Effect of Ph on the Functional Properties of Arthrospira (Spirulina) Platensis Protein Isolate

Food Chemistry 194 (2016) 1056–1063

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Food Chemistry

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Effect of pH on the functional properties of Arthrospira (Spirulina) platensis protein isolate ⇑ Sonda Benelhadj a,b, Adem Gharsallaoui c, , Pascal Degraeve c, Hamadi Attia b, Dorra Ghorbel a,b a Université de Carthage, INSAT (Institut National des Sciences Appliquées et de Technologie), Centre Urbain Nord, B.P. 676, 1080 Tunis, Tunisia b Université de Sfax, ENIS, LAVASA (Laboratoire Valorisation, Analyse et Sécurité des Aliments), BPW 3038, Sfax, Tunisia c Université de Lyon, Université Claude Bernard Lyon 1 – ISARA Lyon, Laboratoire BioDyMIA (Bioingénierie et Dynamique Microbienne aux Interfaces Alimentaires), Equipe Mixte d’Accueil n°3733, IUT Lyon 1, Technopole Alimentec – Rue Henri de Boissieu, 01000 Bourg en Bresse, France article info abstract

Article history: In the present study, a protein isolate extracted from Arthrospira platensis by isoelectric precipitation was Received 9 November 2014 evaluated for its functional properties. The maximum nitrogen solubility was 59.6 ± 0.7% (w/w) at pH 10. Received in revised form 25 July 2015 The A. platensis protein isolate (API) showed relatively high oil (252.7 ± 0.3 g oil/100 g API) and water Accepted 29 August 2015 (428.8 ± 15.4 g of water/100 g of API at pH 10) absorption capacities. The protein zeta potential, the emul- Available online 31 August 2015 sifying capacity, the emulsion ageing stability, the emulsion microstructure and the emulsion opacity as well as the foaming capacity and the foam stability were shown to be greatly affected by pH. Especially, Keywords: emulsifying and foaming capacities were positively correlated to the protein solubility. Moreover, the API Arthrospira platensis was able to form ﬁlms when sorbitol (30% (w/w)) was used as plasticizer and to form gels when the API Protein isolate pH concentration exceeded 12% (w/w). Functional properties Ó 2015 Elsevier Ltd. All rights reserved. Pigment–protein complexes

1. Introduction Arthrospira platensis (common name: Spirulina) is a blue-green algae (Cyanobacterium) belonging to the family of Oscillatoriaceae. Microalgae are one of the most interesting sources of food It forms unbranched, multicellular helicoidal filaments of ingredients and functional food products. They can be used to 200–300 lm length and 5–10 lm widths (Hedenskog & Hofsten, enhance the nutritional value of foods due to their richness in com- 1970). The culture medium of the blue-green algae should have pounds with benefic attributes (Gouveia, Marques, Sousa, Moura, & alkaline characteristics (pH 8.5–11) which may also be interesting Bandara, 2010). Therefore, the use of microalgae as a source of to prevent the proliferation of most pathogenic microorganisms. functional foods is a priority area in algal technology permitting The remarkable protein content (60–70% (w/w) (dry weight basis)) establishment of a cost effective microalgae production system of A. platensis has attracted the scientists’ attention, as well as that with environmental and health-related beneficial effects. In recent of the manufacturer. Moreover, this microalga has interesting years, some research has been carried out regarding the develop- nutritional properties such as content in essential amino acids, ment of many healthy food products prepared from microalgae. some vitamins (particularly vitamin B12) as well as numerous Traditional food products, like biscuits (Gouveia et al., 2008), pasta minerals. The amino acid composition of Arthrospira proteins is (Rodríguez De Marco, Steffolani, Martínez, & León, 2014), gelled generally well balanced reflecting its potential as a human food desserts (Batista, Gouveia, Nunes, Franco, & Raymundo, 2008), ingredient (Belay, Ota, Miyakawa, & Shimamatsu, 1993). On the and ice cream (Priyanka, Kempanna, & Aman, 2013), have been other hand, the absence of cellulose in the cell wall allows for easy developed with microalgae, making these products more attractive digestion of A. platensis (Belay, 2008). Many studies have shown and healthy. Very little is known about the properties of algal pro- that the consumption of these microalgae may result in significant teins. In general, the amino acid profiles of algal proteins are very therapeutic attributes: a hypolipidemic effect (Narmadha, dependent on the species, but several algal strains contain all the Sivakami, Ravikumar, & Mukeshkumar, 2012), a protective effect essential amino acids (Becker, 2007). against diabetes and obesity (Anitha & Chandralekh, 2010), and an inhibitory effect of anemia (Simsek, Karadeniz, Kalkan, Keles, & Unal, 2009). Algal proteins may be free or bound to pigments. The multi- ⇑ Corresponding author. subunit pigment–protein complex, called phycobilisome, is com- E-mail address: [email protected] (A. Gharsallaoui). posed of heterodimeric phycobiliproteins (the major proportion http://dx.doi.org/10.1016/j.foodchem.2015.08.133 0308-8146/Ó 2015 Elsevier Ltd. All rights reserved. S. Benelhadj et al. / Food Chemistry 194 (2016) 1056–1063 1057 of algal cell proteins) that are assembled with the aid of linker values ranging from 3 to 10. The solutions were stirred for 2 h with polypeptides (Arteni, Ajlani, & Boekema, 2009; Liu, Chen, Zhang, a magnetic stirrer until the proper hydration was reached and the & Zhou, 2005). Cyanobacteria contain three types of pigments: pH was adjusted by adding HCl (1 mol/L) or NaOH (1 mol/L). Fluo- chlorophyll, carotenoids and xanthophylls, and phycobiliproteins rescence spectra at 20 °C were measured while stirring with a LS (Ayyaraju, Murthy, & Prasanna, 2012). 55 spectrofluorometer (Perkin Elmer, France) equipped with FL Apart from nutritional properties that were well studied, the Winlab software. Excitation and emission wavelengths were functional properties of microalgae proteins are not well known. 280 nm and 300–500 nm, respectively. UV absorption spectra at Only few studies have been conducted to evaluate the functional 20 °C of API solutions (0.05% (w/w) in imidazole/acetate buffer properties of Spirulina flour and protein concentrate (27% of pro- (0.005 mol/L, pH 10)), prepared in the same way as for the fluores- teins) from algal cells (Devi & Venkataraman, 1984; Nirmala, cence study, were measured from 200 nm to 350 nm with an Prakash, & Venkatarman, 1992) and the gelation properties of Spir- UV/Vis spectrophotometer (LIBRA, Biochrom, Cambridge, UK) in a ulina protein isolate (Chronakis, 2001). This last study is one of the 1 cm quartz cuvette against imidazole–acetate buffer (0.005 mol/ best sources of information on the functional properties of A. platen- L, pH 10). sis for food applications available to date. So far, to the best of our knowledge, no research has been carried out regarding the modification of pH and its effect on the functional properties of algal pro- 2.5. Zeta potential (ZP) teins. A better knowledge on the impact of pH on these properties may help to improve the valorization of these proteins, for instance The zeta potential (f-potential) of extracted proteins was deter- through food products based on emulsions, foams or gels. The mined using a Zetasizer NanoZS90 (Malvern Instruments, Malvern, objective of this study was therefore to examine the influence of UK). The samples were diluted (0.5% (w/w)) with imidazole/acetate pH on the functional properties of A. platensis protein isolate. buffer adjusted to the suitable pH value. The mean f-potential (ZP) values (±SD (standard deviation)) were obtained from the 2. Materials and methods instrument.

2.1. Raw materials and chemicals 2.6. Nitrogen solubility (NS) A. platensis powder (89.27 ± 0.16 g/100 g dry matter) was purchased from Bioalgal Society (Mahdia, Tunisia). Analytical grade Nitrogen solubility profile was determined according to Nirmala et al. (1992). Briefly, samples of 1 g of API powder were imidazole (C3H4N2), acetic acid, sodium azide (NaN3), sodium hydroxide (NaOH), and hydrochloric acid (HCl) were purchased mixed with 10 mL of water, magnetically stirred for 10 min at from Sigma Aldrich Chimie (Lyon, France). 250 rpm at room temperature, and the suspension pH was adjusted to 2, 3, 4, 5, 6, 7, 8, 9, and 10 by addition of 0.1 mol/L HCI or 0.1 mol/L NaOH. Then, the suspensions were magnetically 2.2. Extraction of A. platensis proteins stirred at 250 rpm for 30 min at room temperature and centrifuged at 8000g for 20 min. A 5 mL aliquot of the supernatant was used A. platensis protein isolate (API) was extracted from algal pow- to quantify the nitrogen content by the Kjeldahl method (conver- der by dissolution in imidazole/acetate buffer (0.005 mol/L, pH sion factor: 6.25). The solubilized nitrogen was calculated and 10). After centrifugation for 30 min at 25 °C and 10,000g, the expressed as a percentage of the total nitrogen. supernatant containing soluble proteins was collected (supernatant A). The pellet was dissolved again in the same buffer and centrifuged under the same conditions and the supernatant was 2.7. Water absorption capacity (WAC) collected (supernatant B). Both supernatants A and B were mixed and adjusted at pH 3 with 0.1 mol/L HCl. The precipitated proteins The WAC of API was determined using the method described by were then collected by centrifugation (10,000g, 30 min, 25 °C) MacConnell, Eastwood, and Mitchell (1974) slightly modified. One and dried under a laminar flow hood overnight at room tempera- hundred milligrams of API were added to 10 mL of imidazole/acet- ture. The protein isolate residual moisture content after drying ate buffer (0.005 mol/L) adjusted to pH 3, 7, or 10 in a 50 mL cen- was 6.39 ± 0.24 g/100 g of API. The other components were: trifuge tube and stirred overnight at 4 °C using a rotary shaker. proteins (69.62 ± 0.75 g/100 g of dry matter); ash (17.79 ± Then, the mixture was centrifuged at 1400g for 20 min and the 0.44 g/100 g of dry matter); carbohydrates (8.90 ± 0.88 g/100 g of supernatant was removed by tilting the tube gently to avoid losing dry matter), and fats (3.70 ± 0.35 g/100 g of dry matter). All these proteins. WAC was expressed as g of absorbed water per 100 g of analyses were determined according to AOAC International (1995). sample.

2.3. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) 2.8. Oil absorption capacity (OAC)

SDS–PAGE was performed by the method of Laemmli (1970), The OAC of API was measured using the method described by using a 12% polyacrylamide gel and a Bio-Rad apparatus (Mini- Lin, Humbert, and Sosulski (1974) slightly modiﬁed. Fifteen millili- Protean Tetra Cell). Proteins were stained with Coomassie Brilliant ters of corn oil were added to 0.5 g of API powder in a 50 mL cen- Blue R-250. The SDS–PAGE analysis of proteins was carried out in trifuge tube and then stirred at 100 rpm at room temperature the presence and absence of beta-mercaptoethanol as reducing during 30 min using a rotary shaker. The obtained mixture was agent. centrifuged at 1600g for 25 min. After centrifugation, a part of the oil was absorbed by proteins at the bottom of the tube, and 2.4. Fluorescence and UV spectra the other part remained free in the form of a clear layer at the top of the tube. This free oil (not absorbed by the protein isolate) The extracted API was suspended at a 1% (w/w) concentration was removed carefully by tilting the tube to get rid of the last oil in imidazole/acetate buffer (0.005 mol/L) adjusted to suitable pH drop. OAC was expressed as g of oil held per 100 g of sample. 1058 S. Benelhadj et al. / Food Chemistry 194 (2016) 1056–1063

2.9. Emulsifying properties 2.10. Foaming properties

2.9.1. Emulsifying capacity (EC) 2.10.1. Foaming capacity (FC) Emulsifying capacity (EC) was determined according to the The ability of API to form foams was determined using the method described by Yasumatsu, Sawada, Moritaka, Toda, and method described by Sciarini, Maldonado, Ribotta, Perez, and Ishii (1972) with some modifications. Five grams of API were Leon (2009). Solutions containing 1% API in imidazole/acetate buf- weighed, transferred into a beaker, mixed with 45 mL of imida- fer (0.005 mol/L) adjusted to pH values from 2 to 10 were whipped zole/acetate buffer (0.005 mol/L, pH 2–10), and magnetically stir- at 20,000 rpm for 2 min with a homogenizer (Ultra-Turrax T25, red at 250 rpm for 8 h at room temperature. The pH was then IKA, Staufen, Germany). Foam volumes were recorded after 10 s adjusted with 0.1 mol/L NaOH or 0.1 mol/L HCl and the buffer vol- and foam capacity was calculated according to Eq. (3). ume was completed to 50 mL. The protein suspension was then added to 50 mL of sunflower oil and the mixture was blended at Ifv FCð%Þ¼ 100 ð3Þ 22,000 rpm for 2 min with an Ultra-Turrax T25 blender (IKA, Stau- Tsv fen, Germany) to form an oil-in-water emulsion. A fixed emulsion volume (40 mL, total volume (Tv)) was poured into a 50 mL cen- where Ifv is the initial foam volume and Tsv is the total suspension trifuge tube and centrifuged at 1475g for 5 min. The volume of volume. the emulsified fraction (Ev) after centrifugation was recorded. EC was reported as (Eq. (1)):

ECð%Þ¼ðEv=TvÞ100 ð1Þ 2.10.2. Foam stability (FS) According to Sciarini et al. (2009), the foam stability of API solutions (imidazole/acetate buffer, 0.005 mol/L) at pH 3; 7; or 10 was 2.9.2. Emulsion ageing stability (EAS) expressed as the reduction of foam volume (%) as a function of Freshly prepared emulsions (10% oil; 0.5% API (w/w)) contain- time. ing 0.044 wt% NaN3 (as an antimicrobial agent) were transferred into a cylindrical glass test tube (internal diameter 15 mm, height 180 mm) until an emulsion height of 135 mm, tightly sealed with a 2.11. Film forming properties (FFP) plastic cap, and then stored at room temperature. The extent of creaming was characterized by a creaming index (CI) that repre- Protein films were prepared from 6% API solutions in imidazole/ sents the serum layer formed at the bottom of the tubes expressed acetate buffer (0.005 mol/L) adjusted to pH 10. This pH value was as a percentage of the total emulsion volume in the tube chosen because it corresponds to the maximum protein solubility. (McClements, 1999) (Eq. (2)). The solutions were stirred for 2 h with a magnetic stirrer until the proper hydration was reached and the pH was adjusted by adding CI ¼ðHs=HeÞ100 ð2Þ HCl (1 mol/L) or NaOH (1 mol/L). In relation to the dry matter, 10%, 15% or 30% glycerol or sorbitol amounts were added as plasticizers where Hs is the height of the serum layer and He is the total emul- and the mixtures were magnetically stirred at 250 rpm for 30 min. sion volume. Each film was prepared by weighing 25 g of film-forming solution The creaming index provided indirect information about the and this mass was spread evenly over a plastic Petri dish (90 mm). extent of droplet aggregation in an emulsion: the higher the Films were formed by drying overnight at 25 °C under laminar creaming index, the lower the emulsion stability. Measurements hood and dry films were peeled from the casting surface. were carried out on three separate samples and reported as the mean and standard deviation. 2.12. Least gelation concentration (LGC) 2.9.3. Emulsion microstructure Optical microscope Axiovert 25 CFL (Prolabo, France) was used The minimum API concentration that allowed gel formation to observe (with a 100 magnification objective) the oil droplets of was determined using the method described by Abbey and Ibeh oil-in-water emulsions stabilized by 0.5% API and containing 10% (1988). Arthrospira protein isolate samples were mixed with 5 mL oil. The microscope was connected to a camera (Nikon F90X). of distilled water in a centrifuge tube to obtain 2; 4; 6; 8; 10; 12; 14; 16; 18 or 20% (w/w) concentrations. The centrifuge tubes were heated for 1 h in a boiling water bath, cooled rapidly at 20 °C and 2.9.4. Emulsion color further cooled for 2 h in a refrigerator at 4 °C. The least gelation Emulsion (10% oil; 0.5% API (w/w)) surface color was measured concentration was regarded as the concentration at which the using a Minolta Chromameter CR-200. Absolute measurements are sample from the inverted tube did not fall or slip. displayed as Lab tristimulus values (L, a⁄, b⁄ color space). L (from black to white) is the lightness variable, a⁄ (from green to red) and b⁄ (from blue to yellow) are the chromatic coordinates. For each 2.13. Statistical analysis replicate, five measurements were performed at random positions by putting emulsions in a glass vessel to determine L, a⁄, and b⁄. Results were expressed as mean (±standard deviation) of tripli- cate analyses. They were statistically analyzed by a Duncan test 2.9.5. Emulsion heat stability (EHS) using SPSS (version 19) software to determine any significant dif- Emulsion stability to heating treatment was determined ferences between the average values obtained by the different according to Mahajan, Neetu, and Ahluwalia (2010). After prepara- pHs, at 95% confidence. The data obtained for the comparison of tion, emulsions (1% API; 10% oil; pH 3, 7 or 10) were heated at 80 °C color, emulsion thermal stability and WAC were analyzed statisti- for 30 min in a water bath. Then, heated emulsions were immedi- cally by Student’s t-test using Excel software (Microsoft corpora- ately cooled to 25 °C. The remaining emulsifying capacity after tion, USA) to establish significance of difference between the heating was then measured as indicated in Section 2.9.1. samples at the level of p < 0.05. S. Benelhadj et al. / Food Chemistry 194 (2016) 1056–1063 1059

3. Results and discussion A 900 pH 3 800 3.1. Structural properties 700

The distribution and molecular weights of proteins present in A. 600 platensis protein isolate (API) were analyzed by SDS–PAGE (Fig. 1). pH 2 500 In the presence of b-mercaptoethanol (a reducing agent), as well as 400 sodium dodecyl sulfate (SDS) (a surfactant), protein complexes pH 4 were separated into subunits. Under these conditions, the SDS– 300 pH 5

PAGE electrophoresis gel of the API features several bands with (AU) Emission intensity pH 6 200 corresponding molecular weights in the range 10–50 kDa (10; pH 7 100 pH 8 13; 15; 30; and 45 kDa). The main bands may correspond pH 9 to biliproteins: C-phycocyanin (a subunit: 19,500 Da and b subunit 0 pH 10 21,500 Da) and allophycocyanin (19,600 Da and 17,700 Da for a 300 350 400 450 500 and b subunits, respectively) (Moreno et al., 1997). Wavelength (nm) The fluorescence emission spectra of API soluble fraction 1% (w/ B w) after being excited at 280 nm have a peak signal at 340 nm 2.4 whatever the pH (Fig. 2A). This mainly arises from the tryptophan moiety fluorescence which dominates over the fluorescence of other residues such as tyrosine and phenylalanine in this emission 1.8 region (Palmer, Keely, Breslin, & Ramanujam, 2003). For pH 2, 3 and 4, a second peak with an emission wavelength of 450 nm is 1.2 also present in the fluorescence emission spectra: it could be assigned to Nicotinamide Adenine Dinucleotide Phosphate (NADPH), a primary product of photosynthesis (Dartnell et al., 0.6 Absorbance (AU) 2011). The blue color of these three solutions can be attributed to the presence of phycocyanin which has been reported to be 0.0 + bound to ferredoxin: NADP oxidoreductase in A. platensis. Indeed, 200 220 240 260 280 300 320 340 + in photosynthetic organisms such as A. platensis, ferredoxin: NADP Wavelength (nm) oxidoreductase is known to provide NADPH for CO2 assimilation (Korn, Ajlani, Lagoutte, Gall, & Sétif, 2009). It was recently observed Fig. 2. Effect of pH on the fluorescence emission intensity (300–500 nm) of A. that purified C-phycocyanin was slightly more stable under acidic platensis protein isolate (API) (1% (w/w)) excited at 280 nm at 20 °C (A) and UV ° rather than under neutral or alkaline conditions (Martelli, absorption spectrum of API (0.05% (w/w); pH 10; 20 C) (B). Folli, Visai, Daglia, & Ferrari, 2014). Then, pH increase could be responsible for a molecular change, which could explain the fluo- The UV absorption spectra of API exhibited two characteristic rescence properties modifications generating a color change. protein peaks (Fig. 2B). The first peak was located in the far UV region (228 nm) and reflected the amino acid side chains (particularly Tyr, Trp, Phe, His, and Met) that absorb light strongly in this region of the spectrum (below 250 nm) but the most important contributor is the peptide bond (amide chromophore) (Wu, Wang, & Xu, 2007). The weak absorption peak in the near UV region (240–300 nm) is generally attributed to the aromatic amino acids residues (Trp, Tyr, and Phe) and the disulfide linkage that constitute the chromophores that absorb in this region. As shown in Fig. 2B, the maximal absorption wavelength was at 228 nm, not at 280 nm and this indicated that peptide bonds were the major absorbing group in the ultraviolet region (Wu et al., 2007). Electrical mobility measurements could be used to detect com- positional variations and structural changes in food macromolecules as well as their functional properties. Many factors, such as chemical composition, concentration of charged com- pounds, pH and temperature can have a significant effect on zeta potential values (McClements, 1999). In order to quantify the electrostatic interactions between protein molecules, zeta potential values of API suspensions were measured at different pHs (Fig. 3). The f-potential of the protein particles was slightly positive at pH 2 (+2.4 mV), became less positive with increasing pH until it reached zero (around pH 3), then became increasingly negative as the pH was further increased, until it reached a value of 35.6 mV at pH 10. The decrease in electrostatic repulsions at the pI is a property that was used in this work to extract API by isoelectric precipitation. According to these results, protein solubility could increase at pH values above the pI (and to a lesser extent below the pI, since it is acidic) due to the increase in electrostatic repul- Fig. 1. SDS–PAGE analysis of A. platensis protein isolate (API). From the left, samples deposited in each lane were as follows: 10–200 kDa molecular weight marker (Bio sions and ionic hydration forces between the protein molecules Basic Inc) (Lane 1); API (Lane 2); and API with beta-mercaptoethanol (Lane 3). (Zayas, 1997). 1060 S. Benelhadj et al. / Food Chemistry 194 (2016) 1056–1063

10 Zeta potenal Nitrogen solubility 70 of pH on the emulsion capacity of API is shown in Fig. 4A. The EC evolution as a function of pH showed that API had a minimum 0 60 emulsifying capacity (44.1% ± 0.9) in a pH region close to its iso- 50 electric point (pH 3) which also corresponds to its minimal solubil- -10 ity (Fig. 3). Higher or lower pH values resulted in an increase of API 40 emulsifying capacity: the emulsion capacities at pH 2 and 4 were -20 30 52.1 ± 0.4% and 46.0 ± 0.8%, respectively. In fact, since the lowest -30 protein solubility occurred at the isoelectric point, proteins could

Zeta potenal(mV) Zeta 20 not move rapidly to the oil/water interfaces, and the protein net -40 10 charge could be minimized at this pH. This surface charge is related

Nitrogen solubility(% Nitrogen (w/w)) to the balance between the dissociation of the carboxylic and -50 0 amino groups of protein molecules. The low electric charge inten- 1234567891011 sity at the oil/water interfaces decreases the repulsion intensity pH between the droplets and therefore the association of many droplets to form aggregates is increased. In addition to electric Fig. 3. Effect of pH on the zeta potential and nitrogen solubility of A. platensis charges, the dependence of emulsion capacity on pH can be also protein isolate (API) at 20 °C. explained by the fact that protein surfactant properties depend 3.2. Nitrogen solubility, water and oil absorption capacities on the hydrophilic–lipophilic balance, which can be affected by pH. The behavior of API emulsion capacity was well correlated with Amongst the functional properties of proteins, solubility is crit- the variation, as a function of pH, of zeta-potential of the proteins ical since many functional performances of proteins depend on (Fig. 3). their ability to hydrate and solubilize in water. The nitrogen solu- The ageing stability of emulsions stabilized by API at different bility at different pHs of A. platensis protein isolate is shown in pHs is shown in Fig. 4B. At pH 3, emulsion creaming began to occur Fig. 3. A. platensis proteins presented an U-shaped solubility curve immediately after their preparation with a complete phase separa- with a minimum solubility (N 6.25 = 6.2% (w/w)) at pH 3, which tion being observed after 10 min. Creaming is the upward move- corresponds to the protein isoelectric point, as previously observed ment of droplets due to the fact that their density is lower than when measuring the f-potential values (Fig. 3). The maximum sol- that of the surrounding liquid (McClements, 1999). This destabi- ubility (N 6.25 = 59.6% (w/w)) was at pH 10, i.e. at the highest pH lization could certainly be due to the low solubility of proteins at value tested which is also the pH value with the highest difference their isoelectric pH, their poor hydration, and consequently the with A. platensis proteins pI. These maximum and minimum solu- lack of electrostatic repulsive forces. Ageing stability of emulsions bility values were exploited in the A. platensis proteins extraction at pH 7 and pH 10 were similar and these emulsions were rela- step, for their solubilization and precipitation, respectively. tively stable to creaming and droplet aggregation, presumably Chronakis (2001) showed that the viscosity of A. platensis proteins because the electrostatic repulsion between the droplets was suf- was dependent on pH. At high pH (>8), the viscosity of the protein ficiently strong to prevent droplet aggregation for these two pHs. suspensions decreased due to increased solubility. The pH effect on emulsion stability is usually attributed to ioniza- The functional properties of proteins depend on water solubility tion of polar groups of surface active molecules which induce suf- but also on water retention capacity that is an important parame- ficient electrostatic repulsive interactions to break down the ter to be considered in the formulation of many foods, such as meat interfacial film cohesion (McClements, 2004). This low difference products in which proteins play an important role for their texture. between the stability at pH 7 and that at pH 10 may be explained The water absorption capacity (WAC) of API was measured at three based on the zeta potential measurement results (Fig. 3). Indeed, different pHs (3, 7, and 10). The highest WAC (428.8 ± 15.4 g of according to this figure, it can be seen that as the pH increases, water/100 g of API) was obtained at pH 10. However, there was the zeta potential decreases progressively until it reaches a plateau no significant difference between WAC at pH 7 and pH 3 from pH 7. Thus, it can be assumed that the intensity of the electric (p < 0.05): the measured values were 334.9 ± 19.9 and charges at the droplets surfaces for both pHs is almost the same. 325.2 ± 7.1 g of water/100 g of API, respectively. In fact, pH changes This is manifested by a similarity of ageing stability profiles. can affect the conformation of proteins resulting in exposure or In order to better elucidate the effect of pH on the API emulsi- burial of the water binding sites. Consequently, with an increased fying properties, microscopic and macroscopic aspects of emul- polarity and electric charge of proteins, mainly due to the ioniza- sions are presented in Fig. 4C. Acidic emulsions were composed tion of amino acid groups, the amount of bound water increased. of polydisperse droplets with a large diameter ranging between The obtained results showed that API had a lower WAC than that 23 and 73 lm as estimated from the microscopic observations. of commercial soy protein isolates Supro500EÒ (856 g of Smaller droplets with diameters ranging between 3 and 23 lm water/100 g) (Liadakis, Tzia, Oreopoulou, & Thomopoulos, 1998). and a homogeneous distribution were formed at pH 7 and pH 10. The oil absorption capacity (OAC) of API was 252.7 ± 9.9 g of Direct naked-eye observations of the different emulsions revealed oil/100 g of protein isolate. This interesting OAC could result from that their color characteristics also varied with pH (Fig. 4C). the presence of non-polar residues side chains of proteins. The API Thus, differences between emulsions colors at different pHs showed a higher OAC than soy protein isolates (119–154%) were evaluated by measuring the L, a⁄ and b⁄ parameter values (Kinsella, 1979). According to Kinsella (1979), the ability of pro- (Table 1). On one hand, the emulsion at acidic pH (the two phases teins to bind fat is very important for applications such as meat were slightly mixed before measurements) had the lowest light- replacement or meat extenders, mainly because it enhances flavor ness (L) and the intense color of acidic emulsion was consistent retention and improves mouth feel. with the highest value of a⁄. On the other hand, for each of these two parameters, there was no significant difference between 3.3. Emulsifying properties emulsion at neutral and alkaline pH (p < 0.05). For the three studied pHs, the positive values of b⁄ could be attributed to the color of The emulsifying capacity (EC) reflects the ability of a sample to oil, and the highest value of b⁄ was obtained at pH 7. rapidly adsorb at the oil/water interfaces during the formation of In addition to pH, other environmental conditions such as the an emulsion by preventing flocculation and coalescence. The effect presence of some molecules, ionic strength, as well as heat treat- S. Benelhadj et al. / Food Chemistry 194 (2016) 1056–1063 1061

AB70 70 pH 3 65 60 60 50 pH 7 55 40 30 50 pH 10

Creaming index (%) Creaming index 20

Emulsifying capacity (%) capacity Emulsifying 45 10 40 0 1234567891011 0100200 pH Time (min)

Fig. 4. Effect of pH on the emulsifying capacity of A. platensis protein isolate (API) (A), ageing stability (B), and visual appearance and microscopic observations (C) of 10% (w/w) oil-in-water emulsion stabilized by 0.5% (w/w) A. platensis protein isolate at 20 °C.

Table 1 erties modiﬁcations of proteins under neutral and alkaline condi- Lab chromatic coordinates of 10% (w/w) oil-in-water emulsion stabilized by 0.5% tions which may play a role in the aggregation of the droplets (w/w) A. platensis protein isolate (API) at different pHs. Each value is expressed as ⁄ ⁄ upon heating. mean ± SD (n = 3). For each parameter (a , b , L), the values followed by identical letters are signiﬁcantly similar (p > 0.05). 3.4. Foaming properties pH 3 pH 7 pH 10

a b b L 47.82 ± 0.15 62.77 ± 0.26 64.94 ± 0.10 The foaming capacity (FC) of a protein refers to the amount of a⁄ 1.98 ± 0.15a 1.43 ± 0.18b 1.56 ± 0.18b b⁄ 22.27 ± 0.29a 27.67 ± 0.13b 21.53 ± 0.30a interfacial area that can be created by the protein during foaming, while foam stability (FS) refers to the ability of protein to stabilize air bubbles against gravitational stress. The obtained results revealed that the foaming properties of the studied algal proteins ments can modify the structure and the properties of proteins. For depended on pH (Fig. 5A and B). API showed minimum FC at pH the effect of heat on the emulsion capacity (Table 2), there was no 3 whereas, the maximum value was obtained at pH 10 (Fig. 5A). significant difference between neutral and alkaline pHs (p < 0.05). The high FC at pH 10 is likely due to the increased net charges of This might indicate that the thermal aggregation of the small sized the proteins, which weakened the hydrophobic interactions but oil droplets was slower than that of the large sized ones obtained increased protein flexibility. This likely allowed the protein to dif- at the isoelectric point (pH 3). The higher heat stability observed fuse more rapidly to the air–water interface to encapsulate air bub- at neutral pH may be mainly due to the ability of proteins to gen- bles and to enhance the foam formation (Aluko & Yada, 1995). erate repulsive interactions (e.g., steric and electrostatic) between Indeed, a strong positive correlation (R2 = 0.97) was found between the oil droplets and to form an interfacial membrane that is resis- solubility and FC. A cyanophyceae powder was reported to have a tant to rupture and that plays an important role in stabilizing the very high foaming capacity, especially if the sample was defatted droplets against flocculation and coalescence (McClements, (Nirmala et al., 1992) and even after chemical treatment 2004). This might also result from the fact that the thermal stabil- (Mahajan et al., 2010). Devi and Venkataraman (1984) indicated ity of adsorbed proteins could depend on pH. In fact, thermal treat- that a protein concentrate of A. platensis (27% proteins) exhibited ment of protein stabilized emulsions could induce droplets foaming properties comparable to soybean meal. flocculation because the denatured adsorbed proteins held the dro- The effects of pH on the FS of API at different pHs are shown in plets together. Indeed, when emulsions were subjected to heat Fig. 5B. The foam volume recorded after 30 min at pH 10 treatment, the droplets held together and the flocculation could (56.5 ± 0.7%) was higher than that at pH 7 (35.5 ± 0.7%) and at pH occur because the modification of the balances between polar 3 (2.0 ± 0.3%). An improvement in FS of the proteins at alkaline and non-polar residues. There is a statistical difference between pH could likely be due to increased solubility and surface activity the heat stability of emulsions at pH 7 and pH 10. This difference of the soluble proteins. Regardless of the pH of the solution, FS of could be explained by the effect of the structural and surface prop- the API significantly decreased with time. This low foam stability could be attributed to the fact that API was not able to form cohe- Table 2 sive interfacial air/water films with satisfactory mechanical Heat stability of 10% (w/w) oil-in-water emulsion stabilized by 0.5% (w/w) A. platensis properties. protein isolate (API) at different pHs. Each value is expressed as mean ± SD (n = 3). The values followed by identical letters are significantly similar (p > 0.05). 3.5. Film forming properties Emulsifying capacity pH 3 8.13 ± 0.62b Currently, there is an increasing interest accorded to edible pH 7 13.24 ± 0.12a films made from renewable and natural polymers such as protein, a pH 10 11.04 ± 1.12 polysaccharides, and lipids. Among them, protein-based edible 1062 S. Benelhadj et al. / Food Chemistry 194 (2016) 1056–1063

A 300 B 300

250 250 pH 10

200 200 pH 7 150 150 pH 3 100 100 Foaming capacity (%) capacity Foaming Foam volume (%) volume Foam 50 50

0 0 1234567891011 0204060 pH Time (min)

Fig. 5. Foaming capacity of A. platensis protein isolate (API) (A) and stability of A. platensis protein isolate (API) stabilized foams (B) at different pHs at 20 °C.

0% 10% 15% 30%

Glycerol

Sorbitol

Fig. 6. Effect of the plasticizer type and concentration on the A. platensis protein isolate (API) based ﬁlms obtained at pH 10.

films are the most attractive because of the good film-forming structure against dissociation. Furthermore, strong hydrophobic properties of these macromolecules. The objective here was to interactions, as well as hydrogen bonding and intermolecular investigate the effect of the plasticizer type and concentration on disulfide bonds, were found to impact the thermal gelation behav- the API film-forming properties at pH 10. In the absence of plasti- ior of algal proteins (Chronakis, 2001). cizer, as shown in Fig. 6, the films obtained with API were fragile and crumbly and, therefore, difficult to use for any application. Glycerol was the first plasticizer tested. A breakable film was 4. Conclusion obtained by adding 10% (w/w) of glycerol to the API. An increase in the amount of glycerol made a film which adhered to the Petri The search for new sources of functional molecules remains an dish. The film formulations containing sorbitol gave films easy to important goal in the food industry. To cope with the high cost of peel and the use of 30% (w/w) of sorbitol allowed to obtain the best animal proteins for a long time, researchers and industry have used film structure (Fig. 6). At the present stage of this study, it is very plant proteins such as soy or wheat. However, proteins extracted difficult to speculate on the difference between the plasticizing from microalgae are a good alternative combining both a remark- effects of the two used polyols. However, we can assume that the able nutritional value, a good availability, but also, as it was obtained results are related to the interactions and compatibility demonstrated in this study, good functional properties. Therefore, between polyols and API. Further studies at the molecular level A. platensis protein isolates could in future be used as ingredients should now be performed to explain these results. These film form- for the formulation of food products in the form of emulsions, ing characteristics will be studied in a forthcoming study to design foams or gels if their functional properties are characterized active edible coatings capable of releasing natural antimicrobial according to various physicochemical conditions. In addition, the agents to increase perishable foods shelf life. ability of these proteins to form films could allow development of edible coatings with new features.

3.6. Gelling properties References The minimal gelation concentration of API was 12% (w/w) in distilled water. In another study, Chronakis (2001) had obtained Abbey, B. W., & Ibeh, G. O. (1988). Functional properties of raw and heat processed a gel with a lower concentration of microalgae proteins, it was cowpea ﬂour. Journal of Food Science, 53, 1775–1791. Aluko, R., & Yada, R. Y. (1995). Structure–function relationships of cowpea (Vigna 2.5% (w/w), but in the presence of calcium dichloride (CaCl2) unguiculata) globulin isolate: inﬂuence of pH and NaCl on physicochemical and because calcium ions have a stabilizing effect on the quaternary functional properties. Food Chemistry, 53, 259–265. S. Benelhadj et al. / Food Chemistry 194 (2016) 1056–1063 1063

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