Food Chemistry 194 (2016) 1056–1063 Contents lists available at ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem 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 modifi- cation 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 pur- chased 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 8000Âg 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 10,000Âg, the expressed as a percentage of the total nitrogen. supernatant containing soluble proteins was collected (super- natant 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 (10,000Âg, 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 1400Âg 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).