Unlocking Antioxidant, Antibacterial and Anti-Inflammatory Peptides of Spirulina Platensis Through Bromelain Hydrolysis

Abstract

Spirulina platensis (Arthrospira platensis) is a protein-rich microalga widely recognized for its nutritional value; however, the bioactivity of its native proteins is limited. This study aimed to investigate whether bromelain-assisted enzymatic hydrolysis could enhance the antioxidant, antibacterial, and anti-inflammatory activities of S. platensis proteins. Proteins were extracted by freeze–thawing and sonication, followed by bromelain-mediated hydrolysis at pH 7 and 65 °C for 4 h. The resulting hydrolysates were characterized in terms of yield, degree of hydrolysis, molecular weight distribution, amino acid composition, and functional groups using standard analytical techniques. Bioactivities were assessed through ferric reducing antioxidant power (FRAP), disk diffusion antibacterial assays against Staphylococcus aureus and Escherichia coli, protein denaturation inhibition, and lipopolysaccharide-induced nitric oxide production in RAW 264.7 macrophage cells. The hydrolysis process yielded low-molecular-weight peptides (≤15 kDa) with an increased degree of hydrolysis and protein content compared to the crude extract. The protein hydrolysates exhibited measurable ferric reducing activity in the FRAP assay; however, their antioxidant reducing power was lower than that of the crude protein extract and therefore considered moderate rather than enhanced. In contrast, the hydrolysates demonstrated enhanced antibacterial activity compared with the non-hydrolyzed protein. In anti-inflammatory assays, the hydrolysates significantly inhibited protein denaturation and suppressed nitric oxide production in a concentration-dependent manner while maintaining high cell viability. These findings indicate that bromelain-assisted hydrolysis effectively enhances the bio-functional properties of S. platensis proteins. Thus, S. platensis protein hydrolysates show potential as natural sources of multifunctional bioactive compounds for food and biomedical applications.

HIGHLIGHTS

• Bromelain-mediated hydrolysis effectively converted Spirulina platensis proteins into low–molecular weight peptides (≈6 - 7 kDa), protein yield (67.5%) and degree of hydrolysis (60.515 ± 1.058%).
• Structural (FTIR, SDS-PAGE) and compositional analyses confirmed extensive protein conformational changes and enrichment of bioactivity-related hydrophobic and polar amino acids after hydrolysis.
• Bromelain-derived protein hydrolysates exhibited enhanced antibacterial activity against coli and S. aureus, with lower MIC and MBC values compared to crude protein.
• The hydrolysates have anti-inflammatory activity, strongly inhibiting BSA denaturation and suppressing nitric oxide production in LPS-stimulated RAW 264.7 macrophages (IC₅₀ = 10 µg/mL) without cytotoxic effects.
• An integrated in vitro evaluation demonstrated the multifunctional antibacterial and anti-inflammatory potential of bromelain-hydrolyzed platensis proteins, supporting their application as sustainable bioactive agents.

GRAPHICAL ABSTRACT