). On the other hand, it has been seen that the degree of sulphation is related to bioactivity in concordance with protein or polysaccharides. It is unknown if more significant bioactivity is present in this species related to sulfating of organic compounds. Carrageenan-based delivery systems present excellent performance in the delivery of bioactive ingredients, which can improve the stability and bioavailability of bioactive ingredients (Huang et al., 2021).

Generally, the R-PE yield depends on the solvent of extraction. However, the yield decreased above 20 min of exposition amplitude wave. This situation can be attributed to the pigment denaturation due to the amplitude wave, which increases the extracted temperature (48°C; non shown data). In this sense, more than 20 min is not recommended for S. skottsbergii, assuming that chloroplast is slightly damaged, explaining R-PE’s low release (Pereira et al., 2020). Similar conclusions were reported by Simovic et al. (2022), who found a reduction of R-PE over 45°C from macroalgae Porphyra purpurea (Rhodophyta) when using the high-pressure method. Knowing each variable’s individual and combined effects on R-PE extraction, a model was constructed to predict the optimum conditions at which higher R-PE yields can be extracted. The range optimizes the response is an amplitude between 60-90%, 10-20 min of extraction time, and distilled water as solvent. At the same time, the optimal predicted point is 68% of amplitude ultrasound, 18 min with distilled water. These observations agree with Pereira et al. (2020), who found that between 15-20 min under an ultrasound probe would result in a high yield of phycoerythrin from the red algae Gracilaria gracilis. Although they did not include the evaluation of amplitude wave, the extraction was done using buffer phosphate. This suggests the effects of extraction yield depend on the extraction solvent and the species of the macroalgae used. Mittal et al. (2017) analyzed different green technologies and observed that sustainable yield of the phycoerythrin extraction was obtained by a combination of the ultrasound probe with homogenization under buffer phosphate solvent.

However, the extractions yields are reduced (0.16 mg of R-PE g DW^-1) compared to this study. As a solvent of extraction, the distilled water is the primary factor that enhances the extraction of phycoerythrin and is less influenced by the combined effects of ultrasound wave (%) and time in S. skottsbergii (Figures 2A, B; Table 2). These observations are in agreement to Jubeau et al. (2013), Sudalkar et al. (2015), and Tan et al. (2020), who there reported higher levels when using distilled water for phycoerythrin extraction. The literature suggests that the UAE has the advantage of direct scalability due to its ability to generate progressively high-intensity cavitation zones and, therefore, suitable for scale-up of the industrial process (Le Guillard et al., 2015; Rodrigues et al., 2018). Previous studies reported the successful extraction of phycobiliproteins using ultrasonic waves from other types of algae, such as the microalgae Porphyridium purpureum (formerly Porphyridium cruentum) (Rhodophyta) and the macroalga Dasysiphonia japonica (formerly Heterosiphonia japonica) (Rhodophyta) (Román et al., 2002; Benavides and Rito-Palomares, 2006; Sun et al., 2009). In this sense, Sharma et al. (2020) found that the duty cycle variables and electrical acoustic intensity have significant exposure for enhancing the C-phycoerythrin from Oscillatoria sp. (Cyanobacteria) whereas Ardiles et al. (2020) found the maximum R-PE yield was obtained under optimal US conditions (Amplitude wave 100% and 15 min for Porphyridium cruentum (Rhodophyta). However, when larger periods of treatment were used (>20 min), a degradation of phycobiliproteins was observed (Figures 2A, B) (decrease in maximal absorbance), in agreement with other studies (Rodrigues et al., 2018; Pereira et al., 2020).

Using high-pressure homogenization (HPH), we observed that the distilled water as a solvent of extraction increases the R-PE yields with pressure and number of passes as a combined effect. Similar results were found by Sudalkar et al. (2015) and Jubeau et al. (2012), showing that water as solvent extraction can increase the yield of phycoerythrin extracts from red algae, as was observed in this study with S. skottsbergii. Our results suggest that the release of phycoerythrin can be promoted with distilled water due to accelerated molecule diffusion and solubility of other proteins. However, heat can also induce the degradation of the extracted bioactive compounds. The high amplitude of UAE (between 75-90% wave amplitude) and HPH (>450MPa) produces a higher temperature (average of 40°C; non shown data).

The macroalgae studied usually grow in the cold water (<10°C) from the Magallanes coast of Chile; thus, the bioactive components could be susceptible to increased heat. These might explain why high pressure and high ultrasound caused a decrease in the phycoerythrin content of the extract. Similar results were observed by Pereira et al. (2020), where the pressure at 600 MPa induced an increase in the extracted temperature, promoting pigment denaturation. Simovic et al. (2022) reported that contrary to elevated temperature, high-pressure (HP) treatment showed significant concentration at 450 MPa had a less destructive effect on R-PE color intensity from macroalgae Porphyra purpurea (Rhodophyta). However, the yields are less even if worked in a range similar to our range optimization of 100-300 MPa. This difference can be attributed to the different species of red algae and geographical latitude because the morphology of the cell wall and accumulation of metabolites can vary despite R-phycoerythrin being the pigment with the highest proportion in red algae.

On the other hand, the lyophilization ice crystals are formed during the freezing step (Soni et al., 2006) that, upon thawing, break down the cellular walls and release the intracellular content directly (Hardouin et al., 2014). Therefore, the treatment with freeze biomass can be used to achieve higher yields. Similar results were observed by Pereira et al. (2020), where the samples that were frozen at -80°C obtained higher R-PE yields than that at room temperature. Ghosh and Mishra (2020) found that the interaction between buffer molarity (0.1 to 1 M) and a number of freeze-thaw cycles (-70 to 27°C) was most significant for modeling the responses optimization of the (C-PE) from Lyngbya sp. (Cyanobacteria). Tan et al. (2020) showed similar results with the combination of freezing at −80°C (2 h) and thawing at 25°C (24 h) as optimal temperature and extraction time to obtain the highest amount of phycobiliproteins for Arthrospira sp. (Cyanobacteria). However, several studies have shown that this increase was not significant due to the high content of cellulose deposited in the cell wall (Kannaujiya et al., 2017; Thoisen et al., 2017).

Regarding other species of red macroalgae, previous studies showed that the extractions by using mortar maceration of Gracilaria corticata and Gracilaria canaliculata (formerly Gracilaria crassa) yielded 0.78 mg R-PE g^-1 DW and 0.5 mg R-PE g^-1 DW, respectively. These values are still lower than that by using the HPH method but similar to our maceration extraction (0.65 mg R-PE g^-1 DW). Previous results by Astorga-España et al. (2017), in the same species of S. skottsbergii by using buffer phosphate under the maceration method showed yields ranging from 0.057-0.078 mg g^-1 DW. These differences can be attributed to the extraction method, solvent (phosphate or water), seasonal harvest, and red macroalgae species. However, our work demonstrates high yield levels found in distilled water either by HPH or UAE. In this sense, we believe that the sulfated polysaccharides are released into the matrix because of the increase in temperature, which increases the viscosity of polysaccharides.

Consequently, the cell shear stress is high in this water, allowing the release of a greater quantity to the phosphate buffer. The next step of this work is to study the degree of shear stress at the level of the extraction matrix and comparison between extraction solvents. Therefore, this is a new report of a commercial species where different green extraction methods and solvents for extraction are compared. Considering that S. skottsbergii is a commercial species, the cost of extraction using distilled water is lower than that of buffer phosphate, previous to carrageenan extraction processes, visualizing a possible cost-effective biorefinery of this macroalgae in local industry from Chile.

From the observations in Figures 2, 3, our results could be helping to improve the pigment, which remains at a desirable level, considering its commercial importance and potential applications in the functional food sector or supplement market.

Most of the S. skottsbergii proteins are more soluble in the extracts A₁ and B₁ than A₂ and B₂, but not phycoerythrin, which is found in more water solutions (Jubeau et al., 2013; Sudhakar et al., 2015). This explains why the purity ratios are higher in distilled water than in phosphate buffer. The soluble proteins detected reached 2.6 ± 0.32% and 1.73 ± 1.1% using buffer phosphate and distilled water, respectively (p<0.05; Table 5). This result is in agreement with Jubeau et al. (2013) report, where a higher purity ratio (0.79) in distilled water than in a culture medium applying a two-step high-pressure process in microalgae Porphyridium purpureum was detected. Purification yields obtained here and for Denis et al. (2009) were different because of the biomass treatment, but the concentration of the R-PE was the same effect under 30KDa PES membrane. The permeate flux obtained during the filtration on 30 kDa PES membrane was similar in all treatments, allowing for retaining all R-PE without significant accumulation of unwanted molecules. Moreover, from an industrial point of view, polyethersulfone (PES) membranes are more widely used than cellulose regenerated ones due to their lower fragility and higher resistance to chemicals, pH, and temperature variations (Denis et al., 2009; Mittal et al., 2017).

When using HPH in distilled water (pH 7.0), the levels are higher than in phosphate buffer (pH 6.5). It is precisely in distilled water where the high levels of phycoerythrin are found (treatment B1) and chlorophyll and total carotenoids (treatment A1). This could be related to an increase in temperature and the release of polysaccharides that tend to increase the viscosity of the matrix, and so could change the pattern of the cells towards the pressure and the amplitude of the wave, as well as the solubility of PE, being higher in water as solvent by booth methods (UAE and HPH) compared to other solvents.

The R-PE in S. skottsbergii is influenced by HPH with water solvent reached 5.67 mg g⁻¹ by dry biomass. The content pigment in red algae is highly dependent on the perceived light intensity and quality of light (Castro-Varela et al., 2021). However, we suggest the application of HPH to release more content of the R-PE without decreasing the stability, as shown in the purity index (Table 3; Figure 3). Thus, these results reinforce using phycobiliproteins (aqueous extracts) from a new source of S. skottsbergii for nutraceutical or pharmacology applications (Jung et al., 2016; Mittal et al., 2019).

There are still few studies that evaluate the impact of the extractive technique on the bioactive, which makes it difficult to compare our results with other works on pigments process optimization. In this aspect, we consider that our work is the first approach to the state of the pigmentary material of this red macroalgae after method extractions, of high interest as several authors have valued for food applications (Le Guillard et al., 2015; Khanra et al., 2018; Liu et al., 2019; Mittal et al., 2019).

Biliproteins showed a positive correlation with the antioxidant activity in different red algae and cyanobacteria (Sekar and Chandramohan, 2008; Pagels et al., 2019). The pigments and antioxidant capacity of the water extract from the S. skottsbergii show a good relation. Biliproteins concentrations can vary depending on environmental factors like irradiance, light quality, pH or nutrients (Korbee et al., 2005; Celis-Plá et al., 2014; Astorga-España et al., 2017; Pagels et al., 2019). The extraction can also influence the content; e.g., a great number of authors used the freezing and thawing method (Niu et al., 2006). However, our observations showed significant antioxidant properties, which were increased with the enhancement of R-PE concentration in water solvent by UAE and HPH method. Although our study does not use a chromatographic technique for purification and determinate the polyphenols and polysaccharides, the concentred extract of R-PE could be part of a synergy of these compounds in the redox activity can be attributed to this macroalgae.

The extraction of R-PE from S. skottsbergii was optimized by RSM, using UAE and HPH. RSM proved to be useful for PE extraction in the tested range of optimization, providing a model with a good agreement between the experimental and predicted results. HPH obtained the most efficient extraction yielding 5.7 mg R-PE g^-1 DW biomass at the optimal conditions (300 MPa, 2 passes with distilled water), 50-60% higher than the PE yields obtained with UAE. The most crucial variable in the extraction process was the pressure level, with higher concentrations of R-PE using distilled water. S. skottsbergii revealed an excellent source of R-phycoerythrin and antioxidant activity, visualizing its application as a bioactive ingredient could be suggested for the food industry.

The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

PC-V, PC-P, FF, and MR conceived and designed the experiments; PC-V performed the experiments; all authors analysed the data and co-wrote the paper. All authors contributed to the article and approved the submitted version.

The research leading to these results has received funding from National Agency of Development and Research of Chile (PhD Scholarship N°21180257) and Andalusian government, Spain (Project FACCO, UMA18-FEDER JA-162).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.