Comprehensive metabolite profile of Chlorella vulgaris and metabolic signature of its immunostimulating effect in Nile tilapia [Oreochromis niloticus (Linnaeus, 1758)]

Chlorella vulgaris is a green, single-celled, spherical freshwater microalga belonging to the family Chlorellaceae of the division Chlorophyta. C. vulgaris is currently used as food supplement and medicinal agent due to its carotenoids, chlorophylls and proteins content. Chlorella vulgaris is incorporated in fish feed as a growth and immune stimulant. Tilapia is one of the most important cultured fish species with numerous advantages, contributing more than USD 10 billion to the world’s seafood market annually. Despite the important features of tilapia, disease outbreaks tend to cause massive mortalities in fish farms which impose serious negative impact to economies worldwide. Most tilapia mortalities were reported as a resultant stress due to overcrowding coupled with bacterial infection. Streptococcus agalactiae is one of the most virulent bacteria causing most of tilapia mortalities recently. The use of immunostimulants like microalgal supplementation in fish feed holds a promising future in mitigating tilapia fish disease. Nevertheless, the common amounts used (≥12%) in this practice is considered uneconomical by fish farmers, considering the high price of microalgal biomass procurement. In addition, scientific reports regarding the association of metabolites with pathways involved in the stimulation of immune system of tilapia fish (Oreochromis niloticus) exerted by consumption of C. vulgaris are still limited. Hence, the main aim of this study was to investigate the immunestimulating role of bioactive metabolites from C. vulgaris on the tilapia fish at lower concentrations using proton nuclear magnetic resonance (1H-NMR) metabolomics approach. The first part of the study dealt with the characterization of the metabolite composition of the cultured samples of C. vulgaris via 1H-NMR, gas chromatography mass spectrometry (GCMS) lipid analysis and liquid chromatography mass spectrometry (LC-MS/MS) analysis. 1H-NMR data obtained from six different organic solvent