Vinegar is a traditional fermented condiment in most countries and has been commercially produced and sold for approximately 5000 years (Budak et al., 2014). In China, vinegar has traditionally been produced through solid-state fermentation using grains such as millet, rice, and wheat, which is also referred to as Chinese Traditional Cereal Vinegar (Zhang et al., 2022a, Zhang et al., 2022b). Zhejiang rice vinegar is a renowned traditional Chinese vinegar, distinguished by its reddish color and unique flavor, which result from semi-solid fermentation in ceramic jars. In southeastern China, the rice soaked for 1–5 days, is steamed, and cooled at air temperature, and then transferred into a ceramic cylinder. The saccharification is driven by Monascus and lasts about 20 days, after which water is added to initiate alcoholic and acetic acid fermentation. After approximately 3 months of fermentation, the vinegar will be aged for 1 year before the final products are obtained (Fig. S1) (Fang et al., 2021; Zhang et al., 2022b).

Bioaugmentation has proven to be an effective strategy for enhancing fermentation efficiency, flavor, and function of traditional fermented condiment by modulating microbial composition and metabolism (Chantarot et al., 2022). Unlike Zhenjiang aged vinegar and Shanxi aged vinegar, the fermentation of Zhejiang rice vinegar basically takes place from May to September (Jiang et al., 2013). Optimization of microbial species and inoculation levels has shown promise in improving fermentation efficiency and product quality. For instance, adding Aspergillus niger to Baoning vinegar improved starch utilization, organic acid levels, and free amino acid content, stabilizing quality across fermentation cycles (Liu et al., 2020; Liu et al., 2021). Similarly, incorporating Monascus in Chuanfu vinegar reduced microbial diversity, inhibited pathogens, and increased lactic and acetic acid bacteria, enhancing organic acid, ester, and alcohol accumulation (Ai et al., 2018). Aspergillus flavus SU-16 has been widely used in Chinese liquor fermentation for over 60 years, while Aspergillus species hold significant biotechnological value, Aspergillus oryzae and Aspergillus sojae are commonly used in the industrial production of fermented foods such as sake, soy sauce, and other traditional products. In addition, other species such as Aspergillus tamarii, Aspergillus niger, and Aspergillus avenaceus are employed industrially for the production of enzymes (e.g., amylases, proteases, and cellulases) and bioactive compounds, including insecticidal agents and antibiotics (Sun et al., 2022; Kjærbølling et al., 2020).

Furthermore, mixed fermentation using Trichoderma koningii, Aspergillus oryzae, and Lactobacillus casei can enhance the total phenolic content (TPC) and antioxidant capacity of navel orange peel residue (Yu et al., 2025), suggesting their potential for vinegar fermentation. Several microbial strains have been introduced to shorten fermentation from five months to 72 days and enhance raw material conversion in Zhejiang rosy vinegar, such as Aspergillus niger AS3.4309, Rhizopus species, and Saccharomyces cerevisiae K (Jiang et al., 2013), however, their impact on microbial succession and metabolism is still insufficiently explored. With the development of culture-independent and multi-omics techniques, there is increasing exploration to correlated among microbial communities, metabolic profiles, and sensory attributes in fermenting vinegar (Zhang et al., 2022b). Esters are the dominant aroma compounds, while furfural, ethyl acrylate, and tetramethylpyrazine serve as aging markers, reflecting the evolution of flavor during fermentation. In addition, organic and amino acids contribute not only to the nutritional value but also to the overall flavor profile (Zhang et al., 2022b). HS-SPME/GC–MS and amplicon sequencing identified Acetobacter and Lactobacillus as key contributors to flavor, with ethanol, titratable acidity, and reducing sugars influencing microbial succession (Fang et al., 2021).

This study aims to improve rice vinegar fermentation to reduce the cycle, and identify key flavor and quality factors by inoculating with Aspergillus flavus SU-16 and Monascus purpureus (AfMp). The microbial community composition and dynamics during saccharification, alcoholic fermentation, and liquid-state acetic acid fermentation in rice vinegar were comprehensively characterized using high-throughput sequencing (HTS). Moreover, key physicochemical parameters, including organic acids, volatile flavor compounds, and antioxidant activity, were systematically investigated using high-performance liquid chromatography (HPLC), gas chromatography–mass spectrometry (GC–MS), and spectrophotometric assays. Furthermore, the effect of final AfMp and ZRV vinegar on cell viability and anti-inflammatory injury of IEC-6 cell were compared, as well as untargeted metabolomic profiling. This integrated approach provides valuable insights into the microbial succession and quality evolution during fermentation, offering a scientific basis for using bioaugmentation to modernize and improve the quality of traditional vinegar.