Preliminary MSPD experiments
Before MSPD optimisation, preliminary analysis to evaluate the influence of the elution solvent on the extraction efficiency of fatty acids, carotenoids, and carbohydrates was performed. Ethyl acetate (EtAc), ethyl lactate (lactate), and ethanol (EtOH) recognised by the Food and Drugs Administration (FDA) as GRAS solvents were tested and compared with acetone (AcOH), methanol (MeOH), and MTBE. Figure 2 illustrates the relative overall MSPD extractive profile for the three solvent groups considered from red stage H. pluvialis wet paste.
All tested solvents showed fairly similar results in terms of their carotenoid and fatty acid extraction efficiency. Amongst GRAS solvents, ethanol showed the highest extraction efficiency, only 4% lower than that of MTBE for carotenoids and 2% lower than that of methanol for fatty acids. To understand the behaviour of the different solvents tested, an analysis was then performed for each family of compounds: carotenoids, fatty acids, and carbohydrates. The solvent behaviour analysis for each family of compounds is detailed below.
Once the overall carotenoid profile has been reported, an approach towards the individual response of these bioactive compounds is necessary. H. pluvialis in its red phase is characterised by a rich carotenoid content (up to 6%), with astaxanthin having the highest concentration (Cohen 1999). However, only 5% of the total astaxanthin is found in its free state, being generally present in esterified form, mainly coupled to palmitic, oleic, and linoleic acids (Shah et al. 2016). Figure 3a shows the individual response of the astaxanthin characterised in the preliminary study, as well as its respective monoesters (AME) coupled to C18:1 (oleic acid), C18:2 (linoleic acid), and C18:4 (stearidonic acid) acids for the selected solvents.
In a second approach other carotenoids were identified in H. pluvialis paste: canthaxanthin, diadinoxanthin, and phoenicoxanthin, which extraction efficiency in the tested solvents is depicted in Fig. 3b. The response of the neoxanthin/violaxanthin and zeaxanthin/lutein isomers is plotted together.
The relative response for the most relevant fatty acids in H. pluvialis wet paste was compared. Figure 4a shows the results obtained for the saturated fatty acids (SFA) and Fig. 4b those for the monounsaturated (MUFA) and polyunsaturated (PUFA) groups. The extraction efficiency presents a general trend: MeOH > EtOH > AcOH > MTBE > EtAc > lactate. To highlight the extractive behaviour of each solvent with respect to the overall mean of the respective compound, an average line is drawn.
Haematococcus pluvialis under stress conditions (e.g., temperature variations, nutrient starvation, light stress, and acidity) stored around 40% w/w of the total cellular carbohydrate content (Shah et al. 2016). To assess in detail the affinity profile of the solvents studied in the carbohydrate extraction, two main sugars, which represent the main carbohydrate content in the microalgae, are shown in Fig. 5. Hence, the high affinity for methanol, ethanol, and acetone is highlighted, in contrast to solvents such as ethyl lactate which generates an extract highly refined in these carbohydrate compounds.
MSPD extraction optimisation design
The diverse extraction efficiency shown by the tested solvents for the fatty acids and carotenoids creates a compromise situation. It was necessary to select a solvent with the highest efficiency to extract the bioactive compounds being suitable for nutraceutical purposes. Compared to the non-GRAS solvents, ethanol showed an overall response 2% lower than the best solvent (methanol), presenting a uniform performance for the considered compounds, plus a stable profile within the analytes in each family. Thus, an optimisation procedure for extraction of wet red stage H. pluvialis via MSPD was carried out using ethanol as the extraction solvent.
To analyse the response of red stage concentrated paste (25% w/v) of H. pluvialis to the modification of the main extraction parameters by MSPD, an optimisation design was performed starting from the microalga in its wet state. In this fine design, the critical variables affecting extraction, sample mass, dispersant/sample ratio, and extraction solvent volume, were optimised. They were established based on previous experience in MSPD extractions using wet organic samples (Rubio et al. (2018) and Castillo et al. (2020)). Extraction (solvent elution) time and disruption time were set at 20 min and 10 min, respectively (Hoff and Pizzolato 2018). Table 1 summarises the factors and their selected levels.
In the case of the ratio dispersant/sample mass, a minimum value of 4 was established, due to the wet nature of the sample. Figure 6 shows that ratios lower than 4:1 resulted on a heterogeneous and agglutinated mixture, unsuitable for its treatment in the MSPD extraction column.
The main effect plots are depicted in the Pareto charts of Fig. 7, revealing statistical significance of the parameters A: sample size and C: extraction solvent volume, for both fatty acids and carotenoids.
Although the dispersant:sample mass ratio does not show a statistically significant effect on the extraction of the compounds, a ratio 8:1 operationally improved both compaction and extractive bed size. However, a larger bed size generates a larger solvent path (higher tortuosity). This phenomenon is depicted in Fig. 8, which shows the effect of each parameter studied with the response obtained for carotenoids and fatty acids. In this last case, increasing the dispersant:sample mass ratio generated only a discrete improvement compared to its adverse effect on carotenoid extraction.
MSPD extraction efficiency
The optimised MSPD-based extraction process was able to generate a multicomponent extract from wet paste of H. pluvialis in its red stage without requiring any pre-treatment of the microalgae. The extraction efficiency of the proposed method was compared with reference methods for the isolation and recovery of carotenoids and fatty acids from microalgae. The results of the comparison, as well as the characterisation of the polyphenols and the evaluation of the antioxidant activity of the extract, are detailed below.
The reference method (RM) selected to evaluate the efficiency of MSPD to extract carotenoids from wet H. pluvialis paste was that proposed by Kim et al. (2013) for the recovery of carotenoids from D. tertiolecta, adapted for the strong cell wall disruption of H. pluvialis. From Fig. 9, which provides a comparative graph of the extraction results achieved by both methods for free astaxanthin, canthaxanthin, beta-carotene, and the total of zeaxanthin/lutein isomers, it is clearly shown the improvement in the extraction of carotenoids when MSPD is applied. An average difference of 55% in the recovery between both methods has been obtained, showing a similar behaviour for all compounds. This better efficiency of MSPD for carotenoid extraction was also found in our previous study using lyophilised H. pluvialis (Castillo et al. 2020).
The extraction efficiency of fatty acids by MSPD was compared to the Bligh and Dyer (1959) RM. This method has been tested in H. pluvialis for different lipid chains (from C12 to C20) extraction, generating the highest global results compared to methodologies such as Folch, hypochlorous acid treatment, and acid-catalysed hot-water extraction (Otero et al. 2017). Besides, the option of a prior saponification of H. pluvialis was discarded, as its effect has been proved to be indifferent or disadvantageous on the overall lipid recovery (Otero et al. 2017). The result of the comparative analysis is shown in Fig. 10.
The fatty acids were divided into 2 groups according to the concentration in the microalgae. In addition, a blue-coloured surface is plotted to highlight the absolute difference of the recoveries obtained by both methods. This surface reveals a higher relative extraction by the MSPD as a function of the RM when the unsaturation in the lipid compounds increases. In the case of palmitic acid (C16:0), the recovery by RM is 15% lower, decreasing slightly for linoleic acid (C18:2) with 22%. However, for linolenic acid (C18:3), the difference is even more marked, with MSPD showing a 68% improvement in extraction.
Polyphenolic comprehensive analysis
In a first approach, the total polyphenol content (TPC) of the extract was evaluated by the Folin-Ciocalteu method, obtaining a total of 24.65 mgGAE g−1 DW for the MSPD ethanol extract of the wet paste red stage H. pluvialis. A remarkable aspect of the phenolic content of H. pluvialis is that, although a proportionality with the concentration of carotenoids is known, there is limited information regarding the phenolic compounds responsible for the bioactivities of the microalga (Deniz 2020). Thus, to uncover the polyphenolic profile of H. pluvialis, an untargeted analysis of the extract was performed, aimed to the characterisation of polyphenols.
The criteria used for the untargeted identification and confirmation of the compounds follows a precise validation algorithm previously used for the analysis of H. pluvialis (Castillo et al. 2020), as well as extracts of natural origin via ESI-QToF (Ríos et al. 2015). The process starts with the analysis of the analyte intensity, which must be above the confidence limit of the instrument. If approved, the candidate must meet two main parameters: its isotopic standard and mass deviation. The isotopic pattern, reflected as mSigma, shows the effective ratio of the experimental mass fragmentation to the theoretical basis. Likewise, the mass deviation expresses the deviation between the detected experimental mass and the theoretical mass. As upper limit values, 50mSigma and 5mDa are set, which generates an efficient screening that is then supported by the targeted analysis. In this way, applying the algorithm described above, the phenolic compounds phloroglucinol, p-coumaric acid, gallic acid, and catechin, were identified (Table 2).
Due to their low concentration in the extract, catechin and gallic acid presented a profile that did not allow mSigma values below the established limit, and their identification was initially considered as tentative. In a subsequent targeted analysis, the phenolics compounds (phloroglucinol, p-coumaric acid, gallic acid, and catechin) were identified and quantified by comparison with their respective standards.
Phloroglucinol and p-coumaric acid stand out amongst the reduced number of studies dealing with the polyphenolic profile of H. pluvialis. These compounds have not been previously reported in red stage of H. pluvialis, although a study by Goiris et al. (2014) focused on the search for different compounds of the family of flavonoids, cinnamic acids and simple phenols, revealed that phloroglucinol represented around 99% (81 μg g−1) of the total phenolic compounds in the green phase of the microalga, with more than 70% (0.6 μg g−1) of the remaining amount accounted by p-coumaric acid. The results found for the red stage paste show a similar profile (Fig. 11), although the phloroglucinol concentration was higher (2.3 mg g−1), with 32.0 μg g−1 of p-coumaric acid, and much lower of catechin (10.0 μg g−1) and gallic acid (7.8 μg g−1).
For the antioxidant activity evaluation of MSPD wet paste H. pluvialis extracts, the DPPH analysis was initially performed. It is common to quantify the antioxidant activity of carotenoids using the DPPH radical at a maximum wavelength of 515 nm. However, only one study reports the possible interference between the absorbance of carotenoids contained in H. pluvialis (400–500 nm) with the DPPH reagent (Santoyo et al. 2010). Several dilutions of the H. pluvialis extract in methanol were carried out to check such interference. As shown in supplementary Fig. S3, and considering that absorbance is an additive property, from a dilution factor range from 140 up to 500 (v/v), a marked interference is observed at 515 nm in the absorption of the extracts related to that of DPPH radical. From a dilution factor of 1000, a measurement without significant interference is obtained, but at this dilution the extract is unable to give an appreciable response in the free radical inhibition. Hence, despite the common practice of performing the DPPH procedure to measure the antioxidant activity of carotenoids, and due to the proven impossibility of measuring the antioxidant activity with this methodology, the ABTS radical method was alternatively used. ABTS cation has its maximum absorption at 645, 734, and 815 nm when ethanol is used as the working solvent, which does not interfere with the absorption of the sample analysed (Re et al. 1999). Accordingly, the antioxidant value obtained for red stage H. pluvialis wet paste by the proposed MSPD extraction method was 1.58 mmolTE g−1 DW.