Sea Grape (Caulerpa racemosa) Kombucha: A Comprehensive Study of Metagenomic and Metabolomic Profiling, Its Molecular Mechanism of Action as an Antioxidative Agent, and the Impact of Fermentation Time

Sea grape kombucha has been known to exhibit high antioxidant activity due to its ele-vated total polyphenol content. This study aims to identify and characterize the active microbial community involved in the fermentation of kombucha using sea grapes (C. rac-emosa) as the primary substrate. Furthermore, it evaluates the effects of different Symbi-otic Culture of Bacteria and Yeast (SCOBY) starter concentrations on the physicochemical properties and antioxidant activity of sea grape kombucha. Our results showed that the pH of the kombucha was higher after 7 days of fermentation compared to later time points. The microbial community was composed of 97.08% bacteria and 2.92% eukaryotes, divided into 10 phyla and 69 genera. The dominant genus in all samples was Komaga-taeibacter. Functional profiling based on 16S rRNA data revealed that metabolic functions accounted for 77.04% of predicted microbial activities during fermentation. The most en-riched functional categories were carbohydrate metabolism (15.70%), cofactor and vita-min metabolism (15.54%), and amino acid metabolism (14.24%). At KEGG Level 3, amino acid-associated pathways, particularly alanine, aspartate, and glutamate metabolism (4.24%), were predominant. The fermentation process in sea grape kombucha is primarily driven by carbohydrate and amino acid metabolism, supported by energy-generating and cofactor biosynthesis pathways. Our findings indicate that different metabolic pathways lead to variations in kombucha components, and distinct fermentation stages result indifferent metabolic reactions. For instance, early fermentation stages (Day 7) are dominated by amino acid metabolism, whereas the late stages (Day 21) show increased activity in carbohydrate and sulfur metabolism. Metabolomic analysis revealed that increasing the SCOBY starter concentration significantly influenced pH, soluble solid content, vitamin C, tannin, and flavonoid content. These variations suggest that fermentation duration and microbial composition significantly influence the spectrum of bioactive metabolites, which synergistically provide functional benefits such as antimicrobial, antioxidant, and metabolic health-promoting activities. For example, sample K1 produced more fatty acids and simple sugar alcohols, sample K2 enriched complex lipid compounds and phytosterols, while sample K3 dominated the production of polyols and terpenoid compounds.