fractionation and emulsifying properties

Accepted Manuscript Haematococcus pluvialis soluble proteins: extraction, characterization, concentration/fractionation and emulsifying properties Fatou Ba, Alina Violeta Ursu, Céline Laroche, Gholamreza Djelveh PII: DOI: Reference:

S0960-8524(15)01407-8 http://dx.doi.org/10.1016/j.biortech.2015.10.012 BITE 15637

To appear in:

Bioresource Technology

Received Date: Revised Date: Accepted Date:

17 July 2015 24 September 2015 1 October 2015

Please cite this article as: Ba, F., Ursu, A.V., Laroche, C., Djelveh, G., Haematococcus pluvialis soluble proteins: extraction, characterization, concentration/fractionation and emulsifying properties, Bioresource Technology (2015), doi: http://dx.doi.org/10.1016/j.biortech.2015.10.012

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.

Haematococcus pluvialis soluble proteins: extraction, characterization, concentration/fractionation and emulsifying properties Fatou Ba(1), Alina Violeta Ursu(1), Céline Laroche(1) *, Gholamreza Djelveh(2) (1)

Clermont Université, Université Blaise Pascal, Institut Pascal UMR CNRS 6602, axe

GePEB, 2 Avenue Blaise Pascal, TSA 60206, CS 60026, 63178 Aubière CEDEX, France (2)

Clermont Université, ENSCCF, Institut Pascal UMR CNRS 6602, axe GePEB, 4

avenue Blaise Pascal, TSA60206, 63178 Aubière, France

*Corresponding author [email protected]

1

Abstract A water-soluble matrix was extracted from green vegetative H. pluvialis through highpressure cell disruption either at native pH (5.7) or with pH shifting to neutral (7). The resulting supernatant is mainly composed of carbohydrates and proteins, with the highest yield of proteins obtained at neutral pH (73±2% of total biomass proteins). The key emulsification properties of the proteins isolated in neutral supernatant (emulsification capacity (EC): 534±41 mL oil.g-¹ protein, emulsification stability (ES): 94 ±3% and emulsification activity index (EAI): 80±1 m².g-¹) were comparable to the native supernatant values (EC: 589±21 mL oil.g-¹ protein, ES: 84±3% and EAI: 75±1 m².g-¹). Confronted to sodium caseinate (EC: 664±30 mL oil.g-¹ protein, ES: 63±4%, and EAI: 56±4 m².g-¹) these results highlighted the strong potential of proteins isolated from H. pluvialis as emulsifier agent. Moreover, experiments have shown that the stability of emulsions obtained from supernatants is due to the proteins rather than the carbohydrates. Keywords: Haematococcus pluvialis, proteins, ultrafiltration, emulsification

2

Introduction Microalgae are eukaryotic photosynthetic microorganisms, source of bioactive compounds such as lipids, proteins, carbohydrates and pigments. They are mostly studied as potential raw materials in biofuel production (Gerde et al., 2013). However, the economic production cost of this sustainable energy source is still too high. In order to reduce the biofuel production cost, a more efficient approach for the sustainable valorisation of microalgae biomass is the integrated production of energy and bio based value added products (Batista et al., 2013; Schwenzfeier et al., 2011). However, attention has to be paid to reducing the number of unit operations sources of additional costs. The green microalgae Haematococcus pluvialis belongs to the group of chlorophyceae. This freshwater organism has an atypical life cycle in that it is able to go from a vegetative cell state rich in chlorophylls and proteins to an encysted state under stress. At this encysted state, H. pluvialis is surrounded by a thick cell wall and can produce high amounts of secondary metabolites including carotenoids, especially astaxanthin (Mendes-Pinto et al., 2001) which has attracted considerable attention in recent years. However, the vegetative form is equally worthy of attention due to the amount of proteins (27%) and carbohydrates (40%) (Lorenz, 1999). Proteins play an important role in some food or cosmetic formulations, especially for their good emulsifying properties. Thus egg-white proteins, sodium caseinate and plant proteins extracts such as soy and whey proteins isolates are incorporated in recipes. They participate in the formation of oil-in-water or water-in-oil emulsions and stabilize them. For proteins extracts from microalgae hardly any information is available on their properties. But lately, they have

3

attracted a growing interest. Indeed they have the ability to maintain some properties especially to enhance the solubility in a wide range of pH conditions where functional agents from plants failed. They also displayed promising emulsifying properties (Gerde et al., 2013; Schwenzfeier et al., 2013; Ursu et al., 2014). Regrettably, H. pluvialis proteins functionality has not yet been studied. The present paper intends to study the functionality of water-soluble matrix from H. pluvialis without further purification. The optimal conditions for extraction of these water-soluble compounds were first investigated. The supernatant was quantified and characterized before evaluation of matrix functional properties in terms of emulsification properties, compared to referenced commercial emulsifier. 1. Materials and methods 1.1. Haematococcus pluvialis strain Vegetative-state Haematococcus pluvialis biomass (20% dry matter) was purchased from Microphyt (Baillargues, France). The biomass elementary composition was determined by SCA-CNRS (Solaize, France). After biomass mineralization the inorganic elements were quantified by inductive coupled atomic emission spectroscopy (ICP) using an ICAP Thermo Fisher Scientific device. The percentage of nitrogen used to calculate the Total Protein (TP) content was quantified with a thermal conductivity detector. 1.2. Extraction/solubilization The impact of a mechanical treatment (high pressure cell disruption) on the extraction yield of Water-soluble Proteins (WSP) was determined. Suspensions of 2% dry weight

4

were prepared from frozen paste of microalgae. Cellular lysis was carried out by highpressure cell disruption using a TS HAIVA (Constant systems LTD) device. Each suspension was submitted to a pressure range from 0.5 to 2.7 kbar. The effect of number of passages (1, 2 or 3) was also evaluated at a given pressure (2 kbar). H. pluvialis mash was centrifuged at 10,000 g for 30 min at 20 °C. The pellet was discarded and the supernatant that contained the solubilised compounds, especially proteins and carbohydrates, was used for the rest of the study. 1.3. pH shifting and proteins isoelectric point (pI) Proteins precipitation was carried out by progressively adding HCl (1 or 5 M) to the alkaline supernatant (pH=12). pH was switched from 12 to 2. The obtained precipitates were function of the proteins isoelectric point. The amount of precipitated proteins was expressed as a percentage of Total Protein in Alkaline supernatant (TPA). 1.4. Proteins analysis Total and solubilised proteins were quantified using the methods described below. Quantification of water-soluble proteins WSP content was quantified by the Lowry method (Lowry et al., 1951) using bovine serum albumin as a standard. The procedure involves a reaction of proteins with cupric sulphate and the formation of copper complexes. The addition of Folin-Ciocalteu reagent leads to the formation of molybdenum blue proportionaly to the protein concentration and which absorbs at 750 nm.

5

Aminogram and Nitrogen To Protein factor (NTP) calculation Amino acid composition of proteins was determined within the initial biomass and also the supernatant. They were quantified using ion exchange chromatography combined with a post-column ninhydrin (Hitachi 8900). The samples were pre-treated with 5.5M HCl prior to amino acids analysis. The nitrogen to protein factor was calculated for each sample by the ratio of the amino acid residues to total nitrogen as in Lourenço et al. (2002). Molecular weight distribution (MWD) Molecular weight distribution was evaluated in non denaturing conditions by size exclusion chromatography (SEC). SEC was carried out on an AKTA FLPC chromatograph (Amersham Biosciences, UK) equipped with an UV-VIS detector. The Superdex 200 10/300GL column (Amersham Biosciences, UK) was calibrated using a standard proteins solution ranging between 670 and 1.35 kDa (bovine thyroglobulin: 670kDa; bovine γ-globulin: 158 kDa; chicken ovalbumin: 44 kDa; horse myoglobin: 17 kDa; vitamin B12: 1.35 kDa). This standard proteins mix allowed us to determine void volume, column volume, and to establish a calibration curve between elution volume and molecular mass. Samples containing 1 g.L-¹ of proteins were filtered at 0.2 µm, and 0.1 mL of those was injected. The elution was carried out at 0.5 mL.min-¹ with sodium phosphate buffer (50 mM) at pH=7. The proteins eluted through the column were detected at 280 nm. Two other wavelengths, 661 and 470 nm for chlorophylls and carotenoids respectively (Lichtenthaler and Buschmann, 2001), made possible to assess the impact of mechanical/chemical treatments on the pigments release.

6

1.5. Carbohydrates analysis Quantification of water-soluble carbohydrates Total carbohydrates were also quantified by a colorimetric assay that uses a reaction between resorcinol, sulphuric acid and carbohydrates at 90°C. The absorbance was read at 450 nm against glucose as a standard (Monsigny et al., 1988). 1.6. Concentration and fractionation of proteins Ammonium sulphate (AMS) Precipitation To a given volume of supernatant, the appropriate amount of solid ammonium sulphate was added to reach 25, 40, 70 and 90% saturation. During, and also one hour after the addition, the mixture was stirred and kept at 4°C. The solution was then centrifugated at 10,000 g for 30 min at 4°C to pack the precipitated components. The supernatant was submitted to carbohydrates and proteins quantification. The pellet was re-suspended in water and washed with deionised distilled water to reduce the salinity using an Amicon stirred cell associated with a 1 kDa polyethersulfone Millipore membrane. The influence of the initial concentration of proteins on their precipitation yield was also evaluated. Ultrafiltration Native and neutral pHs supernatants (pH=5.7 and 7 respectively) were filtered through glass porous filters (Duran, Germany) with nominal maximum pore size of 10-16 μm in order to eliminate any remained particulates. The pre-filtered supernatant was then concentrated and fractionated by tangential flow ultrafiltration (UF). The experiments were performed using a pilot-scale Cogent M1 unit (Merck Millipore) equipped with a

7

flat membrane of regenerated cellulose with a nominal molecular weight limit (NMWL) of 1 kDa, a filtration surface of 0.1 m², and a permeability to water of 3.2±0.3 L.h-¹.m².bar-¹ at ˷23°C. The operating parameters transmembrane pressure (TMP) and pressure drop (ΔP) were optimized prior to filtration to enhance the process performance. In order to determine the optimal TMP, permeate flow rates were measured for different TMP and constant ΔP. Once the optimal operating parameters had been determined, the volume concentration factor VCF (the ratio of initial supernatant volume to final retentate volume) was fixed at 10-fold and the corresponding Protein Concentration Factor PCF (the ratio between the protein concentration in retentate and in feed) was calculated after protein quantification. All experiments were performed at room temperature (˷23°C). 1.7. Emulsifying properties Emulsification capacity (EC) Emulsification capacity (EC) was evaluated using the procedure given by Thakur et al. (2008). Rapeseed oil was continuously added to the protein solution homogenized by an Ultra-Turrax device (T25, Ika-Werke GmbH, Germany). At the same time, the conductivity of the emulsion was monitored using a CDM210 conductimeter (Radiometer Analytical, France) until destabilization of the emulsion. EC corresponds to the maximal oil volume emulsified per gram of proteins.

8

Emulsification activity index (EAI) Emulsification activity index (EAI) was quantified using the procedure given by Pearce and Kinsella (1978). Twenty milliliters of a 0.1% (w/v) protein solution at pH=7 was homogenized with 6.6 mL of rapeseed oil for one minute under agitation by UltraTurrax device. Immediately after homogenization, 50 µL of the emulsion were diluted in a 5 mL SDS solution at 0.1%. The absorbance (A0) of the solution was determined at 500 nm and EAI was deduced by the following equation:

m .g

.

d

where A0 is the optical density measured after emulsion homogenization, L is the absorbance path length (L=10-² m), C is the protein concentration (C=1 g.L-¹) before dilution in SDS,

is the oil volume fraction in the emulsion (

. 5), and d is the

dilution factor (d=100). Emulsification stability (ES) Emulsification stability (ES) was measured using the method given byYasumatsu et al. (1972). A protein solution with an equivalent volume of rapeseed oil was homogenized by the Ultra-Turrax device. An aliquot of the emulsion was placed in a tube and heated at 80°C for 30 minutes. The sample was then cooled under tap water and kept at room temperature for 24 hours. ES was expressed as a ratio of the height of the remaining emulsified layer to initial height. All analyses were done in triplicate to estimate the standard deviation.

9

2. Results and discussion The main components of H. pluvialis biomass are subdivided as follows: 30% of carbohydrates, 12% of lipids, 3% of pigments (67% of chlorophylls) also ashes and fibers. The crude proteins content determined from the amino-acids composition as 26% of H. pluvialis biomass (on a dry matter basis) is close to previous data reported by Lorenz (1999) for the same microalgae. The elementary composition of vegetative H. pluvialis biomass is reported in Table 1. The amino acids quantification (Table 2) joined to total nitrogen (7.6%) allowed estimation of the nitrogen to protein factor (NTP) at 3.4. Compared to the data of NTP reported in the literature for red encysted Haematococcus (Safi et al., 2012), the value obtained is distinctly lower (3.4 vs 6.25). This difference between the vegetative and the encysted forms could be attributed to the culture conditions especially to the media composition which can result in a significant difference in the cellular physiology like cells morphology and composition. The composition of amino-acids is crucial for the nutritional quality of proteins. The profile revealed a large panel of amino-acids in the biomass in which the essential amino-acids reach almost 40% of the total content. Glutamic acid (3.4%) was the main component followed by the essential amino acid leucine (2.6%) and then aspartic acid (2.4%). The total nitrogen content and the amino-acids profile of the extracted proteins from the supernatant (data not shown) allowed us to obtain a NTP for the WSP very close to the one of the biomass (3.6 vs 3.4).The WSP and the initial biomass have the same aminoacid profile, but exhibit a slight decrease in essential amino-acids (3%).

10

2.1.pH shifting and proteins isoelectric point (pI) The proteins were separated according to their isoelectric point by a pH shifting procedure. Adjusting pH from 12 to 7 leads to the precipitation of only 8% of water soluble proteins in alkaline supernatant (TPA). The percentage of precipitated proteins reached 71% when pH dropped to 6. Below 4, pH did not significantly impact, and the final amount of precipitated proteins from alkaline supernatant was equal to ∼ 83% at pH=2. The pI of intracellular proteins from H.pluvialis is poorly documented. Wang et al. (2004) achieved a two-dimensional gel electrophoresis on cell wall proteins of H. pluvialis, estimating that a majority of them has an isoelectric point between pH=7 and pH=5. The results obtained in this work on intracellular proteins suggest that most of the H. pluvialis proteins had an isoelectric point between pH=7 and pH=5 with a majority pelleted at pH=6. This value of pI is close to the one exhibited by current plant proteins (5.5-6.5) used in food applications. However this is not always the case. As an example the pI of intracellular proteins from the chlorophyte Tetraselmis suecica is between pH=5 and 6 and the one for phycobiliproteins from cyanobacteria between 4.7 and 5.3. These results imply that the isoelectric point is rather strain-dependent (Glazer and Cohen-Bazire, 1971; Schwenzfeier et al., 2011). Once precipitated, proteins lost their solubility and even by bringing the pH back to 12, less than 30% of proteins were resolubilized. pH shifting appears to denature the proteins and ultimately render them less soluble. This could be a major problem if the recovered proteins need to have a good solubility for further applications.

11

2.2.Influence of high pressure (HP) and pH on proteins extractability H. pluvialis proteins isoelectric point determination showed that it was not a suitable choice to conduct the extraction at high pHs, referring to the loss of its protein solubility probably due to their denaturation. Thus, the extraction at high pressure was conducted at neutral (pH=7) and at native pH (pH=5.7). The yield of extracted proteins without high pressure treatment amounted 23±1% from total proteins (figure 1). The value of extracted proteins is higher than the 6.5 % reported by Safi et al. (2012). This yield gap could be due to a difference in cell wall structure/composition (encysted or not) but also to the method used for microalgae cells conservation. The initial form of the biomass, dried for Safi et al. (2012) and frozen for this study, could modify the cell behaviour, as cellular lysis could have occurred during the conservation treatment. Figure 1 also illustrates the influence of pressure on protein extraction from a suspension of H. pluvialis at native pH (5.7).The yield of extracted soluble proteins increased by applying 0.5 kbar pressure on H. pluvialis cells and increased from 34±5% to 56±4% when pressure was raised from 0.5 to 2 kbar. It indicates that pressure alters further the cell structure, enabling the water travel trough the membrane. Note that increasing HP above 2 kbar failed to further enhance the amount of WSP and thus the influence of number of passages was tested at this pressure. Increasing the number of passages at 2 and 3 for 2 kbar improves the proteins extraction yield (56±4 vs 64±1 and 72±4). Nevertheless, the condition of extraction was settled at 2 passages and 2 kbar. In a global valorisation approach, yields of other components of interest, like pigments and lipids, were taken into account through this choice. Their yields did not improve when the number of passages increased from 2 to 3

12

and the proteins yield enhancement was too low to justify the extra cost derived from energy and time consumption. Shifting pH from native (pH=5.7) to neutral (pH=7) with no pressure treatment leads to the extraction of 27±3% from the total proteins, compared to the yield of proteins extracted at native pH without HP treatment (23±1%). Taking into account the standard deviation, the yields of proteins release at pH=7 and pH=5.7 are equivalent. The influence of pressure at neutral pH (7) has the same trend than cell disruption at native pH (5.7) (data not shown). Associated to HP treatment (2x2 kbar), a pH shift to 7 enabled us to improve the proteins extraction/solubilization yield from 64±1% (2x2 kbar at native pH) to 73% (2x2 kbar at neutral pH) from total proteins of the biomass. It seems that the association of these two operations accentuated the weakening of the cell wall. For microalgae like H. pluvialis in which the cell wall is mainly composed of proteins (75%) and sugars (19%) (Hagen et al., 2002), a pH enhancement favors the cell wall permeabilization and subsequent proteins extraction as noticed by microscopic observations. 2.3.Supernatant Proteins: Characterization and emulsifying properties The SEC profile of proteins extracted under native pH showed a broad distribution of molecular masses from 584 down to 17 kDa. Other small molecules (< 10 kDa) were also detected. They should correspond to peptides, even free amino acids as revealed by analyses achieved on a Superdex peptide 10/300 GL column (data not shown). The SEC profile of proteins obtained under neutral pH revealed the presence of a cluster with a molecular weight higher than 600 kDa, composed of proteins, chlorophyll (661

13

nm) and carotenoids (470 nm). It could be a protein-pigment aggregate formed from the combination of mechanical stress and chemical changes applied during extraction. Ursu et al. (2014) found the same pattern when studying Chlorella vulgaris proteins extracted at alkaline pH. Apart from this, the SEC analysis showed the same pattern than in the native pH extraction, with large amount of small molecules at elution volume above 22 mL. The initial composition of the supernatants revealed that they had a similar amount of lipids, while the native supernatant contained 11% more carbohydrates and 18% less proteins than the supernatant at pH=7 (Table 3). To quantify the emulsifying properties, proteins extract lyophilized powders from supernatants, were solubilised up to their maximum of solubility corresponding to 6 g.L-1for proteins. Results were compared to those obtained with sodium caseinate at the same concentration. Table 3 shows that the ECs of WSP from green H. pluvialis remained very similar between sample types (supernatants at native or neutral pH) and were slightly lower than the EC obtained using sodium caseinate. EC of native and neutral supernatants were 16% and 19% lower than the caseinate EC, respectively. The higher EC of sodium caseinate could be related to their dissociated and naturally unfolded structure. The fact that the percentage of hydrophobic amino-acids to total proteins from caseinate was slightly more important than from H. pluvialis WSP (40% calculated from Sindayikengera and Xia, 2006 vs 36%) may explain its higher EC. Indeed there is a strong positive correlation between EC and high hydrophobicity i.e apolar aminoacids of proteins (Zayas, 1997).

14

However, Table 3 also highlights that after 24 hours, supernatant obtained at pH=7 had a higher stability than the supernatant at native pH (5.7). Both H. pluvialis extracts led to significantly higher ES than sodium caseinate, 25% more for the native pH WSP and 33% for the neutral one. The difference in stability, advantageous to H. pluvialis supernatants, can be explained by the presence of high-molecular-weight proteins in WSP. These macromolecules visibly enhanced the steric forces that enable the formation of stable emulsions. In addition, high-molecular-weight molecules are also responsible for reducing the coalescence and acts against the emulsion degradation (Damodaran, 2005, Choplin et al., 2006). The important EAI value associated with stability reflects a large interfacial area that is probably maintained stable by the high weighted molecules to decelerate the coalescence-driven destabilization of the emulsion (Darine et al., 2010; Dickinson and Stainsby, 1982). An absolute comparison of protein emulsifying properties is sensitive because the methods used are not standardized yet. However, the same trend was noticed with the EC and ES of Chlorella vulgaris and Tetraselmis seucica. The EC of the supernatant extracted at pH=7 from Chlorella was 20% lower while its ES was 26% greater than the reference (Ursu et al., 2014). The results reported by Schwenzfeier et al. (2013) stated that whey isolate proteins (WPI) is a better emulsifier than Tetraselmis soluble proteins while the latter stabilized the emulsion in a larger range of pH. Comparison between the two water-soluble matrixes, at native and neutral pHs, seems to strengthen the neutral extract conditions. Indeed the combination of mechanicalchemical methods allowed the extraction of an interesting proteins molecular weight distribution. Beyond these previous considerations, these results prove the interest in

15

using Haematococcus pluvialis extracts as emulsifying agent because they displayed emulsifying properties as good as or even better than the reference. 2.4.Protein fractionation and concentration 2.4.1. Ultrafiltration Based on the molecular weight distribution (MWD) obtained by SEC, proteins concentration was performed using a 1 kDa membrane. The UF operating parameters TMP and ΔP were fixed at . bar and

bar, respectively. t should be noted that the

flow rate of permeate remains a linear function of TMP regardless of whether ΔP was fixed at 1, 1.4 or 2 bar. The experiment was performed using approximately 5 L of supernatant, and VCF (the ratio of initial supernatant volume to final retentate volume) was fixed at approximately 10, taking into account the dead volume of the membrane unit. The ultrafiltration process was conducted on both supernatants (pH=5.7 and pH=7). Once UF was completed, protein concentration was measured in the final permeates and in the final retentates using the Lowry method. The PCF (ratio between the protein concentration in retentate and in feed) amounted 3.5 and 3.3, thus the UF carried out with a 1 kDa UF membrane appears appropriate for concentration. Permeate, retentate and supernatant for the two pH conditions of extraction were subjected to SEC. SEC profile for permeates revealed the presence of low molecular weight proteins (< 10 kDa), likely the peptides. The SEC profile of the two retentates overlapped the profile of the initial supernatant, the main difference lies in the peaks corresponding to molecular weight proteins > 20 kDa. As to the profile of native extracts, the snapshot of low volume elution (0 to 16 mL) highlighted a higher intensity 16

of the peaks corresponding to high-molecular-weight proteins. The retentate at pH=7 showed a lower intensity for high molecular weight proteins than the neutral supernatant profile. It is due to the retention of molecular aggregates during the prefiltration of the sample before injection. These profiles pointed out a partial fractionation of proteins with the majority of the small peptides located in the permeate. The functional properties were studied using powdered extracts of proteins from retentate and permeate. The proteins obtained from permeate and retentate were soluble up to 10 g.L-1 and 6 g.L-1 respectively, using supernatant at pH=5.7. However, the proteins solubility obtained from retentate using supernatant at pH=7 was reduced to 2 g.L-1. The presence of sterically hindered components like pigments-proteins aggregates are likely to compete with proteins in solute-water interaction. Moreover a lack of electrostatic repulsion forces associated with an increase of protein-protein interaction after an UF would negatively affect protein solubility (Damodaran,1996). As the emulsion capacity of proteins decreases when the protein concentration increases, to compare their EC three samples from supernatant, permeate and retentate with the same concentration (2 g.L-1) were used for supernatant obtained at pH=7. It has been shown that while the emulsion capacity of retentate (2479 mL oil.g-1 protein) and supernatant (2106 mL oil.g-1 protein) are of the same order of magnitude, the permeate displayed a lower EC (989 oil.g-1 protein). The emulsion stability of the retentate (77±1%) was slightly higher than the one of the supernatant (75±3 %) and more than the double of the permeate ES (30±4%). EAI follows the same pattern than the emulsion stability: 119±10, 101± and 27±3 m².g-1 for

17

the retentate, supernatant and permeate respectively. As for the supernatant at neutral pH, the presence of high MW components could explain these properties. When using a protein concentration equals to 6 g.L-1 (supernatant extracted at pH=5.7) ECs remained very similar between sample types (supernatant, retentate or permeate). All samples displayed almost the same trend but with lower ECs due to the increase in the protein concentration (558±7,589±21, 561±15 mL oil.g-1 protein). However, results highlight that after 24 hours, supernatant and retentate led to significantly higher ES than the permeate (84% versus 2.6%). This low stability could be due to the molecular weight of their proteins. Indeed small molecules have negligible steric forces, reducing their ability to maintain their emulsion stable. The high value of EAI of the native permeate, 66±2 versus supernatant at 55±1 and retentate at 75±1 m².g-1, is not contradictory to the prior statement. An important EAI associated with low stability reflects a consequent interfacial area which leads to a faster destabilization of emulsions by coalescence. 2.4.2. Ammonium sulphate (AMS) precipitation Since carbohydrates could modify the techno-functional properties of microalgae protein extracts (Schwenzfeier et al., 2013), it seemed important to verify their influence on emulsifying properties of a water-soluble matrix. In the native supernatant, the sugars to proteins ratio is estimated at 1.4. The ammonium sulphate salting out effect was used to concentrate native supernatant (2x2 kbar) proteins and separate them from sugars. Different amounts of ammonium sulphate saturation were applied (25%, 40%, 70% and 90%) seeking for a compromise between proteins purity level and recovery

18

yield. The highest amount of precipitated proteins achieved was ∼65±1% from the initial protein content in the supernatant at 90% of AMS saturation. It had also been noticed that the higher the saturation of ammonium sulphate, the more sugars are precipitated along with proteins. Thus, 19% of the initial content of carbohydrates was precipitated at 90% AMS vs 3% at 25% AMS, 4% at 40% AMS and 15% at 70% AMS saturation. However, compared to initial supernatant, the ratio of carbohydrates to proteins was almost 50% lower, resulting in the conjugated effect of the concentration of proteins associated with the elimination of the carbohydrates. Therefore, at 90% AMS saturation the ratio amounted 0.65 vs 1.4 for initial native supernatant. The SEC chromatogram of precipitated proteins displayed the presence of high molecular weight proteins, whereas those with molecular weights lower than 10 kDa are almost inexistent. This lack of small peptides and free amino acids, as compared to the SEC profile of initial supernatant, could be due to the desalting of sample using Amicon filtration cell, as the membrane cut-off was 1kDa. As ammonium sulphate precipitation separated the water-soluble carbohydrates from proteins and also fractionated them by allowing the recovery of high molecular weighted proteins, the emulsification properties of the proteins concentrate were then tested. The EC difference between the AMS proteins extract and the other samples at native pH seems to be related to the MWD of proteins. Indeed the purified proteins from AMS precipitation are exempt of small proteins compared to those from retentate, permeate and supernatant. Those small proteins are known for their ability to promote emulsifying capacity. Their absence could reduce the ability of the proteins extract to

19

emulsify more oil and lead to a decrease in the EC value. This is the case with the EC of AMS proteins extract which was estimated at 451±30 versus 558±7 from the native supernatant. However, it may not be precluded that the carbohydrates separated by AMS precipitation had a possible influence in the emulsifying capacity. The stability of the emulsion prepared from AMS proteins extract is similar to native supernatant even if the ratio carbohydrates/proteins is twice lower due to the decrease in carbohydrates content. The absence of carbohydrates seems not to influence the stability of emulsion obtained from vegetative H. pluvialis. As a conclusion, AMS permitted to concentrate and purify proteins extracts. Decreasing the content of carbohydrates and pool high molecular weighted proteins from the native supernatant did not improve the functional properties of the precipitated proteins. Thus, the use of the whole water-soluble matrix as emulsifying agent seems to be more interesting.

Conclusion Extraction of water-soluble biomolecules from H. pluvialis was achieved by mechanical treatment alone or combined with pH shifting (7). Consequently, the yield was enhanced from 64% under pH 5.7 to 73% at pH 7. Emulsion capacities of both watersoluble matrixes were comparable whereas the emulsion stability was higher for an extraction under pH 7 (94±3%) compared to an extraction under pH 5.7 (84±3%) and always better than sodium caseinate (63±4%). Tangential UF made possible the concentration of the supernatant by a factor close to 8 with the advantage of using the concentrated solution directly to the recipe for food formulation.

20

Supplementary materials The SEC chromatograms relating to this study (water soluble proteins extracted at pH=5.7 and pH=7, retentates, permeates, and after ammonium sulphate precipitation) are available as supplementary materials. Acknowledgments This work has been sponsored by the French government research program Investissements d'Avenir through the IMobS3 Laboratory of Excellence (ANR-10LABX-16-01), by the European Union through the program Regional competitiveness and employment 2007-2013 (ERDF – Auvergne region) and by the Auvergne region.

21

References 1.

Batista, A.P., Gouveia, L., Bandarra, N.M., Franco, J.M., Raymundo, A., 2013. Comparison of microalgal biomass profiles as novel functional ingredient for food products. Algal Res. 2, 164–173.

2. Choplin, L., Marchal, P., Sadtler, V., Della Valle, D., 2006. Emulsification, In: olonna, P., Della Valle, D. ( ds.), Mise en œuvre des matières agroalimentaires 1. Lavoisier, Paris, pp. 190-302. 3. Damodaran, S., 2005. Protein Stabilization of Emulsions and Foams. J. Food Sci. 70, 54–66. 4. Damodaran, S., 1996. Amino Acids, Peptides and Proteins, In: Fennema, R.O., (Ed.), Food Chemistry, 3rd Edition, CRC Press, New York, pp.321-416 5. Darine, S., Christophe, V., Gholamreza, D., 2010. Production and functional properties of beef lung protein concentrates. Meat Sci. 84, 315–322. 6. Dickinson, E., Stainsby, G., 1982. Colloids in food. Applied Science publishers, London. 7. Gerde, J.A., Wang, T., Yao, L., Jung, S., Johnson, L.A., Lamsal, B., 2013. Optimizing protein isolation from defatted and non-defatted Nannochloropsis microalgae biomass. Algal Res. 2, 145–153. 8. Glazer, A.N., Cohen-Bazire, G., 1971. Subunit Structure of the Phycobiliproteins of Blue-Green Algae. Proc. Natl. Acad. Sci. 68, 1398–1401.

22

9. Hagen, C., Siegmund, S., Braune, W., 2002. Ultrastructural and chemical changes in the cell wall of Haematococcus pluvialis (Volvocales, Chlorophyta) during aplanospore formation. Eur. J. Phycol. 37, 217–226. 10. Lichtenthaler, H.K., Buschmann, C., 2001. Chlorophylls and Carotenoids: Measurement and Characterization by UV-VIS Spectroscopy, in: Wrolstad, R.E., Acree, T.E., Decker, E.A., Reid, D.S., Schwartz, S.J., Shoemaker, C.F., Smith, P., Sporns, D.M. (Eds.), Handbook of food analytical chemistry, water, proteins, enzymes, lipids, and carbohydrates. Wiley, New York, pp. 153-200. 11. Lorenz R.T., 1999. A technical review of Haematococcus algae. NatuRoseTM Technical Bulletin #060, Cyanotech Corporation, Kailua-Kona, Hawai, pp. 1-12. 12. Lourenço, S.O., Barbarino, E., De-Paula, J.C., Pereira, L.O. da S., Marquez, U.M.L., 2002. Amino acid composition, protein content and calculation of nitrogen-to-protein conversion factors for 19 tropical seaweeds. Phycol. Res. 50, 233–241. 13. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein Measurement with the Folin Phenol Reagent. J. Biol. Chem. 193, 265–275. 14. Mendes-Pinto, M.M., Raposo, M.F.J., Bowen, J., Young, A.J., Morais, R., 2001. Evaluation of different cell disruption processes on encysted cells of Haematococcus pluvialis: effects on astaxanthin recovery and implications for bio-availability. J. Appl. Phycol. 13, 19–24.

23

15. Monsigny, M., Petit, C., Roche, A.C., 1988. Colorimetric determination of neutral sugars by a resorcinol sulfuric acid micromethod. Anal. Biochem. 175, 525–530. 16. Pearce, K.N., Kinsella, J.E., 1978. Emulsifying properties of proteins: evaluation of a turbidimetric technique. J. Agric. Food Chem. 26, 716–723. 17. Safi, C., Charton, M., Pignolet, O., Silvestre, F., Vaca-Garcia, C., Pontalier, P.Y., 2012. Influence of microalgae cell wall characteristics on protein extractability and determination of nitrogen-to-protein conversion factors. J. Appl. Phycol. 25, 523–529. 18. Schwenzfeier, A., Helbig, A., Wierenga, P.A., Gruppen, H., 2013. Emulsion properties of algae soluble protein isolate from Tetraselmis sp. Food Hydrocoll. 30, 258–263. 19. Schwenzfeier, A., Wierenga, P.A., Gruppen, H., 2011. Isolation and characterization of soluble protein from the green microalgae Tetraselmis sp. Bioresour. Technol. 102, 9121–9127. 20. Sindayikengera, S., Xia, W., 2006. Nutritional evaluation of caseins and whey proteins and their hydrolysates from Protamex. J. Zhejiang Univ. Sci. B 7, 90– 98. 21. Thakur, R.K., Villette, C., Aubry, J.M., Delaplace, G., 2008. Dynamic emulsification and catastrophic phase inversion of lecithin-based emulsions. Colloids Surf. Physicochem. Eng. Asp. 315, 285–293.

24

22. Ursu, A.-V., Marcati, A., Sayd, T., Sante-Lhoutellier, V., Djelveh, G., Michaud, P., 2014. Extraction, fractionation and functional properties of proteins from the microalgae Chlorella vulgaris. Bioresour. Technol. 157, 134–139. 23. Wang, S.-B., Hu, Q., Sommerfeld, M., Chen, F., 2004. Cell wall proteomics of the green alga Haematococcus pluvialis (Chlorophyceae). Planta 220, 17-29. 24. Yasumatsu, K., Sawada, K., Moritaka, S., Misaki, M., Toda, J., Wada, T., Ishii, K., 1972. Whipping and Emulsifying Properties of Soybean Products. Agric. Biol. Chem. 36, 719–727. 25. Zayas, P.D.J.F., 1997. Emulsifying Properties of Proteins, in: Functionality of Proteins in Food. Springer Berlin Heidelberg, pp. 134–227.

25

Figure 1 Effect of extraction procedure on the yield of water-soluble proteins (WSP; Yaxis) extracted from Total Proteins (TP) of H. pluvialis initial biomass as a function of high pressure (HP; X-axis). At 2 kbars, effect of number of passages was evaluated: 1(

), 2 (....) or 3 (- - - ) passages. Error bars correspond to standard deviation for

triplicate experiments

26

Table 1 Elementary composition of vegetative (green) Haematococcus pluvialis microalgae Elements

C

H

O

N

K

S

P

Mg

53.1

7.3

26.8

7.6

0.1

0.7

0.9

0.3

H. pluvialis (%)

27

Table 2 Amino acids profile of vegetative H. pluvialis initial biomass

Amino acids

Initial biomass (w/w %)

aspartic acid

2.4

threonine *

1.9

serine

1.3

glutamic acid

3.4

glycine

1.4

alanine

2.1

valine*

1.5

isoleucine*

1.1

leucine*

2.6

tyrosine

1.0

phenylalanine*

1.5

histidine

0.6

lysine*

1.4

arginine

2.1

proline

1.5

“ ”essentials amino acids

28

Table 3 Supernatants initial composition and emulsifying properties emulsification capacity (EC, mL oil.g-1protein), emulsification stability (ES, %) and emulsification activity index (EAI, m².g-1) Components (w/w %)

Supernatant Proteins

a

Emulsifying properties

Carbohydrates

Lipids

Pigments

EC

ES

EAI

pH=5.7

26

37

18

-

558±7

84±3

55±2

pH=7

44

26

16

0.2

534±41

94±3

80±1

CaNa

96

664±30

63±4

56±4

n.d.a

n.d. = not determined

29

80 passage 3 passage 2

70

passage 1 60

% WSP/TP

50 40 30 20

10 0 0-7

0-5.7

0.5

1 1.5 Pressure (kbar)

2

2.5

2.7

Figure 1

30

Highlights  Extraction and solubilization of proteins from green H. pluvialis biomass  73±% of total proteins soluble at neutral pH (7) versus 64±1% at native pH (5.7)  Ultrafiltration achieved proteins concentration  Supernatants emulsifying properties equivalent to sodium caseinate  Valorization of soluble proteins from green H. pluvialis as emulsifying agent