## 3 Results
### Functional Microorganisms and Volatile Compounds Identified Based on O2 PLS-DA Model  
The O2 PLS-DA model was used to explore the correlation between the active microorganisms and the volatile flavors during NLS fermentation. A total of 420 active genera were analyzed as X, and 181 volatile flavors were analyzed as Y. The R2X and R2Y values in the model were 0.9823 and 0.9323, respectively, indicating that the O2 PLS-DA model was well fitted for analysis and prediction. Then, the top 20 genera with the greatest influence on the formation of flavor compounds were displayed according to the order of the loading scores. They included 9 bacterial genera (_Streplococcus_, _Lactobacillus_, _Pediococcus_, _Thermotoga_, _Faecalibacterium_, _Canyylobacter_, _Yersinia_, _Weissella_ and _Acinetobacter_) and 11 fungal genera (_Saccharomyces_, _Talaromyces_, _Aspergillus_, _Mixia_, _Rhizophagus_, _Gloeophyllum_, _Scheffersomyces_, _Kluyveromyces_, _Paracoccilioides_, _Clavispora_ and _Colletotrichum_) (Figure 5A). Meanwhile, the top 20 volatile metabolites with the great impact on the microbial community were chosen, and they comprised 10 esters including ethyl acetate, ethyl hexanoate, ethyl (2S)-lactate, ethyl octanoate, ethyl hydroxycaproate, ethyl decanoate, ethyl linoate, ethyl oleate, diethyl succinate and ethyl hydrogen succite, 3 organic acids including acetate, 2-methylpropanoate and 3-methylbutanoic acid, 2 alcohols including ethanol and 3-methylbutanol, 2 aldehydes including acetaldehyde and 3-methylbutal, 1 alkene (\(\beta\)-caryophyllene), and other metabolites of ether and benzene (Figure 5B). Subsequently, the variable sorting was provided according to the importance of the model interpretation by VIP\({}_{\text{O2 PLS}}\) profiles for the top 20 genera. The value of VIP varied from 0.45 to 19.11. A total of 14 microbial genera, including 8 bacterial genera and 6 fungal genera with a value of VIP > 1.0, had the most important effects on the volatile flavor generation (Figure 5C). In short, genera of _Streptococcus_, _Lactobacillus_, _Saccharomyces_, _Pediococcus_, _Talaromyces_, _Aspergillus_, _Thermotoga_, _Faecalibacterium_, _Camylobacter_, _Yersinia_, _Mixia_, _Rhizophagus_, _Weissella_, and _Gloeophyllum_ were the important contributors to the volatile flavor generation of NLS.  
Accordingly, the O2 PLS-DA model was used to analyze the association between the active species and the volatile compounds. The results indicated that the R2X and R2Y values were 0.9924 and 0.9272, respectively. Then, the top 20 species with the greatest influence on the generation of flavor metabolites were composed of 14 bacterial species, including _S. mutans_, _L. otakiensis_, _L. lelveticus_, _P. damnosus_, _Thermotoga_ sp. TBYP3.1.4.1, _L. acettolerans_, _F. prasunitzii_, _L. pasteurii_, _C. jejuni_, _L. brevis_, _Y. pestis_, _P. pentsaceus_, _W. cibaria_ and _L. delbrueckii_, and 6 fungal species including _S. cerevisiae_, _A. niger_, _T. marneffei_, _M. osmundae_, _R. irregularis_, and _G. trabeum_ (Figure 6A). The top 20 volatile metabolites with the greatest impact on species were displayed (Figure 6B). There were 15 bacterial species and 9 fungal species with a value of VIP > 1.0 among all top 25 species (VIP varied from 0.99-24.33) (Figure 6C). Therefore, except for the top 20 species based on the loading score, four species, including _L. reuteri_, _T. stipitatus_, _S. stipites_, and _K. marxianus_, were the most important species for flavor generation during NLS fermentation.  
Figure 5: O2 PLS-DA model based on the metatranscriptomics and metabolomics at the genus level during NLS fermentation. (**A**) Top 20 genera influencing the flavor compounds based on HS-SPME-GC-MS. (**B**) Top 20 compounds influencing the active genera. (**C**) VIP plot of microbiota at the genus level.