**Background:** Fish sauce is a traditional amber-colored condiment fermented from low-value marine fish, with a production cycle of 6–18 months for natural fermentation. Rapid fermentation technologies (koji fermentation, microbial fermentation, compound protease fermentation, heat preservation) have emerged as alternatives, but the microbial mechanisms underlying flavor formation in these processes remain poorly understood. This study aimed to identify characteristic flavor compounds and establish the metabolic network linking microorganisms to flavor generation across different fish sauce fermentation approaches.
**Methods:** Four fish sauce fermentation processes were conducted using anchovies (body length 75–140 mm, weight 5–20 g) from the East China Sea: (1) Natural fermentation (WQ): 30% sea salt, fermented 7 months at 20±5°C outdoors; (2) Koji outdoor fermentation (YQ): minced fish, deionized water, sea salt (10:5:2 w/w/w) plus 20% soya meal koji (Aspergillus oryzae), fermented 7 months at 25±5°C outdoors; (3) Heat preservation with koji (BWQ): same as YQ but fermented 5 months at 35°C then 2 months at 55±5°C; (4) Heat preservation with enzyme (BWE): minced fish with deionized water (1:1 g:mL), 0.5% composite protease at 50°C for 2.5 h, then 0.7% flavor protease at 55°C for 2.5 h, 15% salt, fermented 5 months at 35°C then 2 months at 45°C. Samples were collected at 3 and 7 months. Volatile compounds were analyzed by HS-SPME-GC–MS. Metagenomic DNA was extracted using PowerSoil DNA Isolation Kit, sequenced on Illumina platform, and annotated against NCBI NR and KEGG databases. O2PLS-DA and VIP values were used to identify differential flavor compounds.
**Key Results:** A total of 114 volatile flavor compounds were quantified across eight sample groups (four processes × two time points), including 21 aldehydes, 6 alcohols, 19 esters, 6 acids, 35 hydrocarbons, 10 ketones, 14 nitrogen-containing compounds, and 3 sulfur-containing compounds. The O2PLS-DA model showed excellent discrimination (R²(X)=83.3%, R²(Y)=99.7%, Q²(cum)=95%). At the early fermentation stage (3 months), 15 compounds with VIP >1.2 were identified as differential markers; at the late stage (7 months), 28 compounds were identified. Key differential markers for natural fermentation included 3-ethenyl-cyclopentene, 2-methyl-2-pentenal, benzyl alcohol, octanoic acid ethyl ester, nonane, and 2,6,11-trimethyldodecane, while propanal and methyl myristate were characteristic of rapid fermentation processes. Metagenomic sequencing revealed that genes belonging to metabolism processes were most abundant (76.51–78.77% across samples). Carbohydrate metabolism was the largest category (24.49–28.83% at 3 months). A total of 56 enzymes related to flavor metabolism were identified. Key findings include: (1) In carbohydrate metabolism, pyruvate production involved multiple genera including Aspergillus, Halococcus, Virgibacillus, Paenibacillus, Tetragenococcus, and Halanaerobium; (2) For amino acid metabolism, aromatic amino acid metabolism was highest (19.17–23.64%), and phenylacetaldehyde formation from phenylalanine was catalyzed by aromatic-amino acid transaminase (EC 2.6.1.57) annotated to Achromobacter, Stenotrophomonas, Acinetobacter, Agrobacterium, Pseudomonas, Enterobacter, and Phenylobacterium; (3) In lipid metabolism, lipoxygenase (EC 1.13.11) and hydroperoxide lyase (EC 4.1.2.-) were key enzymes, with Staphylococcus involved in lipoxygenase pathways and Halanaerobium involved in complete β-oxidation. The top 10 bacteria with the highest number of flavor-associated enzymes were Lactobacillus, Staphylococcus, Enterobacter, Aspergillus, Tetragenococcus, Halanaerobium, Mycobacterium, Psychrobacter, Stenotrophomonas, and Paenibacillus.
**Clinical Implications:** While this study does not have direct clinical applications, it provides fundamental insights into food fermentation microbiology that could inform the development of safer, higher-quality fermented fish products. The identification of key microorganisms and metabolic pathways enables targeted starter culture design for rapid fermentation, potentially reducing fermentation time from 6–18 months to 7 months while maintaining or improving flavor quality. The finding that heat preservation fermentation (BWQ and BWE) significantly reduced trimethylamine and indole content—compounds associated with spoilage and off-flavors—suggests these methods may improve product quality and safety. The metabolic network established in this study provides a framework for future research using transcriptomic, metabolomic, and proteomic approaches to further optimize fish sauce production.