Proteins and their Enzymatic Hydrolysates from the Marine Diatom Nitzschia laevis and Screening for their in vitro Antioxidant, Antihypertension, Anti-Inflammatory and Antimicrobial Activities

Abstract

High-value products from microalgae, which can be used in various applications (nutritional, pharmaceuticals and cosmetic) can considerably increase the commercial value of microalgal biomass. This research focuses on the following: - Green extraction of proteins from the marine diatom Nitzschia laevis (N. laevis) and comparing them with those obtained from well-studied species such as Spirulina (Arthrospira) platensis (S. platensis) and Chlorella vulgaris (C.vulgaris). - Protein extracts obtained were subjected to three different protease enzymes including Trypsin, Alcalase®CLEA and Flavourzyme® to produce “bioactive hydrolysates”. - Screening of anti-oxidative, anti-hypertension, anti-inflammatory and antimicrobial activities in vitro. - Fractionation and characterization of bioactive proteins and hydrolysates produced from the microalgal extracts using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Soluble proteins and other major constituents (lipids and carbohydrates) from N. laevis were extracted using ultrasound, then compared to other well-investigated microalgae (Spirulina and Chlorella). N. laevis cellular lysates contained 21.09±2.00% protein, whereas Spirulina contained 54.20±10.73%, and Chlorella 14.01±9.51% (w/w). The aqueous protein extracts of Nitzschia contained 11832.5±3.53 μmol/L α-amino groups and were of high nutritional valuable due to the presence of most of the essential amino acids. In addition, Nitzschia’s proteins exhibited high thermal stability (84.31℃) after Chlorella proteins (87.49℃) at neutral pH. Protein hydrolysates from these microalgae were produced using Alcalase®CLEA™ from Bacillus subtilis, Flavourzyme® from Aspergillus oryzae, and trypsin from bovine pancreas, independently. During the first assessment of protein extract and their enzymatic hydrolysates, it was noticed that the antioxidant activities were enhanced by the hydrolysis process in most cases, , especially those obtained using the commercial enzymes Alcalase®CLEA™ and Flavourzyme® at 1% enzyme to substrate ratio. N. laevis showed the highest (in a fixed concentration of 2 mg mL-1) total phenolic content/reducing capacity (2.40±0.02 mg Gallic Acid Equevalent (GAE) 100g-1) after 90 minutes of hydrolysis with Alcalase®CLEA™, followed by Spirulina after 90 minutes of hydrolysis and Chlorella after 120 minutes of hydrolysis (2.30±0.04 and 1.90±0.05 mg GAE 100g-1, respectively). In the second assessment, the starting concentration of protein extracts and hydrolysates were increased to 4 mg mL-1, then 2-fold diluted in all assays for IC50 studies. Only the most antioxiadtive hydrolysate were used in the second assessment. They include Alcalase®CLEA™ hydrolysates at 120, 90, and 120 minutes from N. laevis, S. platensis and C. vulgaris, respectively; Flavourzyme® hydrolysates at 30, 60, 120 minutes from N. laevis, S. platensis and C. vulgaris, respectively; and Trypsin hydrolysates at 90 minutes from N. laevis, 120 minutes from S. platensis and C. vulgaris. Antioxidant activities, including DPPH, ABTS, and superoxide anion scavenging potentials, Oxygen radical absorbance capacity (ORAC), and xanthine oxidase inhibition were tested on aqueous protein extracts and the selected hydrolysates from each enzyme. All protein extracts and hydrolysates showed dose-dependant scavenging activities of ABTS and DPPH stable radicals. Flavourzyme® hydrolysates of N. laevis and S. platensis produced the highest ABTS scavenging activities (84.37±2.09% and 80.77±3.08%, respectively), with IC50 0.85±0.00 and 0.92±0.03 mg mL-1, respectively. These were followed by Alcalase®CLEA™ and trypsin hydrolysates, although the differences weren’t singinficant (p> 0.05). The protein extracts alone did not show significant ABTS scavenging acitvities. DPPH scavenging activities observed where mild, and Flavourzyme® hydrolysates from all species produced the most DPPH radical scavenging activities, although the differences were not significant between data sets. The highest DPPH scavenging activity was found in N. laevis hydrolysates obtained by Flavourzyme® (39.23±0.84%), followed by S. platensis (31.23±2.00%), with IC50 2.12±0.01 and 1.71±0.04 mg mL-1, respectively. ORAC values were measured for all samples and N. laevis protein extracts exhibited the highest GAE and TE (trolox equevalent) compared to S. platensis and C. vulgaris (4.06±2.89, 3.56±1.24 and 2.97±1.31 mg GAE g-1 sample, and 1989.17±579.3, 1806.17±249.7 and 1590.17± 263.9 μM TE g-1 sample, respectively) (p< 0.05). The calculated IC50 for protein extracts from N. laevis, S. platensis and C. vulgairs were 1.36±0.17, 5.96±2.49 and >6 μM TE respectively. The IC50 for N. laevis, S. platensis and C. vulgairs Alcalase®CLEA™ hydrolysates were 0.67±0.05 , 0.13±0.05 and 1.03±0.04 μM TE respectively. The IC50 of Flavourzyme® hydrolysates of N. laevis, S. platensis and C. vulgairs were 0.34±0.05 , 1.38±0.08 and 1.66±0.06 μM TE and using trypsin hydrolysates, the IC50 were 0.67±0.05 , 7.17 and 2.89±0.06 μM TE, respectively. Superoxide anion radical scavenging activity of trypsin hydrolysates from N. laevis were the highest achieved (118.45±0.00% and IC50 was 0.24±0.00 mg mL-1), followed by Alcalase®CLEA™ (112.62±0.01% and IC50 was 0.70±0.00 mg mL-1) and Flavourzyme® (93.84±0.00% and IC50 was 0.30±0.00 mg mL-1). S. platensis proteins and hydrolysates in this study did not show significant superoxide radical scavenging activities, and the highest scavenging activity achieved was with trypsin hydrolysates (58.99±0.06% and IC50 was 1.21±0.00 mg mL-1). C. vulgaris protein extracts showed better superoxide scavenging activity than hydrolysed samples (115.44±0.00% and IC50 was 0.24±0.00 mg mL-1), followed by trypsin hydrolysates (90.61±0.02% and IC50 was 0.30±0.00 mg mL- 1), Alcalase®CLEA™ (88.68±0.01% and IC50 was 0.37±0.00 mg mL-1) and lastly Flavourzyme® hydrolysates (76.70±0.01% and IC50 was 0.24±0.00 mg mL-1).The highest xanthine oxidase inhibition activity was achieved by Flavourzyme® hydrolysates of N. laevis at 156.14±7.58% and IC50 was 2.12±0.00 mg mL-1. S. platensis protein extracts showed the highest xanthine oxidase inhibitory activity (103.48±6.90% and IC50 was 2.07±0.03 mg mL-1), C. vulgaris protein extracts also showed better xanthine oxidase inhibition activity compared to the hydrolysed samples (85.35±7.32%, IC50 was 2.01±0.06 mg mL-1) (p> 0.05). In addition, protein extracts and hydrolysates showed antagonist effect toward acetylcholinesterase (AChE) and angiotensin-I converting (ACE) enzymes. The highest inhibitory activity of AChE was of obtained for Spirulina proteins (46.30±0.03 %, IC50 2.05±0.03 mg mL-1) followed by Nitzschia (32.32±0.12 %, IC50 1.09±0.11 mg mL-1) and Chlorella (20.98±0.02 %, IC50 2.97±0.01 mg mL-1). Flavourzyme® hydrolysates showed better AChE inhibitory activity, especially those of N. laevis (52.64±0.61%, IC50 1.59±0.00 mg mL-1). ACE was highly inhibited by Nitzschia proteins (138.08±0.01 %, IC50 0.40±0.13 mg mL-1) followed by Chlorella (90.15±1.32 %, IC50 1.55±2.00 mg mL-1) and Spirulina (83.79±0.11 %, IC50 0.27±0.04 mg mL-1). Trypsin hydrolysates of protein extracts from all three phyla showed better ACE inhibitory activities compared to other enzyme hydrolysates (88.03±7.47 % and IC50 1.63±0.01, 95.17±12.82 and IC50 0.68±0.06, and 61.36±23.42 and IC50 2.63±0.01 mg mL-1 for N. laevis, S. platensis and C. vulgaris trypsin hydrolysates, respectively) (p> 0.05). Protein extracts and hydrolysates did not show antibacterial or bactericidal activities against the tested organisms with the used range of concentrations. Aqueous protein extracts and hydrolysates from Nitzschia, Spirulina and Chlorella have shown in vitro antioxidant, anti-angiotensin I-converting enzyme and anti-acetylcholinesterase activities, suggesting potential new sources of bioactive proteins and peptides of different phyla with nutraceutical and pharmaceutical potentials. Until now, most of the biological activities of microalgal-derived bioactive proteins and hydrolysates have been observed in vitro. Therefore, further ex vivo research studies are needed in order to investigate their potential market as nutraceuticals and pharmaceuticals based on their therapeutic potential shown in this study.