Extraction of astaxanthin from microalga Haematococcus pluvialis in red phase by using generally recognized as safe solvents and accelerated extraction

Accepted Manuscript Title: Extraction of astaxanthin from microalga Haematococcus pluvialis in red phase by using Generally Recognized As Safe solvents and accelerated extraction Authors: Antonio Molino, Juri Rimauro, Patrizia Casella, Antonietta Cerbone, Vincenzo Larocca, Simeone Chianese, Despina Karatza, Sanjeet Mehariya, Angelo Ferraro, Evangelos Hristoforou, Dino Musmarra PII: DOI: Reference:

S0168-1656(18)30536-4 https://doi.org/10.1016/j.jbiotec.2018.07.010 BIOTEC 8215

To appear in:

Journal of Biotechnology

Received date: Revised date: Accepted date:

17-1-2018 4-7-2018 4-7-2018

Please cite this article as: Molino A, Rimauro J, Casella P, Cerbone A, Larocca V, Chianese S, Karatza D, Mehariya S, Ferraro A, Hristoforou E, Musmarra D, Extraction of astaxanthin from microalga Haematococcus pluvialis in red phase by using Generally Recognized As Safe solvents and accelerated extraction, Journal of Biotechnology (2018), https://doi.org/10.1016/j.jbiotec.2018.07.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Extraction of astaxanthin from microalga Haematococcus pluvialis in red phase by using Generally Recognized As Safe solvents and accelerated extraction

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Antonio Molino(1,*), Juri Rimauro(1), Patrizia Casella(1), Antonietta Cerbone(1,2), Vincenzo

Larocca(3), Simeone Chianese2, Despina Karatza2, Sanjeet Mehariya(1,2), Angelo Ferraro4, Evangelos

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Hristoforou4, Dino Musmarra2

ENEA, Italian National Agency for New Technologies, Energy and sustainable economic

Department of Civil and Building Engineering, Design and Environment, Università degli Studi della Campania

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Development. Department of Sustainability - CR Portici. P. Enrico Fermi, 1, 80055 Portici (NA), Italy

ENEA, Italian National Agency for New Technologies, Energy and sustainable economic

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3

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“L.Vanvitelli”, Real Casa dell’Annunziata, Via Roma 9, 81031 Aversa (CE), Italy

Development. Department of Sustainability - CR Trisaia SS Jonica 106, km 419+500 - 75026 Rotondella (MT), Italy School of Electrical and Computer Engineering, National Technical University of Athens, Zografou Campus, 9, Iroon

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4

Polytechniou str, 15780 Athens, Greece

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* corresponding author name: PhD Antonio Molino - ENEA, Italian National Agency for New Technologies, Energy and sustainable economic Development. Department of Sustainability - Biotechnologies and Agroindustry Division. CR

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Portici. P. Enrico Fermi, 1, 80055 Portici (NA), Italy. Tel.: +39 081/7723276 E-mail address: [email protected]

Highlights

Astaxanthin extraction by using Accelerated solvent extraction technology;

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Extraction of bio-products from micoralgae;

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Effect of pretreatment on the yield extraction.

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Abstract Solvent Extraction was tested to extract astaxanthin from Haematococcus pluvialis in red phase (HPR), by investigating effects of solvents, extraction pressure and temperature. Astaxanthin isomers were identified and quantified in the extract. The performances of acetone and ethanol, Generally Recognized As Safe (GRAS) solvents, were explored. Negligible effect of pressure was

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found, while with increasing extraction temperature astaxanthin recovery increased till a maximum value, beyond which thermal degradation seemed to be greater than the positive effect of

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temperature on extraction. Furthermore, to maximize the extraction yield of astaxanthin, mechanical pre-treatment of HPR biomass was carried out and several extraction runs were consecutively performed. Experimental results showed that after the mechanical pre-treatment the astaxanthin

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recovery strongly increased while a single extraction run of 20 minutes was sufficient to extract

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more than 99% of total astaxanthin extracted. After pre-treatment, maximum recovery of about 87%

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was found for acetone (pressure = 100 bar; temperature = 40°C; total time = 60 minutes).

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Keywords: Bioprocess, astaxanthin carotenoid, accelerated solvent extraction, antioxidants, GRAS

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solvents

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1. Introduction

Astaxanthin (C40H52O4, 3,3'-dihydroxy-β,β-carotene-4,4'-dione) is a carotenoid belonging to the

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xanthophylls group, which is very attractive for important industrial markets thanks to its interesting properties, such as food grade, colouring and antioxidant agent. It is used for different purpose, for example, in aquaculture field as feed additive for salmons, trouts, and crustaceans to provide the characteristic pink/red colour. Astaxanthin is also appreciated for its antioxidant and beneficial effects on both reproduction and immune systems of bred species (Amaya et al., 2014; Lim et al., 2017) and anti-ageing effect in the cosmetic sector (Ambati et al., 2014). However, its

main use remains the feed additive in the aquaculture for fishes growth and nourishment of ornamental birds (Irwandi Jaswir et al., 2011). Over its anti-aging effect, several literature studies showed its anti-inflammatory, cardioprotective, neuroprotective, gastroprotective, nephroprotective, anti-diabetic, anti-cancer, antiasthmatic and immunoprotective properties. For these reasons, astaxanthin is also currently used in the prevention and control of many pathological conditions on

Kamath et al., 2008; Liao et al., 2016; Masoudi et al., 2017; Wang et al., 2014).

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low oxidative and inflammatory (Bolhassani, 2015; Chuyen and Eun, 2017; Donà et al., 2013;

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Indeed, astaxanthin is directly not produced by fishes, crustaceans and other animals, which must assimilate it through food. In nature, it is mainly synthesized by aquatic and non-acquatic

microorganisms such as bacteria, fungi, yeasts and microalgae. Although it is a natural substance,

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but mostly used in synthetic form due to lower production costs, greater purity and

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stability.Chemical synthesis is carried out through several reactions, such as canthaxanthin

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condensation of dienol-ether (Nguyen, 2013).

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hydroxylation, witting reaction between a C10 dialdehyde and a phosphonium salt or by

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Both colouring and antioxidant properties of astaxanthin are due to its molecular structure, such as the presence of double bonds in the aliphatic chain of 40 carbon atoms. Therefore, it is enable to

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absorb light at wavelengths between 400 and 500 nm, which giving the characteristic pink/red colour. Furthermore, this structural characteristic makes astaxanthin skillful to defend cells from

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oxidative species and free radicals due to the high light exposure (Britton G. et al., 2004). Astaxanthin molecule consists of three different configurations: two enantiomers (3'R 3R and 3'S

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3S) and one meso-form (3R 3'S), and the most widespread natural form is the 3’S 3S configuration (Higuera-Ciapara et al., 2006). On the contrary, synthetic astaxanthin consists of a racemic mixture composed by the three configurations which confer higher stability to the substance (Ambati et al., 2014). Synthetic astaxanthin has a cost of about 1000 $/kg, while the natural one, derived from Phaffia Rhodozyma yeast and/or H. pluvialis microalgae, is in the range of 2,500-7,000 $/kg (Del Campo et al., 2007; Koller et al., 2014). Almost all commercially available astaxnthins are synthetic

(about 99%) and they are produced by big companies such as BASF and Hoffman-La Roche. Only a little fraction of astaxanthin (<1%) currently derives from natural sources. In the last decade, due to its valuable properties astaxanthin has reached an increasing market trend as it is shown in the

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market analysis reported in figure 1.

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Figure 1: Astaxanthin estimated market trend.

The astaxanthin production was about 200 ton with a turnover of 368 million euros in 2014, and the forecasted market size is expected to double up to 729 million euros in 2022 (Industry Experts,

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2015; Lorenz and Cysewski, 2000; Panis and Carreon, 2016).Whilst synthetic astaxanthin is more

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competitive than natural one, because of lower costs, better stability and purity. In the lasts years,

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several projects have been carried out to demonstrate the feasibility of astaxanthin extraction from

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natural sources. In this field, H. pluvialis can be considered a promising source of astaxanthin due

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to its ability to accumulate high quantities with respect to Phaffia Rhodozyma yeast (Global Market Insights, 2017; Li et al., 2011).

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H. pluvialis belongs to Volvocales a Green algae (Cloroficee) class, which lives as single cell in freshwater environments. The life cycle consists of several phases: macrozooid (zoospore),

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microzooids, palmella, and aplanospore (Shah et al., 2016). The macrozooid stage is an elliptical cell equipped with two filaments from 8 to 20 µm long, a thin cell wall and extracellular gelatinous

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matrix (Wayama et al., 2013). As most unicellular organisms, H. pluvialis in many phase of its life cycle may present a rigid cell-wall formed by a mix of proteins and polysaccharides, which surrounds and protects the cellular body. The highly complex and dynamic composition of cell-wall allows H. pluvialis to survive in hostile environment, but on the other hand represents an issue when H. pluvialis biomass has to be processed to extract valuable intracellular compounds such as astaxanthin.

A peculiar characteristic of this microorganisms is evident when they growth under stressed conditions. Indeed, this condition induces a cellular and metabolic transformation in H. pluvialis that loses the two characteristic filaments, increasing body volume as well as the cell wall thickens up to 2 µm and the elliptical cell converts into a spherical form of diameter in the range of 20-40 µm (cyst form) (Wayama et al., 2013). In this phase, called “red phase” because of the colour

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transition from green to red, H. pluvialis increases its astaxanthin synthesis from the lower content in the green phase (0.1% on dry basis) until the range of 1.5% to 3% on dry basis (Cerón et al.,

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2007).

To make astaxanthin extraction from H. pluvialis more competitive than synthetic one and, at the same time, promote its use in the aforementioned markets. Therefore, it is necessary to overcome

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critical points related to the biomass concentration, treatment and degradation reactions that can

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occur compromising both quality and quantity of the extracted astaxanthin (Mercer and Armenta,

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2011).

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In the last years, several extraction techniques have been explored, such as mechanical treatments,

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chemical treatments using solvents, pressurized extraction, ultrasounds and microwaves but there is not yet a clear framework about advantages/disadvantages on their uses (Denery et al., 2004; Haque

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et al., 2016; Kim et al., 2016; Ruen-ngam et al., 2010). Nowadays, several research groups are working on the use of green solvents (or less toxic solvents)

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for the extraction process. Also improving the critical point related to the degradation reactions that can compromise both quality and quantity of the extracted astaxanthin (Mercer and Armenta, 2011).

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Among them, Denery et al. (2004) homogenized H. pluvialis samples and extracted astaxanthin by both acetone and ethanol pressurized extraction solvent technique (Extraction conditions: 1500 psi, 40°C, two 5 min extraction cycles); experimental findings resulted in total astaxanthin extraction yields of 9.5 mg/g and 8.4 mg/g, respectively. Jaime et al. (2010) applied mechanical disruption at freezing temperatures followed by ethanol pressurized extraction solvent (extraction conditions: 10.34 MPa, 100°C, 20 min); experimental findings resulted in a total astaxanthin extraction yield of

20.7 mg/g dry weight. Mendes-Pinto et al. (2001) used a cell homogeniser developed ad hoc and compared several pre-treatment techniques (Autoclave, HCl, NaOH, Enzyme, spray drying and mechanical disruption) by using acetone as extraction solvent (extraction conditions: room temperature, 16 hours); experimental findings highlighted that the highest recoveries of astaxanthin (81%) were found for biomass mechanically pre-treated or autoclaved. In our work, experimental

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tests have been done focused on developing extraction technologies that avoid of above mentioned issues.

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In this work, the effects of several parameters such as time, temperature, pressure and mechanical pre-treatment on the extraction yield of astaxanthin through Accelerated Solvent Extraction were

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investigated. Several solvents, including GRAS solvents (acetone and ethanol), also in comparison

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with more toxic solvent (chloroform), were tested. 20 minutes for every extraction run was used,

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two levels of pressure (50 and 100 bar) were investigated while extraction temperature varied in the

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range 20-100°C. Moreover, astaxanthin isomers (all-trans astaxanthin, 9-cis astaxanthin and 13-cis

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astaxanthin) were identified and quantified by u-HPLC and GC-FID analysis.

2. Materials and Methods

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figure 2.

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The experimental procedure for astaxanthin extraction from HPR is schematically represented in

The procedure includes a mechanical pre-treatment that can be used or not, the solvent extraction at temperature and pressure controlled, and the final astaxanthin separation and quantification.

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A detailed description of the investigation planning and of the experimental procedure, including solvents, reagents, chemical standards and H. pluvialis in red phase, are given in the following sub sections.

Figure 2: Extraction and experimental set-up for astaxanthin recovery from Haematococcus pluvialis red phase.

2.1. Solvents, reagents and chemical standards Acetone, chloroform, methanol, ethanol, hexane and water were purchased from Sigma-Aldrich (Saint Louis, Missouri, USA) as well as the standards used for u-HPLC and GC-FID analysis. All solvents were of u-HPLC grade and each sample were filtered through a PTFE 0.22µm membrane

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filter prior their use. Acetone and ethanol are listed among the so called Generally Recognized As Safe (GRAS) solvents, since toxicological and medical studies show no adverse effects on human

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health over their use in food over a long period (Food and Drug Administration, 2016; Rodríguez et al., 1993). In particular, acetone was used as solvent extraction because it is naturally present in many fruits and vegetables, including grapes, onions and beans. Based on the European Directive

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2009/32/CE for the solvents usable in the food preparation, acetone is also “Generally Recognized

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As Safe” (GRAS) by the FDA when present in beverages, baked goods and desserts in

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concentrations from 5-8 mg/L. At the same time, ethanol, is considered GRAS solvent without any

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limits in terms of its concentration,therefore it was tested as second solvent tested in this study.

2.2. H. pluvialis

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H. pluvialis in Red phase (HPR) (Fig.2) was provided by MICOPERI BLUE GROWTH®, an Italian company. HPR presented a mesh particle sieve lower than 50µm and a total content of astaxanthin

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of 2%wt (20 mg astaxanthin/gr dry biomass), measured by analytical methods from Li et al. (2012), while the total carotenoids content was of about 29 mg/gr dry biomass measured by u-HPLC analysis after

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alkaline hydrolysis with the main scope of avoiding the overlapping of lipids and chlorophills peaks with carotenoids ones. Microalgal biomass was kept at -20°C before extraction tests. Proteins and carbohydrate were analyzed as reported in the international standard method UNI EN ISO 20483 and UNI EN 15086. AOAC method 985.29 was used for the total fiber analysis and AOAC method 920.39 for the quantification of total fats. Moisture and ashes were determined following the conventional method reported in EN ISO 712 and EN ISO 2171. Lipids were

analysed by following the international standard UNI EN ISO 12966; characterization of lipids allowed to identify saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs) as well as polyunsaturated fatty acids (PUFAs):

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Figure 3: Lipids characterization in the H. pluvialis in red phase.

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Results of HPR characterization in Table 1 are reported:

Table 1: Characterization of organic compounds present in H. pluvialis in red phase

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As shown, contents of total fibres, proteins, carbohydrate and lipids close to 58%wt, 25.7%wt,

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6.3%wt and 3.2%wt were found, while total carotenoids, composed by astaxanthin, lutein and

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betacarotene is of about 2.9wt%. According to characterization of lipids, as schematized in Fig. 2,

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2.3 Experimental planning

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linoleic acid and γ-linolenic acid.

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the main fatty acid species contained in HPR microalgae are: palmitic acid, stearic acid, oleic acid,

Two series of experiments were performed in order to quantify the astaxanthin recovery from HPR:

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the former without a mechanical pre-treatment of the microalgal biomass, the latter with a mechanical pre-treatment of the microalgal biomass. Each experimental condition was replicated by three to five times and for each value standard deviation (SD) was calculated.

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The first series of experiments has been planned to evaluate the effect of different solvents and different operative conditions on the astaxanthin extraction. Solvents used were GRAS solvents (acetone and ethanol), a mixture of chloroform/methanol (C/M = 1:1) and hexane. This last solvent is used as reference because it is often used during the industrial extraction processes. The extraction capacity of these solvents were tested at two different extraction temperature 50°C and

100°C and at two different extraction pressure 50bar and 100bar. The extraction time of 20min. was used for all tests. The investigation plan of the first series of experiments is reported in Table 2.

Table 2: Investigation plan of the first experimental setup (without mechanical pre-treatment). Extraction time = 20

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minutes.

The investigation plan of the second series of experiments was designed to maximize the

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astaxanthin recovery from HPR, in particular it includes a mechanical pre-treatment for all the

sample, a temperature investigation in the range of 20°C-100°C and consecutive extraction 20min each (up to six) in order to obtain the maximum astaxanthin recovery. In these experiments pressure

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was kept constant at 100 bar and only GRAS solvents (acetone and ethanol) were used. The

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investigation plan of the second series of experiments is reported in Table 3.

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Table 3: Investigation plan of the second experimental setup (with mechanical pre-treatment). Extraction pressure =

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100 bar; extraction time of each run= 20 minutes.

2.4. Mechanical pre-treatment

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A mechanical pre-treatment finalized to disrupt the hard double-walled red cysts and to break the

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thylakoids where astaxanthin molecules are located in their ester forms using the ball mill Retsch MM400® (Fig. 2) was carried out. The pre-treatment procedure was optimized in terms of quantity of diatomaceous earth as well as pre-treatment time; in particular, 2g of HPR were mixed with 0.8g

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of diatomaceous earth in the ball mill for a run time of 5 min. These conditions were used for all experimental tests of the second series of experiments. 2.5. Astaxanthin extraction A Dionex ASE 200 extractor (Salt Lake City, UT, USA), shown in Fig.2, was used to carry out Accelerated Solvent Extraction (ASE). Stainless steel extraction cells (volume = 11 ml) were filled by four consecutive layers consisting of (from the bottom): cellulose filter (20 µm pore size), inert

diatomaceous earth (2–3 cm3), microalgal sample for extraction and another layer of diatomaceous earth (2–3 cm3). Extraction cells and collection vials were loaded onto the automated carousel. Each cell was heated up for 5 min to reach the required temperature before starting the extraction run. At the end of each extraction run (20 minutes), extracts were collected into 40 mL amber glass vials, by flushing the

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system with 6.6 ml of fresh solvent, and the system was purged for 1 minute with nitrogen (Purity ≥

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99.999%).

2.6 Astaxanthin separation and quantification

Astaxanthin was separated from the other carotenoids by using Agilent Zorbax Eclipse plus C18

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column 1.8 µm. An isocratic mixture of methanol/water 95:5 as solvent was used, while the sample

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in a mixture of methanol/chloroform 90:10 containing 0.1 % BHT as antioxidant agent was

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mL/min and 28°C, respectively.

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solubilized. Flow rate and column temperature, equipped with an oven, were kept constant at 0.4

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Astaxanthin content following its standard characterization methods was measured. Separation, identification and quantification of astaxanthin isomers (all-trans astaxanthin, 9-cis astaxanthin and

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13-cis astaxanthin) by using a chromatographic station composed by an u-HPLC Agilent 1290 Infinity II and a GC Agilent (Fig.2) was carried out. The u-HPLC with a quaternary pump,

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thermostated oven column, and UV diode array detector (DAD) (measuring absorbance at 444-450478 nm) was equipped, while for Flame Ionization detector (FID) a column HP-88

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100mtx0.25mmx0.2µm was used. This chromatographic column produced by Agilent is composed of a High Polarity bis (Cyanopropyl) Siloxane Stationary Phase and was chosen for its high Resolution of Positional and Geometric Isomers of Fatty Acid Methyl Esters. Nitrogen (purity ≥ 99.9999%) with a spatial velocity of 30 cm/sec as carrier was used. Oven temperature at isothermal conditions, temperature of injection and detector were set to 155°C, 240°C and 250°C.

To quantify the concentration of the astaxanthin compounds, linear regression were built injecting five different standard levels: 0.25, 0.5, 1.0, 2.50, 5.00, 10.0 mg/L for all the studied compounds and using both chromatographic methodologies. A typical chromatogram of astaxanthin analysis is

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reported below:

Figure 4: u-HPLC chromatogram of H. pluvialis astaxanthin extracts. Solvent used was acetone at 40°C-100bar (first

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extraction).

Figure 4 shows the u-HPLC chromatogram for the characterization of astaxanthin isomers extracted

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with acetone and fix pressure at 100bar. It is possible to see that the all-trans astaxanthin (retention

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time-RT: 7.880) represent over the 80-85% of the total astaxanthin with respect to other isomers 9-

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cis (RT:11.625) and 13-cis (RT:13.149). The chromatogram also reveals the presence of Lutein

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(RT: 13.489) in the H. pluvialis red phase after the extraction and low quantity of betacarotene (RT: 17.879). Other species of carotenoids family was found but they weren’t reported due to their low

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content.

3. Results and Discussion

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Results of the first series of extraction experiments, designed for the evaluation of both temperature and pressure on the extraction yield, in terms of astaxantin isomers (all-trans, 9-cis, 13-cis of

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astaxanthin) and total astaxanthin, are reported in table 4.

Table 4: Astaxanthin isomers analysis via u-HPLC for each solvent extraction referred to first experimental set up. Without mechanical pre-treatment of H. pluvialis biomass. Note that astaxanthin yield is expressed in μg/g. Ldl= Lower detection limit

The total astaxanthin extraction yield depends on the solvent typology according to the following order chloroform:methanol> ethanol> hexane>acetone. This result follows the trend found by Haque et al. (2016), which investigated the effect of these solvents on astaxanthin recovery from H. pluvialis by using ultrasonicated microalgal biomass, with and without pre-treatment, finding, without out pre-treatment, the following extraction performance order methanol> ethanol> acetone.

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The maximum value of total astaxanthin recovery close to 30 μg TOT Astax/gr dry biomass was found with C/M as extraction solvent at 50°C and 100 bar. By using ethanol, the highest astaxanthin

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recovery was close to 26 μg TOT Astax/gr dry biomass, (100°C and 100bar). At the same time the use of

conventional extraction solvent as hexane reveal that the maximum astaxanthin recovery is obtained at 100°C and 50bar but this value is of about 5.8 μg TOT Astax/gr dry biomass, while by using acetone the

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highest astaxanthin recovery was close to 4.4 μg TOT Astax/gr dry biomass (50°C and 100 bar). In terms of

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astaxanthin isomers, for all-trans of astaxanthin, acetone and C/M were the worst and the best

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extraction media.For ethanol and acetone, 9-cis isomer was virtually absent as it was well below the

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machine’s detection limit, while C/M only resulted effective. For 13-cis of astaxanthin, C/M

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resulted the most effective, with a range of 3.99 – 5.42 μg13-cis astax/gr dry biomass, followed by ethanol, with a range of 2.69 – 5.05 μg13-cis astax/gr dry biomass, and acetone, with a range of 1.50 –

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1.65 μg13-cis astax/gr dry biomass.

Total astaxanthin extracted amount, as well as astaxanthin isomers, were affected by temperature, in

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accordance with literature data (Denery et al., 2004; Liu et al., 2013; Pan et al., 2012), while negligible effect of pressure on extraction yield was found, as reported by several research papers

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(Long and Winefordner, 1983; Quan and Turner, 2009; Sun et al., 2015; Yuan and Chen, 1998; Zhu et al., 2000). In particular, temperature showed opposite effects on C/M and acetone extraction performance with respect to ethanol one: above 50°C, for C/M and acetone extracted amount of astaxanthin reduced, in agreement with Ruen-ngam et al. (2010), while for ethanol the extracted amount of astaxanthin increased, in agreement with Jaime et al. (2010).

Results shows that by using temperature and pressure without pre-treatments, very low astaxanthin recovery was achieved, since a total amount of astaxanthin close to 30 μg TOT Astax/gr dry biomass, corresponds to a recovery close to 0.15%, with respect to a total astaxanthin of 20 mg/gr dry biomass. As pointed out by several research groups, in order to increase carotenoid extraction, H. pluvialis in red phase pre-treatment, implying rupture of the hard double-walled red cysts, is required. Several

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pre-treatments, such as mechanical disruption, sonication, HCl, NaOH and autoclave, followed by solvent extraction, have been investigated (Bubrick, 1991; Denery et al., 2004; Jaime et al., 2010;

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Mendes-Pinto et al., 2001; Sarada et al., 2006).

In order to improve astaxanthin recovery, the second series of extraction experiments was carried out by applying the mechanical pre-treatment detailed in section 2.4. The effect of mechanical pre-

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treatments in terms of total astaxantin recovery in the operative conditions of the first experimental

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setup are reported below:

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Figure 5: Results obtained by using mechanical pretreatment in the same operative condition of the first experimental

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setup (50bar-50°C; 50bar-100°C; 100bar-50°C; 100bar-100°C)

Mechanical pre-treatments allow to increase drastically the astaxantin recovery of three order of

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magnitude. Furthermore, it is possible to see from Fig. 5 that generally recognized as safe solvents as ethanol and acetone have the higher extraction capacity than hexane and C/M. The effect of

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increase the pressure in the range 50-100bar is very limited but has a positive effect and this due to avoid the solvent evaporation. On the basis of these results, the second experimental setup has been carried out fixed the pressure at 100bar and varying the temperature at the end to evaluate the effect

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of temperature on the extraction yield. These experimental tests have been done only for the GRAS solvents that are more promising in terms of environmental sustainability. Results of the second series of extraction experiments using acetone as extraction solvent, in terms of characterization of astaxanthin isomers and total astaxanthin for a number of consecutive extraction runs are showed in table 5. All the extracts have been characterized by u-HPLC.

Table 5: Astaxanthin analysis via u-HPLC for extinction tests carried out at 100bar with acetone as extraction solvent and mechanical pre-treatment. Note that astaxanthin extraction yield is expressed in mg/gr dry biomass

As shown, in each experimental test the residual biomass decolouring was achieved, confirming the

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complete astaxanthin extraction. The minimum amount of  12.5 mgAstax/gr dry biomass, corresponding to an astaxanthin recovery of about 62.5%, at total extraction time of 80 minutes and temperature of

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100°C was obtained; in terms of astaxanthin isomers, all-trans, 9-cis and 13-cis of astaxanthin were characterized, resulting in 10.3 mgall-transAstax/gr dry biomass, 1.2 mg9-cisAstax/gr dry biomass and 1 mg13cisAstax/gr dry biomass,

respectively. The maximum amount of  17.5 mgAstax/gr dry biomass, corresponding

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to an astaxanthin recovery of about 87.5%, at total extraction time of 60 minutes and temperature of

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40°C was obtained; in terms of astaxanthin isomers, all-trans, 9-cis and 13-cis of astaxanthin were

respectively. At an extraction temperature of 20 °C for a total extraction time of

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cisAstax/gr dry biomass,

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characterized, resulting in 14 mgall-transAstax/gr dry biomass, 2 mg9-cisAstax/gr dry biomass and 1.5 mg13-

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80 minutes an amount of total astaxanthin  15.4 mgAstax/gr dry biomass, corresponding to an astaxanthin recovery of  77%, was found; at an extraction temperature of 50 °C for a total

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extraction time of 80 minutes an amount of total astaxanthin  16.8 mgAstax/gr dry biomass, corresponding to an astaxanthin recovery of  84%, was found; at an extraction temperature of 60

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°C for a total extraction time of 60 minutes an amount of total astaxanthin  13.7 mgAstax/gr dry biomass,

corresponding to an astaxanthin recovery of  68.5%, was found. Finally, at an extraction

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temperature of 75 °C for a total extraction time of 80 minutes an amount of total astaxanthin  13.6 mgAstax/gr dry biomass, corresponding to an astaxanthin recovery of  68%, was found. The effect of extraction time on astaxanthin recovery was also investigated. Results showed in figure 6, in which total astaxanthin extraction yield as a function of the extraction time (20-80 minutes) for each extraction temperature (20-100°C) is reported.

Figure 6: Astaxanthin extraction yield versus extraction time by varying temperature extraction and using acetone as solvent at 100 bar.

Figure 6 highlights that the first extraction stage of 20 minutes, after mechanical pre-treatment, was sufficient to recover astaxanthin extraction yield from a minimum of 12.5 mgAstax/gr dry biomass, corresponding to a recovery of about 62% (100 °C), to a maximum of about 17.2 mgastaxanthin/gr dry corresponding to a value of about 86% (40 °C). Recovery was calculated with respect to the

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biomass,

total astaxanthin content in HPR, measured via SPE/UV (Li et al., 2012), corresponding to a value

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of about 20mgAstaxa/gr dry biomass. It is worth highlighting that for all the temperature of extraction, after an extraction time of 40 minutes (second extraction run), the extraction time resulted to be ineffective on the total astaxanthin extraction yield. It is also interesting pointing out that at the

U

temperature of 40°C, an extraction time of 20 minutes was sufficient to recover 98% of the

N

maximum amount of astaxanthin which is possible to extract; as a consequence 20 minutes, 40°C

A

and 100bar may represent optimal extraction conditions for acetone.

M

Results of the second series of extraction experiments using ethanol as extraction solvent, in terms

ED

of characterization of astaxanthin isomers and total astaxanthin for a number of consecutive

PT

extraction runs are showed in table 6.

Table 6: Astaxanthin analysis via u-HPLC for extinction tests carried out at 100bar with ethanol as extraction solvent

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and mechanical pre-treatment. Note that astaxanthin extraction yield is expressed in mg/g

As shown, in each experimental test the decolouring of the residual biomass was achieved,

A

confirming that the extraction of almost total astaxanthin was done. The minimum amount of total astaxanthin  12.1 mgAstax/gr dry biomass, corresponding to an astaxanthin recovery of about 60%, at total extraction time of 80 minutes and temperature of 100°C was obtained; in terms of astaxanthin isomers, all-trans, 9-cis and 13-cis of astaxanthin were characterized, resulting in 9.3 mgalltransastax/gr dry biomass,

1.4 mg9-cis-astax/gr dry biomass and 1.3 mg13-cis-astax/gr dry biomass, respectively. The

maximum amount of total astaxanthin  14.6 mgastax/gr dry biomass, corresponding to an astaxanthin recovery of about 73%, at total extraction time of 80 minutes and temperature of 67°C was obtained; in terms of astaxanthin isomers, all-trans, 9-cis and 13-cis of astaxanthin were characterized, resulting in 11 mgall-transastax/gr dry biomass, 2.6 mg9-cis-astax/gr dry biomass and 1.1 mg13cis-astax/gr dry biomass,

respectively. At an extraction temperature of 50 °C for a total extraction time of

IP T

120 minutes an amount of total astaxanthin  13 mgAstax/gr dry biomass, corresponding to an

astaxanthin recovery of  65%, was found; at an extraction temperature of 58 °C for a total

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extraction time of 80 minutes an amount of total astaxanthin  13.3 mgastaxanthin/gr dry biomass, corresponding to an astaxanthin recovery of  66.5%, was found; finally, at an extraction

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temperature of 75 °C for a total extraction time of 100 minutes an amount of total astaxanthin 

A

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13.3 mgAstax/gr dry biomass, corresponding to an astaxanthin recovery of  66.5%, was found:

M

Figure 7: Astaxanthin extraction yield versus extraction time by varying temperature extraction and using ethanol as

ED

solvent at 100 bar.

As possible to see in Fig. 7, also by using ethanol, during the first extraction stage of 20 minutes,

PT

with mechanical pre-treatment, was sufficient to recover astaxanthin extraction yield from a

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minimum of about 11 mgastax/gr dry biomass, corresponding to a recovery of about 55% (50 °C), to a maximum of about 13.5 mgastaxn/gr dry biomass, corresponding to a value of about 67.5% (67 °C). Recovery was calculated with respect to the total astaxanthin content in HPR, measured via

A

SPE/UV (Li et al., 2012), corresponding to a value of about 20mgastaxa/gr dry biomass. It is worth highlighting that at the temperature of 67°C, an extraction time of 20 minutes was sufficient to recover 92% of the maximum amount of astaxanthin which is possible to extract; as a consequence, 20 minutes, 67°C and 100bar may represent optimal extraction conditions for ethanol. For both solvents, increasing the temperature total astaxanthin recovery increased till a point to achieve maximum value, beyond which a reduction of extraction yield was observed. For acetone,

the maximum value of total astaxanthin recovery (86%) was found at the temperature of 40°C, above that astaxanthin extraction yield reduced. This phenomenon was also observed by Ruenngam et al. (2010), who compared several extraction technologies (maceration, Soxhlet extraction, UAE, and MAE) and may be explained by considering astaxanthin thermal degradation. For ethanol, the maximum value of total astaxanthin recovery (67.5%) was found at the temperature of

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67°C, above that astaxanthin extraction yield reduced, due to thermal degradation which was found to be greater than the positive effect of temperature on extraction, as reported by several authors

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(Chafer et al., 2004; Krichnavaruk et al., 2007; Machmudah et al., 2006; Pan et al., 2012; Sun and Temelli, 2006; Tachaprutinun et al., 2009; Thana et al., 2008; Vasapollo et al., 2004). Results highlight that the proposed mechanical pre-treatment of HPR strongly promoted the

U

recovery of astaxanthin, which increased by three orders of magnitude, passing from  30 μg Astax

A

N

/gr dry biomass (without mechanical pre-treatment) to  17.5 mg Astax /gr dry biomass (with mechanical pre-

M

treatment) for both GRAS solvents. In particular, the use of the suggested mechanical pre-treatment resulted into maximum values of astaxanthin recovery of about 17.5 mg astax /gr dry biomass with an

biomass

ED

extraction time of 60 minutes and temperature of 40°C for acetone and of about 14.6 mg Astax /gr dry with an extraction time of 80 minutes and temperature of 67 °C for ethanol, in view of the

PT

total astaxanthin content in the biomass measured via SPE/UV (Li et al., 2012) equal to 20mgAstax

CC E

/gr dry biomass. For the optimal extraction time of 20 minutes, astaxanthin extraction yield of about 17.2 mgAstax/gr dry biomass and of about 13.5 mgastaxn/gr dry biomass were achieved for acetone and ethanol. The experimental protocols of astaxanthin extraction at 100bar by using ethanol as solvent

A

revealed good extraction capacity force also compared with acetone that is considered as reference standard for this kind of extraction. Since with an extraction time of 20 minutes through ethanol at 67°C, it was possible to recover 78% of the total astaxanthin extracted by acetone. Despite, acetone outcomes more effective than ethanol it is worth pointing out that without the application of mechanical pre-treatment, ethanol resulted more effective than acetone. This could be due to the higher extractive capacity of acetone with ester forms of astaxanthin with respect to ethanol, that

has greater extraction capacity with free forms of astaxanthin. Also, similar result was found by Haque et al. (2016), who investigated the effect of several solvents, such as ethanol and acetone, on astaxanthin recovery from H. pluvialis by using ultrasonicated (25 min) biomass without and with chemical pre-treatment (2M sodium hydroxide). Haque et al. (2016) highlighted that without pretreatment, ethanol resulted more effective than acetone, while with pre-treatment acetone resulted

IP T

more effective than ethanol.

Comparison between experimental findings obtained by H. pluvialis mechanically pre-treated and

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extracted by using acetone with literature data highlights that significant improvements in terms of total astaxanthin yield and recovery were achieved, passing from  9 mgAstax/ gr dry biomass (Denery et

N

U

al., 2004) to  17 mgAstax/gr dry biomass and from 81 % (Mendes-Pinto et al., 2001) to 87%.

A

4. Conclusion

M

Effects of time, pressure, temperature and mechanical pre-treatment on astaxanthin recovery through ASE were investigated, comparing GRAS solvents with more toxic solvent. First

ED

experimental series has shown a very poor, well below 1%, astaxanthin recovery whatever the solvent or operative conditions. On the contrary, in the second series of experiments, the extraction

PT

was performed after a mechanical pre-treatment, a very high astaxanthin recovery was obtained.

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After an extraction time of 20 minutes, maximum recovery of 86% and 67% were reached at 100 bar and 40°C and 67°C for acetone and ethanol, defining conditions avoiding astaxanthin

A

degradation.

Acknowledgments This paper has received funding from the Bio Based Industries Joint Undertaking under the European Union’s Horizon 2020 research and innovation program under grant agreement No 745695 (VALUEMAG).

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Chem. 48, 4097–4102. https://doi.org/10.1021/jf991183f

Value (wt.%)

Proteins

25.69 ±0.2

Carbohydrate

6.30 ±0.1

Total Carotenoids

2.87±0.1

Total Fibers

58.52 ±0.5 4.02 ±0.1

Lipids:

2.60 ±0.05

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Ash

88.30 ±0.1

Saturated Fatty acids (wt% of FA):

28.14 ±0.1

Palmitic Acid (C16-0)

22.0 ±0.05

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Fatty Acids (FA) – wt% of lipids

Stearic Acid (C18-0)

1.83 ±0.1

Myristic acid

Pentadecanoic acid

M

Docosanoic acid (acid Beenico)

A

N

Arachidic acid

2.75 ±0.05 0.44 ±0.05 0.23 ±0.03 0.14 ±0.02 0.18 ±0.02

Tricosanoic acid

0.43 ±0.05

Undecanoic acid

0.10 ±0.02

PT

ED

Heinecosanoic acid

Monounsaturated fatty acids (wt% FA):

23.66 ±0.1

Palmitoleic Acid (C16-1w7)

0.21 ±0.05

cis-10-Hptadecanoic Acid (C17-1c)

1.86 ±0.03

Oleic acid

21.16 ±0.1

cis-11-Eircosenoic acid

0.43 ±0.05

Polyunsaturated Fatty Acids (wt% of FA):

48.20 ±0.1

Linoleic Acid (C18-2w6c)

31.09 ±0.1

γ-Linolenic Acid (C18-3w6 GLA)

8.69 ±0.1

Linoleadic acid

0.61 ±0.1

Arachidonic acid

7.54 ±0.1

CC E A

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Parameters

Table 1: Characterization of organic compounds produced by H. pluvialis in red phase dry basis (moisture content:7.0

A

CC E

PT

ED

M

A

N

U

SC R

IP T

wt%)

Pressure (bar) 50 50 100 100

Acetone Acetone Acetone Acetone

50 100 50 100

50 50 100 100

C/M C/M C/M C/M

50 100 50 100

50 50 100 100

Hexane Hexane Hexane Hexane

50 100 50 100

50 50 100 100

U

SC R

IP T

Ethanol Ethanol Ethanol Ethanol

Temperature (°C) 50 100 50 100

N

Solvent

A

CC E

PT

ED

M

A

Table2: First experimental setup: Evaluation of pressure on H. pluvialis cellular wall disruption.

Number of extraction

Acetone Acetone Acetone Acetone Acetone Acetone

From 1ST extr to 4TH extr From 1ST extr to 3RD extr From 1ST extr to 4TH extr From 1ST extr to 3RD extr From 1ST extr to 4TH extr From 1ST extr to 4TH extr

Temperature (°C) 20 40 50 60 75 100

Ethanol Ethanol Ethanol Ethanol Ethanol

From 1ST extr to 6TH extr From 1ST extr to 4TH extr From 1ST extr to 4TH extr From 1ST extr to 5TH extr From 1ST extr to 4TH extr

50 58 67 75 100

SC R

IP T

Solvent

Table 3: Second experimental setup: Evaluation of the temperature and mechanical pre-treatment on astaxanthin

A

CC E

PT

ED

M

A

N

U

recovery with acetone and ethanol

Pressure (bar) 50 50 100 100

all trans astax (μg /g) 6.86 (±0.10) 20.52 (±0.20) 6.87 (±0.10) 21.20 (±0.20)

9-cis astax (μg/g)
13-cis astax (μg/g) 2.69 (±0.10) 4.92 (±0.10) 2.75 (±0.10) 5.05 (±0.10)

TOT ASTAX (μg/g) 9.55 (±0.10) 25.44 (±0.20) 9.62 (±0.20) 26.25 (±0.20)

Acetone Acetone Acetone Acetone

50 100 50 100

50 50 100 100

2.76 (±0.10) 2.46 (±0.10) 2.78 (±0.10) 2.56 (±0.10)


1.62 (±0.10) 1.50 (±0.10) 1.65 (±0.10) 1.54 (±0.10)

4.38 (±0.15) 3.96 (±0.15) 4.43 (±0.15) 4.10 (±0.15)

C/M C/M C/M C/M

50 100 50 100

50 50 100 100

22.26 (±0.20) 16.18 (±0.20) 23.25 (±0.20) 17.15 (±0.20)

1.29 (±0.05) 1.18 (±0.05) 1.35 (±0.05) 1.25 (±0.05)

5.28 (±0.10) 3.99 (±0.10) 5.42 (±0.10) 4.23 (±0.10)

28.83 (±0.30) 21.35 (±0.30) 30.02 (±0.30) 22.63 (±0.30)

Hexane Hexane Hexane Hexane

50 100 50 100

50 50 100 100

2.75 (±0.10) 3.75 (±0.10) 3.80 (±0.10) 3.90 (±0.10)


1.51 (±0.10) 2.05 (±0.10) 1.55 (±0.10) 1.72 (±0.10)

4.26 (±0.30) 5.80 (±0.30) 5.35 (±0.30) 5.62 (±0.30)

IP T

Ethanol Ethanol Ethanol Ethanol

TEMP (°C) 50 100 50 100

SC R

Solvent

Table 4: Astaxanthin isomers analysis via u-HPLC for each solvent extraction referred to first

U

experimental set up. No mechanical pre-treatment was performed on H. pluvialis biomass. Note that

A

CC E

PT

ED

M

A

N

astaxanthin yield is expressed in μg/g. Ldl= Lower detection limit

29

TEMP (°C)

All-trans Astax (mg/g)

9-cis Astax (mg/g)

13-cis astax (mg/g)

Tot Astax (mg/g)

1ST extr

20

9.970 (±0.10)

1.832 (±0.10)

1.234 (±0.10)

13.036 (±0.25)

2ND extr

20

1.633 (±0.05)

0.174 (±0.01)

0.296 (±0.011)

2.104 (±0.05)

3RD extr

20

0.160 (±0.01)

0.002 (±0.0002)

0.008 (±0.0002)

0.170 (±0.01)

4TH extr

20

0.039 (±0.005)

0.002 (±0.0001)

0.007 (±0.0001)

0.048 (±0.005)

11.803 (±0.15)

2.009 (±0.10)

1.545 (±0.10)

15.358 (±0.30)

TOT

40

13.882 (±0.10)

1.985 (±0.10)

1.414 (±0.10)

17.281 (±0.20)

2ND extr

40

0.115 (±0.01)

0.009 (±0.001)

0.028 (±0.005)

0.152 (±0.01)

3RD extr

40

0.014 (±0.001)


0.003 (±0.0003)

0.017(±0.001)

14.010 (±0.10)

1.994 (±0.10)

1.445 (±0.10)

17.449 (±0.20)

1ST extr

50

13.707 (±0.10)

0.715 (±0.05)

1.294 (±0.10)

15.716 (±0.10)

2ND extr

50

0.839 (±0.01)

0.046 (±0.001)

0.162 (±0.01)

U

TOT

SC R

1ST extr

3RD extr

50

0.032 (±0.001)

0.002 (±0.0005)

0.005 (±0.0003)

0.039 (±0.001)

4TH extr

50

0.011 (±0.001)

0.001 (±0.0005)

0.002 (±0.0003)

0.013 (±0.001)

14.588 (±0.10)

0.764 (±0.055)

1.463 (±0.110)

16.815 (±0.150)

1.383 (±0.05)

60

10.796 (±0.10)

2ND extr

60

0.193 (±0.01)

3RD extr

60

0.027 (±0.005)

75

11.041 (±0.10)

CC E

1ST extr

A

N

1.047 (±0.05)

1.235 (±0.05)

13.414 (±0.10)

0.020 (±0.003)

0.046 (±0.003)

0.260 (±0.01)

0.002 (±0.0003)

0.006 (±0.0003)

0.034 (±0.005)

1.405 (±0.05)

1.287 (±0.05)

13.707 (±0.05)

1.198(±0.05)

1.049 (±0.05)

13.288 (±0.10)

PT

11.015 (±0.10)

TOT

M

1ST extr

ED

TOT

2ND extr

75

0.283(±0.05)

0.013 (±0.005)

0.041 (±0.005)

0.336 (±0.03)

3RD extr

75

0.016 (±0.005)

0.001 (±0.0003)

0.003 (±0.0003)

0.020 (±0.005)

4TH extr

75

0.008 (±0.0003)


0.002 (±0.0003)

0.010 (±0.005)

11.348 (±0.10)

1.211 (±0.05)

1.094 (±0.05)

13.653(±0.13)

A

TOT

1ST extr

100

9.924 (±0.10)

1.210 (±0.05)

0.935 (±0.05)

12.069 (±0.10)

2ND extr

100

0.369 (±0.05)

0.018 (±0.005)

0.050 (±0.005)

0.437 (±0.03)

3RD extr

100

0.017 (±0.005)

0.001 (±0.0003)

0.003 (±0.0003)

0.021 (±0.005)

4TH extr

100

0.007 (±0.0005)

0.002 (±0.0002)

0.007 (±0.0004)

0.016 (±0.005)

10.317 (±0.15)

1.230 (±0.055)

0.995 (±0.055)

12.543 (±0.14)

TOT

30

Solvent extracted

Residual biomass (after extraction)

IP T

Extraction number

Table 5: Astaxanthin analysis via u-HPLC for extinction tests carried out at 100bar with acetone as extraction solvent and mechanical pre-treatment. Note that astaxanthin extraction yield is expressed in

A

CC E

PT

ED

M

A

N

U

SC R

IP T

mg/g

31

TEMP (°C)

All-trans astax (mg/g)

9-cis astax (mg/g)

13-cis astax (mg/g)

Tot Astax (mg/g)

1ST extr

50

8.471 (±0.10)

1.435 (±0.10)

1.170 (±0.10)

11.076 (±0.10)

extr

50

1.364 (±0.10)

0.140 (±0.05)

0.218 (±0.05)

1.722 (±0.05)

3

RD

extr

50

0.102 (±0.02)

0.0040 (±0.0003)

0.012 (±0.005)

0.118 (±0.02)

4

TH

extr

50

0.047 (±0.003)

0.0020 (±0.0003)

0.0060 (±0.0002)

0.055 (±0.002)

5 TH extr

50

0.029 (±0.003)

0.0010 (±0.0002)

0.0040 (±0.0002)

0.034 (±0.002)

50

0.023 (±0.003)

0.0010 (±0.0001)

0.0030 (±0.0002)

0.027 (±0.002)

10.036 (±0.25)

1.583 (±0.15)

1.411 (±0.15)

13.031 (±0.20)

58

8.788 (±0.10)

2.032 (±0.10)

0.903 (±0.05)

11.722 (±0.10)

6

TH

extr

TOT

1ST extr extr

58

1.011 (±0.05)

0.210 (±0.02)

0.134 (±0.01)

1.354 (±0.05)

3

RD

extr

58

0.134 (±0.02)

0.014 (±0.002)

0.018 (±0.002)

0.166 (±0.02)

4

TH

extr

58

0.045 (±0.002)

0.004 (±0.001)

0.006 (±0.001)

0.054 (±0.01)

9.977 (±0.15)

2.259 (±0.13)

1.061 (±0.08)

13.297 (±0.18)

67

10.014 (±0.10)

2.440 (±0.10)

1.001 (±0.05)

13.455 (±0.10)

extr

67

0.756 (±0.05)

0.145 (±0.01)

0.107 (±0.02)

1.009 (±0.03)

3RD extr

67

0.099 (±0.002)

0.010 (±0.002)

0.014 (±0.002)

0.123 (±0.02)

67

0.039 (±0.002)

0.0040 (±0.0002)

0.0050 (±0.0002)

0.0480 (±0.0002)

10.908 (±0.15)

2.599 (±0.11)

1.128 (±0.07)

14.635 (±0.15)

75

9.483 (±0.10)

1.824 (±0.05) 0.045 (±0.01)

TOT

1ST extr 2

ND

extr

75

0.511 (±0.05)

3

RD

extr

75

0.052 (±0.01)

4

TH

extr

75

0.023 (±0.005)

5 TH extr

75

0.016 (±0.005)

1.284 (±0.05)

12.591 (±0.10)

0.107 (±0.01)

0.663 (±0.03)

0.003 (±0.001)

0.007 (±0.001)

0.062 (±0.01)

0.0010 (±0.0005)

0.0030 (±0.0005)

0.027 (±0.005)

0.0010 (±0.0005)

0.0020 (±0.0005)

0.019 (±0.005)

1.873 (±0.06)

1.404 (±0.06)

13.361 (±0.14)

PT

10.085 (±0.16)

TOT

N

extr

A

4

TH

M

2

ND

ED

1ST extr

U

2

ND

TOT

100

8.964 (±0.10)

1.397 (±0.05)

1.278 (±0.05)

11.640 (±0.10)

2ND extr

100

0.340 (±0.05)

0.042 (±0.003)

0.061 (±0.003)

0.443 (±0.05)

CC E

1ST extr

3

RD

extr

100

0.034 (±0.002)

0.0020 (±0.0005)

0.0050 (±0.0005)

0.040 (±0.003)

4

TH

extr

100

0.014 (±0.002)

0.0010 (±0.0005)

0.0020 (±0.0005)

0.017 (±0.003)

9.352 (±0.15)

1.442 (±0.055)

1.347 (±0.055)

12.140 (±0.15)

A

TOT

Residual biomass (after extraction)

SC R

2

ND

Solvent extracted

IP T

Extraction number

Table 6: Astaxanthin analysis via u-HPLC for extinction tests carried out at 100bar with Ethanol as extraction solvent and mechanical pre-treatment. Note that astaxanthin extraction yield is expressed in mg/g

32

700 600 500 400 300 200

IP T

Astaxanthin market size (M€)

800

100 0

SC R

2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020 2021 2022

Figure 1: Astaxanthin estimated market trend.

U

Description of Figure1: A significant increase in the astaxanthin market is expected to reach more than

A

CC E

PT

ED

M

A

N

700 million euro in 2022.

33

HPR residue colourless

Accelerator Solvent Extraction ASE 200

Retsch MM400® Mixer Mill

After extraction cycles

Extraction

Preatreatment

Screw-top grinding jars after HPR milling

Acetone and Ethanol extracts

U N

uHPLC analysis

Zymark TurboVap®

Agilent 1290 Infinity II

A

GC-FID Agilent 7820A

Drying

SC R

Stainless steel cell with diatomaceous earth and HPR

IP T

Haematococcus p. in red phase

M

Figure 2: Extraction and experimental set-up for astaxanthin recovery from H. pluvialis red phase. Despcription of Figure 2: The figure describes the different experimental phases of extraction in ASE

ED

200, drying with Zymark TurboVap®(for GC analiysis) and analysis of carotenoids and fatty acids with uHPLC and GC-FID respectively. H. pluvialis. in red phase was also pre-treated mechanically with

A

CC E

PT

Retsch MM400® mill in the second experimental phase.

34

Fatty acids

Unsaturated FA

Polyunsaturated FA

Linoleic acid(C18-2 ω 6c) γ-Linolenic acid (C18-3 ω 6 GLA) Arachidonic acid (C20-4ω6)

Monounsaturated FA FA

IP T

Palmitic acid (C16) Stearic Acid (C18) Arachidic acid (C20)

Palmitoleic acid(C16-1 ω 7) Heptadecenoic acid cis(C17-1c) Oleic acid(C18-1 ω 9c)

SC R

Saturated FA

U

Figure 3: Main molecules of fatty acids contained in theH. pluvialis in red phase.

Description of Figure 3: Lipids were characterized between saturated and unsaturated fatty acids for the

A

CC E

PT

ED

M

A

N

presence of healthy 3-ω and 6-ω fatty acids in H. pluvialis biomass.

35

IP T

Figure 4: uHPLC chromatogram of H. pluvialis astaxanthin extracts. Description of Figure 4: Solvent used was acetone at 40°C-100bar (first extraction). In the

SC R

chromatogram are well defined the three peaks of astaxanthin isomers (all-trans, 9-cis and 13-cis), the

A

CC E

PT

ED

M

A

N

U

carotenoid lutein is also identified and quantified in H. pluvialis extracts

36

25

20

50bar-100°C 100bar-50°C 100bar-100°C

IP T

15

10

5

SC R

Total astaxanthin recovery (mg/gr dry biomass)

50bar-50°C

0

Hexane

Acetone

U

Ethanol

N

Chloroform:Methanol (1:1)

A

Figure 5: Results obtained by using mechanical pre-treatment in the same operative condition of the first

A

CC E

PT

ED

M

experimental setup (50bar-50°C; 50bar-100°C; 100bar-50°C; 100bar-100°C)

37

100

80 70 60 50

IP T

40 30 20

Acetone 20°C Acetone 50°C Acetone 75°C

10 0 0

10

20

30

40 50 Extraction Time (min)

Acetone 40°C Acetone 60°C Acetone 100°C

SC R

Total Astaxanthine Recovery (%)

90

60

70

80

90

U

Figure 6: Astaxanthin extraction yield versus extraction time by varying temperature extraction and using

N

acetone as solvent at 100 bar.

A

Description of figure 6: Mechanical pre-treatment was performed. The total extraction yield is reported for each temperature showing the trend in relation to the extraction time, which already at 20 minutes is

M

suitable to achieve an excellent yield efficiency. Note that astaxanthin extraction yield is expressed in

A

CC E

PT

ED

mg/g

38

100

80 70 60 50

IP T

40 30 20 10

Ethanol 50°C

Ethanol 58°C

Ethanol 75°C

Ethanol 100°C

0 10

20

30

40 50 60 Extraction time (min)

70

80

90

U

0

Ethanol 67°C

SC R

Total Astaxanthin Recovery (%)

90

Figure 7: Astaxanthin extraction yield versus extraction time by varying extraction temperature and using

N

ethanol as solvent at 100 bar.

A

CC E

PT

ED

M

for each temperature for ethanol as GRAS solvent

A

Description of figure 7: Mechanical pre-treatment was performed. The total extraction yield is reported

39