Spirulina platensis proteins: Functional and physicochemical properties, angiotensin-I converting enzyme inhibitory activity, bioavailability and encapsulation studies

Spirulina platensis is symbiotic, multicellular, and filamentous blue-green microalgae. It is a rich source of some high-value bioactive molecules containing essential amino acids, pigments like β-carotene and phycobiliproteins and polyunsaturated fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Moreover, it is one of the important protein sources with high protein content (60-70% in dry weight). The objectives of this Ph.D. thesis were (i) to optimize ultrasound assisted extraction of protein from Spirulina platensis by using Response Surface Methodology (RSM), to obtain the protein extract with high total phenolic content and antioxidant activity, and to evaluate protein digestibility after in vitro gastrointestinal digestion; (ii) to optimize extraction conditions for extraction of protein from Spirulina platensis by RSM, to obtain protein concentrates with improved functional properties, and to identify the functional groups, to determine of thermal properties and to examine the microstructure of protein concentrate isolated at optimum point; (iii) to evaluate the changes in angiotensin-I converting enzyme (ACE) inhibitory and antioxidant activities of Spirulina platensis tryptic protein hydrolysates obtained from enzymatic hydrolysis during a simulated gastrointestinal digestion; (iv) to determine the influence of pH and protein/chitosan ratio on the complex coacervation between Spirulina platensis protein concentrate and chitosan. To achieve these goals, four different experiments (Chapters 2-5) were conducted. In the first part of the thesis (Chapter 1); research framework and objectives of this Ph.D, and a review on Spirulina platensis and protein/peptides derived from it and coacervation as an encapsulation technique are presented. In the second part of the thesis (Chapter 2); chemical composition and fatty acid and sugar profiles of Spirulina platensis powder (SPP) was determined. Later, the protein extraction of Spirulina platensis by ultrasound assisted extraction (UAE) was optimized for obtaining product with high extraction yield, total phenolic content (TPC), antioxidant activity (AOA), and in vitro protein digestibility by using response surface methodology (RSM). A three factors Box-Behnken design (BBD) of experiments were performed at three levels of pH (7, 8, 9), temperature (25, 35, 45 °C) and time (60, 90, 120 min). The analysis of composition of SPP showed that it contained 65.6±0.12% protein, 14.2±0.62% lipid, 10.7% carbohydrate and 9.5±0.02% ash in dry weight. According to the result of gas chromatography (GC) analysis; the fatty acids of SPP were palmitic acid (56%) and linoleic acid (19.63%), linolenic acid (17.07%), oleic acid (2.98%), palmitoleic acid (2.78%) and stearic acid (1.49%). The sugar profile of SPP, which was determined by ultra performance liquid chromatography (UPLC), consisted of 4 sugars: rhamnose (56%), glucose (22%), galactose (8%), mannose (5%) and unknown sugar (9%). Analysis of variance (ANOVA) indicated that the models showed coefficients of determination (R2) values were 0.91, 0.84, 0.70, 0.46 and 0.72 for extraction yield, TPC, AOA by cupric reducing antioxidant capacity (CUPRAC) and antioxidant activity by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay and in vitro protein digestibility, respectively. According to particularly high R2 for the models (>0.80), except antioxidant activity by CUPRAC and DPPH (0.70, 0.46) and in vitro protein digestibility (0.72), the other polynomial models were suitable to represent the real relationships between variables. Based on the RSM analysis, optimum extraction conditions (temperature: 45 °C, pH: 8.55 and time: 60 min,) were obtained for extraction yield (28.45%), TPC (4.84 mg caffeic acid equivalent/g dry weight), antioxidant activity (12.23 mg Trolox equivalent/g dry weight) and in vitro protein digestibility (90.09%). To determine the optimum process conditions, the experiment was carried out at the optimal conditions generated from the model. The experimental scores were satisfactorily close to the values predicted by the model. In the third part of the thesis (Chapter 3), the optimization of ultrasound assisted extraction of protein from Spirulina platensis to obtain product with improved functional properties by using RSM and determination of physicochemical and thermal properties and characterization of microstructure of the protein concentrate obtained at optimum point were carried out. BBD of experiments was performed at levels of pH (7, 8, 9), temperature (25, 35, 45 °C) and time (60, 90, 120 min). Water absorption capacity (WAC), oil absorption capacity (OAC), foaming capacity (FC), foaming stability (FS), emulsifying activity (EA) and emulsifying stability (ES) were investigated. Additionally, identification of functional groups, determination of thermal properties and examination of microstructure of protein concentrate isolated at optimum point were carried out. Analysis of variance of the fitted second-order polynomial model and R2 for each dependent variable were obtained. The R2 values were 0.87, 0.95, 0.57, 0.37, 0.49 and 0.79 for WAC, OAC, EA%, ES%, FC% and FS%, respectively. According to particularly high R2 for the models (>0.80), except EA % (0.57), ES % (0.37) and FC % (0.49), applied polynomial models were in accordance with experimental results. Based on the RSM analysis, at the optimum extraction conditions (time 60 min, temperature 43.87 °C and pH 7.16) the highest values for WAC (4.41g of water/g of protein), OAC (10.13g of oil/g of protein), FC (300%), FS (83.94%), EA (42.99%) and ES (92.50%) were obtained. In this study, functional properties of SPPC were found higher than other algae and plant proteins including soybean protein isolate as a commercial protein. To determine the optimum process conditions, the experiment was carried out at the optimal conditions generated from the model. The experimental scores were satisfactorily close to the values predicted by the model. Studies characterization of the SPPC obtained at the optimum point were performed. The nitrogen solubility and zeta potential (ζ-potential) of SPPC at a pH ranged from 2 to 10 were determined. The isoelectric point of SPPC was at pH ~3 where the minimum solubility (N×6.25= 11.38 % (w/w)) and nearly neutral charge (0.250 ± 0.29 mV) was observed. The maximum solubility (N×6.25= 59.06 % (w/w)) found at pH 10 among the pH range studied. At all the pH values above pH 3.0, the SPPC solutions remained negatively charged. The fourier transform infrared (FT-IR) spectra was used to determine the major functional groups in the structure illustrated interaction between amide II, amide Ι stretching (N-H) and carboxly group (C=O) for complex appeared at 1533 cm-1, 1631 cm-1 and 2920 cm-1 respectively. According to differential scanning calorimetry (DSC) thermography of SPPC, the peaks of denaturation temperatures were 159.45 and 165.72 °C and denaturation enthalpies of free SPPC were -3983 W/g and -3702 W/g. In the fourth part of the thesis (Chapter 4), the effects of hydrolysis time and enzyme to substrate ratio on the degree of hydrolysis (DH), antioxidant activity and angiotensin-I converting enzyme (ACE) inhibitory activity were determined in tryptic Spirulina platensis protein hydrolysates (SPPH). Moreover, an in vitro gastrointestinal digestion model was used to simulate the human gastrointestinal (GI) digestion system in order to investigate phenolic contents, antioxidant and ACE inhibitory activities of the digests. The initial DH of SPPHs varied between 18.57% and 25.03%. Obvious changes in initial DH were observed depending on the hydrolysis time and E/S ratio (p < 0.05). After pepsin digestion, the DH of the digests ranged from 21.39±0.62 to 29.22±1.48 (p < 0.05). On the other hand, the DH of pancreatic digests became 30.73±3.41-43.12±0.18 after intestinal digestion (p < 0.05). TPC ranged from 23.97±2.56 to 41.11±2.71 mg CAE/g dw was observed for the tryptic protein hydrolysates. The effects of hydrolysis time and enzyme to substrate ratio were found significant on TPC (p < 0.05). The cupric reducing antioxidant capacity of the tryptic SPPHs were ranged between 6.33±1.54 and 13.71±0.15 mg TE/g dw. The antioxidant activity of further digested SPPHs by gastric and intestinal proteases remained generally stable after in vitro treatment. The increase of E/S ratio and hydrolysis time resulted in an increase in the DH and in an improved ACE inhibitory activity of both initial and the GI digested samples (p < 0.05). The effect of stomach digestion on the ACE inhibitory activity of SPPHs was insignificant as a general trend (p > 0.05). However, further digestion in the intestine brought the ACE inhibitory activity of the final GI digests to 24.85±1.01-33.60±2.40 (p < 0.05), indicating that some peptides with stronger ACE inhibitory activity were produced during the simulated GI digestion. Finally, in the fifth part of the thesis (Chapter 5), the effects of pH (2.0-10.0) and protein:chitosan mass ratio (1:1, 2.5:1, 5:1, 7.5:1, 10:1, 15:1, 20:1, 30:1) were determined on the complex coacervation between SPPC and chitosan. Identification of functional groups and characterization of thermal properties of optimized coacervates were also investigated. According to ζ-potential results of individual SPPC and chitosan, the complex coacervation was formed at pH 6.0 for optimization of SPPC-chitosan mass ratio (1:1, 2.5:1, 5:1, 7.5:1, 10:1, 15:1, 20:1, 30:1). The coaservates prepared at ratio of 7.5:1 were nearly stoichiometric equivalence. Additionally, the turbidity of the complex coacervation, prepared at the SPPC-to-chitosan ratio of 7.5:1 and pH 6.0, was determined as the highest value (100-T% = 79.00±0.19, p < 0.05). Later, for further pH optimization, the coacervate mixtures were preparared with 7.5:1 ratio at pHs ranged from 2.5 to 9.5. The coacervate solution obtained at 7.5:1 ratio and pH 5.5, exhibited nearly neutral charge (0.79±0.41 mV) and the highest turbidity value (100-T% = 76.80±0.76, p < 0.05). Furthermore, the pH dependence of the yield of complex coacervate at the optimum ratio (7.5:1) was investigated. According to the results, the highest yield was obtained at pH 5.5 (77.74±0.32%). According to FT-IR spectrum of the coacervate, it had peaks of a combination of the peaks of SPPC and chitosan. The SPPC-chitosan coacervate displayed 1565 cm-1 and 1374 cm-1 attributed to –COO- and -NH3+ groups stretching vibration, indicating the electrostatic interaction between the carboxyl groups of SPPC (-COO-) and amine groups of chitosan (-NH3+). According to the DSC thermogram of SPPC-chitosan coacervate, its the peak denaturation temperature and denaturation enthalpy were 209.5 °C and -3.414 W/g, respectively.