Solubility of extracted proteins from Chlorella sorokiniana, Phaeodactylum tricornutum, and Nannochloropsis oceanica: Impact of pH-value

LWT - Food Science and Technology 105 (2019) 408–416

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Solubility of extracted proteins from Chlorella sorokiniana, Phaeodactylum tricornutum, and Nannochloropsis oceanica: Impact of pH-value

T

Lutz Grossmanna, Jörg Hinrichsb, Jochen Weissa,∗ a

Department of Food Physics and Meat Science, Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstrasse 21/25, 70599, Stuttgart, Germany Department of Soft Matter Science and Dairy Technology, Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstrasse 21, 70599, Stuttgart, Germany

b

ARTICLE INFO

ABSTRACT

Keywords: Glycoprotein Beverages Algae Sustainability Bioeconomy Fraction

Microalgae are a new source of proteins, with yet unknown technofunctionalities. The aim of this work was to study protein solubility – a key technofunctionality - in water in the pH-range of 2–12 of proteins from watersoluble (WSPE) and water-insoluble protein extracts (WISPE) obtained from Chlorella sorokiniana, Phaeodactylum tricornutum, and Nannochloropsis oceanica. Proteins in the WSPEs exhibited an unusual solubility pattern with a solubility ≥39.5 g/100 mL and no complete precipitation in the pH-range 2-12, which was attributed to protein glycosylation and a high amount of hydrophilic amino acids. However, slight aggregation was evident at all pHvalues and no transparent solutions were formed. The highest protein solubility was observed for proteins of Nannochloropsis oceanica, followed by Chlorella sorokiniana, and Phaeodactylum tricornutum. In the WISPEs, strong aggregation and a low solubility (≤22.6 g/100 mL) were observed in the pH-range of 2–11, with an increase at pH 12. Overall, the studied proteins exhibited solubility patterns in the pH-range 2-12 that were different to conventional food proteins, and especially the proteins in the WSPE of Nannochloropsis oceanica exhibited a high solubility (≥58.8 g/100 mL) at pH ≤ 5. Thus, this fraction is quite promising for food applications (e.g. protein-rich acidic beverages) and is of interest for further studies.

1. Introduction Proteins are major ingredients in foods providing several important functions including aroma, texture, nutritional value, biological activity, and technofunctionality (Grossmann, Wefers, Bunzel, Weiss, & Zeeb, 2017; Guichard, 2006; Kilara & Panyam, 2003; Kutzli, Gibis, Baier, & Weiss, 2018; Reichert, Salminen, Badolato Bönisch, Schäfer, & Weiss, 2018). The latter is of importance to food manufacturers since it enables the complex structural design of foods. Key technofunctional properties include amongst others solubility, emulsifying, gelation, and foaming characteristics (Zayas, 1996). Knowledge about the solubility of a protein is of particular importance because it impacts other technofunctional properties as a prerequisite, too. For example, protein solubility directly affects its ability to act as an emulsifier, and an insufficient protein solubility may in turn lead to poor emulsion stability (Griffin, 1949). Solubility is also required for gel formation, as proteins need to be mobile and able to interact with other proteins to form threedimensional networks (Marangoni, Barbut, McGauley, Marcone, & Narine, 2000). Protein solubility was defined as the concentration in a saturated solution, that is in equilibrium with a solid (precipitated)

∗

phase (Walstra, 2003). It may range from almost zero to hundreds of milligram per millilitre (Kramer, Shende, Motl, Pace, & Scholtz, 2012). Sine proteins are zwitterions, solubility in water is influenced by several intrinsic factors, such as e.g. molecular weight, amino acid composition, and α-helix propensity and extrinsic factors such as e.g. pH, ionic strength, and temperature (Diaz et al., 2010; Pelegrine & Gasparetto, 2005). Foods are often subject to a change in pH due to fermentation and acid addition to enhance their shelf life and to provide specific flavours. This may limit the range of use of food proteins though, since many proteins (e.g. soy and casein) have a low solubility at mildly acidic conditions. This is because proteins lose most of their overall net charge around their isoelectric point, which is in the pH region of 4–6 for many common food proteins (Chove, Grandison, & Lewis, 2001; Farrell et al., 2004; Hegg, 1979; Sumner, Nielsen, & Youngs, 1981; Zayas, 1996, pp. 6–75). To meet the globally increasing protein demand, and to comply with consumer demands for more vegan foods, novel protein sources are currently being examined. Microalgae are known to accumulate high amounts of protein, which has been frequently reported to be as much as 60 g/100 g of the cell's dry mass (Becker, 2007), and the proteins are

Corresponding author. E-mail address: [email protected] (J. Weiss).

https://doi.org/10.1016/j.lwt.2019.01.040 Received 19 September 2018; Received in revised form 20 January 2019; Accepted 23 January 2019 Available online 24 January 2019 0023-6438/ © 2019 Elsevier Ltd. All rights reserved.

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therefore of increasing interest to food manufacturers. Microalgae are unicellular photosynthetic microorganisms with cellular sizes of several micrometre, which are less differentiated than land plants and grouped by a polyphyletic approach (Barsanti & Gualtieri, 2014; Chacón-Lee & González-Mariño, 2010; Renaud, Thinh, & Parry, 1999). Three commonly used microalgae from different genera that are of particular interest for food formulations because of their high protein contents were used in this study, namely Chlorella sorokiniana, Phaeodactylum tricornutum, and Nannochloropsis oceanica. The genus of Chlorella belongs to the group of green algae and is one of the most often cultivated microalga. One commonly used Chlorella species is C. sorokiniana, which can be cultivated phototrophically, heterotrophically, and mixotrophically. High growth rates under heterotrophic cultivation conditions and protein contents of 75.3 g/100 g (in dry mass) were reported for this species (Kim, Park, Cho, & Hwang, 2013; Qiao, Wang, & Zhang, 2009). N. oceanica and the diatom P. tricornutum are currently mainly used for lipid and pigment production (Chen, Li, Ren, & Liu, 2016; Zhao, Zang, Xie, Huang, & Wang, 2014). However, appreciable high protein contents of 47.7 g/100 g (N. oceanica) and 49.0 g/100 g (P. tricornutum) were reported for these microalgae as well, which makes them an interesting protein source for various purposes (Skrede et al., 2011). A protein extraction process was previously described for these microalgae, yielding a water-soluble and water-insoluble protein fraction (Grossmann, Ebert, Hinrichs, & Weiss, 2018b). The extracts were a mixture of proteins and polysaccharides, which might be beneficial in terms of protein solubility, due to protein-polysaccharides interactions (Sola & Griebenow, 2009; Zeeb, Grossmann, & Weiss, 2016). So far, only a few studies investigated the solubility of proteins obtained from microalgae. Gerde et al. (2013) investigated the protein extraction efficiency from Nannochloropsis spp. at various pH and found a maximum protein extraction yield at high pH values, indicating increased protein solubility at higher pH values. Furthermore, Schwenzfeier, Wierenga, and Gruppen (2011) found an overall high solubility for proteins from Tetraselmis sp. in the pH-range 5.5 to 8.5 and the study of Grossmann, Ebert, Hinrichs, and Weiss (2018a) showed that soluble proteins extracted from the microalgae Chlorella protothecoides have a high solubility in the pH range of 2–6. The studies thus indicate, that microalgae proteins appear to exhibit unusual protein solubility patterns when compared with other food proteins, such as those in whey or soy. The aim of this study was therefore to elucidate for the first time in more detail the protein solubility and possible underlying mechanisms of proteins extracted from three different microalgae genera over a broad pH range to allow food manufacturers to identify possible use scenarios.

Fig. 1. Protein solubility in water at pH values of 2-12 of proteins in watersoluble (WSPE) and water-insoluble protein extracts (WISPE) expressed as the ratio of protein concentration in supernatant after centrifugation to protein concentration before centrifugation (A, C), and quantification of dispersion turbidity by transmittance measurements at a wavelength of 600 nm before centrifugation (B, D). Plot shows average ± standard deviation (n = 2), standard deviation in some cases smaller than symbol size.

from the soluble proteins by centrifugation (20,000 g, 30 min, 25 °C). Both, the soluble and insoluble proteins were precipitated using ethanol:acetone (1:1 v/v), employing a solvent to sample ratio of 6:1 (v/m) to obtain a powdered extract after solvent evaporation at 25 °C. The obtained extracts (water-soluble protein extract (WSPE) and the waterinsoluble protein extract (WISPE), refer to product number 6 and 7 in Fig. 1 of (Grossmann et al., 2018b)) were stored at 8 °C prior to further use. 2.3. Preparation of protein extract dispersions

2. Materials and methods

The obtained WSPE and WISPE of C. sorokiniana, P. tricornutum, and N. oceanica were dispersed in purified water at a concentration of 0.5 g/ 100 g. To prevent microbial spoilage, 0.075 g/kg of ProClin™ 950 (2Methyl-4-isothiazolin-3-on; Sigma-Aldrich, Steinheim, Germany) was added to the dispersions. All dispersions were stirred over night before further use.

2.1. Materials The following whole microalgae were obtained as dry powder: Nannochloropsis oceanica from allmicroalgae (Lisbon, Portugal), Phaeodactylum tricornutum from Necton (Olhão, Portugal), and heterotrophically cultivated Chlorella sorokiniana from Roquette Frères (Lestrem, France). All chemicals used in this study were of analytical grade and were used without further purification.

2.4. Adjustment of pH of protein dispersions

2.2. Protein extraction: cell disruption, fractionation, and solvent precipitation

To assess the protein solubility at different pH values, the pH of the protein extract dispersions with WSPE and WISPE was adjusted in the range of 2–12 with HCl and NaOH (0.01, 1, 2, and 6 mol/L) under continuous stirring. The pH-value was increased or decreased in pH steps of 1.0 starting from the native pH value of the dispersions with an incubation period of 30 min at each pH value. After incubation, the pH was set to the respective pH with an error of ±0.05. Samples were taken and stored at 6 °C before further analyses. An inoLab pH 720 pHmeter with a SenTix 41 pH-electrode (Xylem Analytics Germany Sales GmbH & Co. KG, Weilheim, Germany) was used for pH measurements.

Precipitated protein extracts were obtained by cell disruption, fractionation, and subsequent solvent precipitation as described previously Grossmann et al. (2018b). Cell dispersions (c = 10 g/100 g) were passed through a high-pressure homogenizer at 180 MPa and a maximum temperature of 22 °C (M110-EH-30, equipped with a F12Y interaction chamber, Microfluidics International Cooperation, Newton, MA, USA). After cell disruption, the insoluble proteins were separated 409

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2.5. Analytical techniques

oxidized at 4 °C using hydrogen peroxide, phenolic formic acid solution, and phenol. Then, hydrochloric acid and phenol were used to hydrolyze the proteins at 113 °C for 24 h. An L-8900 Amino Acid Analyzer was employed to quantify the amino acids with Norleucine as external standard (VWR, Hitachi Ltd, Tokyo, Japan). In this method, asparagine/aspartic acid, and glutamine/glutamic acid cannot be analyzed separately. The tryptophan content was analyzed after an alkaline hydrolysis with barium hydroxide by reverse-phase chromatography and fluorescence detection (Agilent 1100 HPLC, Agilent, Waldbronn, Germany) at an excitation of 283 nm and an emission wavelength of 355 nm. N to protein conversion factor k. Two different nitrogen to protein conversion factors k were calculated according to Mosse (1990). First, kA was calculated as the ratio of the total anhydrous amino acid residue weights to the nitrogen content in the analyzed amino acids (in dry weight). Second, kp was determined as the ratio of the sum of total anhydrous amino acid residue weights to the nitrogen content of the WSPE and WISPE (in dry weight). The nitrogen content in the extract was determined by combustion using the Dumas method (FP-528, LECO Corporation, St. Joseph, MI, USA). The final factor k used to calculate the protein contents was calculated as the average value of kA and kp. Hydrophilicity index. Hopp and Woods (1981) proposed a hydrophilicity value for each amino acid. According to Diaz et al. (2010), the hydrophilicity index (HI) may then be calculated as follows (equation (2)):

2.5.1. Protein solubility Protein solubility in water was determined using a centrifugation method (Morr et al., 1985). Samples were centrifuged for 30 min at 20,000 g and 25 °C. Then, the protein concentration before centrifugation and after centrifugation (supernatant) was determined with the bicinchoninic acid (BCA) assay employing the Pierce™ BCA Protein Assay Kit (Fisher Scientific GmbH, Schwerte, Germany). In this assay, proteins induce a reduction of Cu2+ to Cu1+, which results in a colored chelated complex with two BCA molecules (Smith et al., 1985). In detail, the samples were incubated with the BCA-reaction solution for 30 min at 37 °C and subsequently cooled down for 7 min to room temperature. The absorbance was detected at 562 nm with an UV–Vis spectrophotometer (HP 8453, Agilent Technologies 95–00, Waldbronn, Germany). Bovine serum albumin was used to establish the calibration curve. The concentration of soluble protein was determined using equation (1):

Water

soluble protein Protein concentration in supernatant = x 100 (g/100 mL) Total protein concentration before centrifugation (1)

2.5.2. ζ-potential The ζ-potential of the protein dispersions was measured at each pHvalue with a laser doppler micro-electrophoresis device (Nano-ZS, Malvern Instruments Ltd, Malvern, UK). The method is based on the measurement of the movement of charged particles in an applied electric field. The direction and speed in which the particles move is related to the ζ-potential of the particles, and can be measured by phase analysis of the light scattering data. The ζ-potentials were calculated using the Smoluchowski model and folded capillary cuvettes (DTS 1070, Malvern Instruments Ltd, Malvern, UK) were used for all measurements. Every dispersion was measured in duplicate with 20 readings per measurement.

HI =

AA

nAA

fAA

nAA AA

(2)

where nAA is the amino acid (AA) content in mole per 100 g dry weight, and fAA the hydrophilicity value for every amino acid. For Glutamine/ Glutamic acid and Asparagine/Aspartic acid, the mean values were taken for the calculations. 2.5.7. Gel electrophoresis 2-D gel electrophoresis. Prior to analysis, samples were precipitated with acetone (−20 °C) and stored overnight at −20 °C. After solvent removal, proteins were redispersed in DeStreak™ rehydration solution containing 1.0 g/100 mL IGP buffer (GE Healthcare Europe GmbH, Freiburg, Germany). Proteins were placed onto a rehydrated linear Immobiline™ DryStrips pH 3–10 using a cup-loading method (GE Healthcare Europe GmbH, Freiburg, Germany). Focusing was carried out for 21 h at a maximum voltage of 8000 V and 75 μA using an Ettan™ IPGphor™ 3 (GE Healthcare Europe GmbH, Freiburg, Germany). Subsequently, the strips were transferred in a solution containing 75 mmol/L Tris/HCl pH 8.8, 6 mol/L urea, 30 g/100 mL glycerol, 2 g/ 100 mL SDS, 1 g/100 mL dithiothreitol for 15 min. After incubation, the strips were transferred for 15 min into the same solution containing 2.5 g/100 mL iodoacetamide and no dithiothreitol. Eventually, the second dimension was carried out in a 12 g/100 mL polyacrylamide gel at 12 W per gel for 12 h in an Ettan DALTsix electrophoresis module (GE Healthcare Europe GmbH, Freiburg, Germany). For protein detection, the gels were incubated in colloidal Coomassie or silver nitrate (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) and images were obtained at an excitation wavelength 633 nm without emission filter employing a Typhoon Trio + imaging system (GE Healthcare, Germany). SDS-PAGE. Proteins were solubilized in a reducing sample buffer according to the manufacturer's description (Bio-Rad Laboratories, Hercules, CA, USA) and incubated for 5 min at 95 °C. The proteins were transferred in Mini-Protean® TGX™ Precast Gels (Bio-Rad Laboratories, Hercules, CA, USA) and treated at 200 V for 35 min in a Mini-protean® II module (Bio-Rad Laboratories, Hercules, CA, USA). After separation, staining was performed with colloidal Coomassie (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) and the Pierce™ glycoprotein staining kit (Fisher Scientific GmbH, Schwerte, Germany), which

2.5.3. Transmittance The development of light-scattering aggregates in the visible wavelength range was studied by transmittance measurements at 600 nm. Measurements were performed in polystyrene cuvettes (path length of 10 mm) under continuous stirring to prevent settling of aggregates with an ultraviolet–visible (UV–Vis) spectrophotometer (HP 8453, Agilent Technologies 95–00, Waldbronn, Germany). Measurements are reported from 0 (no transmittance) to 100 (full transmittance). 2.5.4. Microstructure Optical microscopy was carried out to visualize the microstructure of the protein samples. Images were captured with an AxioCam ICc 3 mounted on an Axio Scope optical microscope (A1, A-Plan objective lens with numerical aperture of 0.65, Carl Zeiss Microimaging GmbH, Göttingen, Germany). Images were taken at three different locations per sample using 200-fold magnification. 2.5.5. Gravitational separation Photographic images of test tubes containing the samples were taken to analyze the gravitational separation behavior. Samples were photographed after sample preparation (t = 0) and after t = 24 h at 4 °C. 2.5.6. Amino acid composition, N to protein conversion factor, and hydrophilicity index Amino acid composition. Analysis of amino acids was carried out according to the study of Rodehutscord and coauthors (Rodehutscord, Kapocius, Timmler, & Dieckmann, 2004). The WSPE and WISPE were 410

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Table 1 Amino acid profile (in dry weight d.w., n = 2), hydrophilicity index, nitrogen to protein conversion factor k and protein content (nitrogen x k) of precipitated soluble (WSPE) and insoluble protein (WISPE) extracts. No separate analysis of Glutamine/Glutamic acid and Asparagine/Aspartic acid.

employs fuchsin-sulfite.

4–5.5 (Farrell et al., 2004; Lee, Ryu, & Rhee, 2003). Conversely, we found no such behavior for the examined proteins in the WSPE and a complete insolubility was not observed at any given pH. The proteins in the WSPE of N. oceanica had an overall high solubility of ≥79 g/100 mL in the pH-range 6 to 12. A lower solubility in this pH range was observed for the proteins in the WSPE of C. sorokiniana (≥68 g/100 mL) and P. tricornutum (≥50 g/100 mL). The solubility declined for all proteins in the WSPEs at lower pH-values with a minimum solubility of 45.7 ± 0.3 g/100 mL at pH 4 (C. sorokiniana), 39.5 ± 0.1 g/100 mL at pH 2 (P. tricornutum), and 58.8 ± 1.0 g/100 mL at pH 2 (N. oceanica). Surface charge decreased with decreasing pH (Fig. 2), confirming a decrease in repulsive electrostatic interactions and thus a decrease in protein-protein repulsion at low pH. The lowest charge was observed at pH 2 for all WSPEs except for C. sorokiniana, which exhibited a shift

3. Results and discussion Low solubility of many food proteins at different pH-values is a major challenge in food formulations. Novel food proteins may have unique solubility patterns that differ from those of conventional food proteins (Lam & Nickerson, 2013). Since microalgae have a complex protein composition, the solubility of proteins obtained from microalgae might differ as well (Guarnieri, Nag, Yang, & Pienkos, 2013). Prior to analysis, microalgae cells were disrupted to liberate the proteins from the cells, proteins were separated by centrifugation into a soluble- and insoluble fraction and both fractions were subsequently precipitated employing solvent precipitation, resulting in powders with a protein content ranging from 26.5 g/100 g–68.6 g/100 g (Table 1). 3.1. Soluble protein fractions: solubility and aggregation behavior The overall objective of this study was to investigate the solubility of both protein fractions from different microalgae over the pH range from 2 to 12. This range was chosen because of two reasons: (i) low pHvalues generally decrease the solubility of most proteins due to the fact that acidic amino acids becoming protonated, which results in an overall zero net charge (isoelectric point), and (ii) the highest solubilities are often observed at high pH, which might enhance the solubility of insoluble proteins. Initially, the solubility at different pH values of the proteins in the WSPE was investigated (Fig. 1A). The obtained solubility profile deviates from known solubility curves of widely employed food proteins, such as soy protein and casein. It is well known that these proteins have virtually zero solubility around their isoelectric point in the pH range of

Fig. 2. Electrostatic interactions displayed as ζ-potential at 25 °C of dispersions at pH 2-12 prepared with water-soluble (A, WSPE) and water-insoluble protein extracts (B, WISPE). 411

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Fig. 3. Macroscopic appearance and gravitational separation behavior of dispersions (c = 0.5 g/100 g) prepared with water-soluble (WSPE) and water-insoluble protein extracts (WISPE) extracted from different microalgae at pH 2–12 (as indicated) after 24 h.

at any tested pH-value with proteins of N. oceanica exhibiting the overall highest solubility followed by C. sorokiniana, and P. tricornutum.

from negative to positive charge between a pH of 3 and 4. These results are in contrast to a previously reported solubility of ≥90 g/100 mL in the pH-range 2 to 6 for proteins that were liberated from Chlorella protothecoides cells and not further processed (Grossmann et al., 2018a). Interestingly, the study of Schwenzfeier et al. (2011) revealed a similar solubility behavior of proteins from Tetraselmis sp. compared to those in the present study, with also no complete precipitation in the pH range of 2.5–8.5 (protein solubility of >20 g/100 mL). This indicates that solubility strongly varies from microalga to microalga, and that some species might be quite interesting for food applications where solubility may be an issue (e.g. acidified beverages). However, slight aggregation was evident at all pH-values (Fig. 4) with a transmittance of ≤89% for all samples (Fig. 1B) and an increase in aggregate size at lower pH. These aggregates were likely formed due to a decrease in electrostatic repulsion, which increases attractive protein-protein interactions allowing hydrophobic forces to induce aggregation. This effect could have been enhanced by downstream processing of the microalgae biomass. It is already known that processes such as cell disruption and solvent precipitation can lead to partial protein denaturation and thus proteins are not completely soluble after resolubilization (Schubert & Finn, 1981). Moreover, downstream processing may have resulted in the presence of some completely insoluble proteins, which were not fully separated in the centrifugation process. In order to further understand the observed solubility and aggregation behavior, WSPEs were assessed as well for gravitational separation after 24 h (Fig. 3). All WSPEs showed gravitational separation at low pH-values due to aggregation, with a clear upper phase in the pH range of 2–5 for C. sorokiniana, and for P. tricornutum at a pH-value of 2. For these reasons, these extracts may be more suitable at low pH for foods with medium to high viscosities in which a potential aggregation may not be a hindrance. The WSPE of C. sorokiniana might also be used for food formulations with low viscosities at neutral pH because of a high solubility and no gravitational separation. In contrast, proteins in the WSPE of N. oceanica had an overall high solubility and displayed only slight gravitational separation in the pH-range 2-6, which makes this fraction a promising candidate for low viscous foods that are formulated at pH-values <4 (Reddy, Norris, Momeni, Waldo, & Ruby, 2016). Interestingly, dispersions prepared with WSPEs from N. oceanica showed gravitational separation at pH values of 7–12, but protein solubility stayed almost constant at the same time (Fig. 1A). This discrepancy might be a result of residual nucleic acid in this fraction, which is known to be affected by a change in pH (Li & Breaker, 1999). Clearly more research is necessary to fully understand this phenomenon in this fraction. In conclusion, none of the examined WSPEs completely precipitated

3.1.1. Protein characteristics: isoelectric points and degree of glycosylation Results from the previous chapter showed a high solubility of proteins in the extracts at pH 2–12 especially for N. oceanica, and no clear isoelectric point could be determined for the proteins in the WSPEs. Protein solubility is generally affected by both intrinsic factors (e.g. molecular weight, total number of hydrophobic residues, content of specific amino acids) and extrinsic factors (e.g. pH, ionic strength, temperature). Furthermore, solubility may be enhanced at various environmental conditions due to (i) a high surface hydrophilicity, (ii) an electrostatic stabilized protein-polysaccharide complexation, and (iii) a covalent protein glycosylation (Schwenzfeier, Wierenga, Eppink, & Gruppen, 2014; Sola & Griebenow, 2009; Zhu & Damodaran, 1994). The aim of the following experiments was to reveal the cause of the observed unusual solubility profiles. Previous findings suggested that mainly a high degree of glycosylation and a high content of hydrophilic amino acids is responsible for a high solubility of microalgae protein extracts (Grossmann, Hinrichs, & Weiss, 2018c). For that purpose, amino acid analysis (Table 1), 2-D electrophoresis, and SDS-PAGE with subsequent protein and glycoprotein-staining (Fig. 5) was carried out. Isoelectric points for most proteins in the WSPEs were found to be in the pH-range of 4–7 (C. sorokiniana), 3.5–7 (P. tricornutum), and 4–6.5 (N. oceanica). These results are in line with previous studies that also reported a very diverse isoelectric point profile of proteins from the microalgae C. vulgaris (Ursu et al., 2014). The highly diverse protein profile is most likely related to the diversified cell metabolism of these single cell organisms, which have to carry out all of the necessary metabolism pathways in one cell. Nonetheless, this is in contrast to ζpotential measurements, which only showed a charge shift from negative to positive with decreasing pH for the WSPE of C. sorokiniana. This indicates the presence of negatively charged colloids, who affect the charge of the proteins. This negative charge can be caused by negatively charged sugar acids, such as uronic acids, and was positively correlated to protein solubility in microalgae (Schwenzfeier et al., 2014). This hypothesis was affirmed by a glycoprotein staining that showed glycosylation for all three protein fractions, with the highest degree and no clear bands in C. sorokiniana indicating an overall broad glycosylation (Fig. 5). It is known that covalently bound carbohydrates can increase the solubility of proteins under different environmental conditions by increasing the solvent accessible area (Sola & Griebenow, 2009). Therefore, the Maillard reaction was used in several studies to deliberately generate such structures (de Oliveira, Coimbra, de Oliveira, Zuñiga, & Rojas, 2016; Kutzli et al., 2018). In cells, glycosylation of proteins has several functions including cell-cell identification, 412

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Fig. 4. Presence of aggregates and overall microstructure of water-soluble (WSPE) and water-insoluble (WISPE) protein dispersions (c = 0.5 g/100 g) extracted from Chlorella sorokiniana (Cs), Phaeodactylum tricornutum (Pt), and Nannochloropsis oceanica (No) at pH 2, 6, and 12 visualized by light microscopy. Scale bar = 20 μm.

hormone-receptor binding, interactions between cells, protein targeting, malignant transformation, and phagocytosis (Tanner & Lehle, 1987) and thus specific cells may act as natural source of technofunctional glycoproteins. However, proteins in the WSPEs of P. tricornutum and N. oceanica still exhibited overall high solubility at low pH values (Fig. 1A), while showing a lower intensity in the glycoprotein staining. This might be a result of a high hydrophilicity index of the proteins in these fractions (Table 1), which is related to a high degree of hydrophilic amino acids. These hydrophilic amino acids can be accumulated around the surface of the protein and thereby increase the protein-water interactions of the area that is in contact with the water molecules. However, hydrophilicity values were lower compared to proteins in a WSPE of C. protothecoides, and this might be the reason for a lower solubility around the isoelectric point of these proteins (Grossmann et al., 2018c). This correlation between high solubility over a broad pH-range and hydrophilicity was also reported for other proteins, such as native whey protein (Zhu & Damodaran, 1994). Thus, it is suggested that the observed solubility patterns of the proteins in the WSPEs at different pHvalues is mainly related to a high degree of glycosylation and a high degree of hydrophilic amino acids, albeit other causes such as the

formation of electrostatically stabilized soluble complexes cannot be completely ruled out. 3.2. Insoluble protein fractions: solubility behavior The proteins in the WSPEs exhibited interesting solubility patterns, especially at low pH-values. However, to fully exploit the entire proteinaceous microalgae biomass, WISPEs should be considered as well. It should be noted though that insoluble proteins of N. oceanica had to be excluded from this part of the study because of an overall low cell disruption (Grossmann et al., 2018b), which would have interfered with the protein solubility determination method. An overall low solubility (≤22.6 g/100 mL) was observed for all examined proteins in the WISPEs over the tested pH range of 2–11 with an increase in solubility at pH 12 for all tested microalgae (Fig. 1C). For example, a solubility of 52.5 ± 11.1 g/100 mL was obtained at pH 12 for C. sorokiniana. This effect is related to the increasing charge of the proteins at higher pH values. Proteins possess the highest charge at high pH-values, because of the charged side chains of arginine (pKa 12) and lysine (pKa 10.7–11.2) (André, Linse, & Mulder, 2007), which leads to a maximum negative protein charge at pH values above their respective 413

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of several micrometers (Fig. 4). However, additional microscopy analyses are required to reveal further details about microstructural variations between these aggregates. A high degree of aggregation in these fractions could have been promoted during cell disruption by high pressure homogenization, which can lead to aggregation of released cell particles; a phenomenon that has also been described by other authors (Shene, Monsalve, Vergara, Lienqueo, & Rubilar, 2016). Further insights into the solubility and aggregation behavior of the WISPEs was obtained by transmittance measurements (Fig. 1D). The size of the protein aggregates resulted in a high turbidity of the samples and low transmittance values with an increase at higher pH, which correlated well with the solubility measurements. 3.2.1. Gravitational separation behavior of WISPEs The purpose of this set of experiments was to gain further insights into the aggregation behavior of the WISPEs by determining their gravitational separation behavior. Therefore, the gravitational separation behavior was monitored over a 24 h period for the WISPEs (0.5 g/ 100 g) (Fig. 3). Considerable phase and gravitational separation were observed after 24 h for all WISPEs and these fractions are therefore more suitable for foods with medium to high viscosities where colloidal sedimentation is hindered. According to Stokes' law, the sedimentation velocity depends on the density difference between the particles and the fluid, the size of the particles, and the viscosity of the fluid (McClements, 2007). As shown above, the WISPEs had particle sizes of several micrometers (Fig. 4), which leads to a rapid sedimentation compared to dispersed protein monomers. Furthermore, the high sedimentation speed suggests an overall high density of the protein aggregates, which is in line with the observed low solubility of these proteins. However, sedimentation behavior varied with pH. A complete sedimentation with a transparent upper phase was observed at a pH of 2–4 (C. sorokiniana, P. tricornutum), indicating minimal protein-solvent interactions at these pH values. In order to better understand these observations, ζ-potential measurements were performed (Fig. 2B), which are an indicator of the surface charge of the proteins. The proteins in the WISPE of C. sorokiniana exhibited a shift from positive to negative charges between pH 3 and 4. Dispersions of P. tricornutum had a minimal charge at pH 2, with a gradual decrease with increasing pH. Furthermore, the ζ-potential measurements supported the observed phase and gravitational separation behavior. A complete gravitational separation at low pH values was obtained in the WISPEs where the surface charge of the proteins is low or shifting from negative to positive, which can result in aggregates that do not dissolve again due to hydrophobic interactions. Interestingly, the WISPE of C. sorokiniana had an appreciable high charge at low pH-values. The same is true for the WISPE of P. tricornutum at high pH-values although the solubility stayed low, which seems to be contradictory as a high surface charge should result in high protein-water interactions. However, because of their large size, the surface charge is not high enough to generate enough protein-water interactions to keep the proteins dispersed. This assumption is supported by previous studies of other authors, which reported high surface charges in biopolymer particles produced from water-insoluble proteins such as zein and gliadin, which are virtually insoluble in water (Davidov-Pardo, Joye, & McClements, 2015; Hu & McClements, 2015).

Fig. 5. Determination of isoelectric points by 2-D gelelectrophoresis with Coomassie (blue), silver (grey) protein staining and detection of glycoproteins by SDS-PAGE (bottom) with subsequent protein (blue) and glycoproteinstaining (pink) of water-soluble proteins (WSPE) from Chlorella sorokiniana (Cs), Phaeodactylum tricornutum (Pt), and Nannochloropsis oceanica (No). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

4. Conclusions The proteins of the tested microalgae had different solubility patterns over a broad pH-range compared to commonly employed food proteins. In case of WSPEs of C. sorokiniana, P. tricornutum, and N. oceanica, ≥39.5 g/100 mL of the proteins stayed in solution at pH 2–12, with proteins of N. oceanica exhibiting the overall highest (≥58.8 g/ 100 mL), and P. tricornutum the lowest solubility with no single clear isoelectric point due to the high diversity of different proteins. This

pKa. The overall observed low solubility of the proteins in the WISPEs could also be due to the presence of protein aggregates in the dispersions. This assumption is supported by the microstructure, which showed strong aggregation at all pH values with aggregates having sizes 414

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Table 2 Suggested applications of employed microalgae protein extracts (WSPE = water-soluble protein extract; WISPE = water-insoluble protein extract) in foods depending on pH and viscosity of the final food product. low pH (≤5)

high pH (≥5)

WSPE

WISPE

WSPE

WISPE

low to medium viscosities

Nannochloropsis oceanica

-

–

medium to high viscosities

Nannochloropsis oceanica Phaeodactylum tricornutum Chlorella sorokiniana

Nannochloropsis oceanica Phaeodactylum tricornutum Chlorella sorokiniana

Nannochloropsis oceanica (
effect was attributed to the high degree of glycosylation and/or high amount of hydrophilic amino acid in the proteins, which enhances the water-protein interactions. However, all WSPEs showed aggregation and no clear solutions were formed (transmittance ≤ 89%). In contrast, the pH had only minor effects on the proteins in the WISPEs. Proteins were strongly aggregated and remained virtually insoluble across the investigated pH range with an increase in solubility at pH 12. Based on these results, possible applications in foods depending on pH and viscosity may be suggested, as listed in Table 2. Especially the proteins in the WSPE of Nannochloropsis oceanica exhibited a high solubility at pH ≤ 5 and thus are promising for food applications (e.g. protein-rich acidic beverages). More general, extracts with a high solubility and low gravitational separation speed are especially suited for foods with low to medium viscosities while extracts that are prone to gravitational separation are more suitable for foods with medium to high viscosities. However, more studies are needed to fully elucidate the effect of glycosylation in microalgae proteins on protein solubility and to functionalize the insoluble proteins.

Nannochloropsis oceanica Phaeodactylum tricornutum Chlorella sorokiniana

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