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Lai et al. Microbiome Res Rep 2024;3:21 https://dx.doi.org/10.20517/mrr.2023.76 Page 7 of 15

Figure 1. (A) Overview pattern of the metabolite differences based on PLS-DA score plots; (B) heatmap of metabolites. All of the
metabolites were calculated in the PLS-DA plot, and the heatmap shows metabolites whose relative content was in the top fifty.

[27]
related compounds . In addition, nucleotide metabolism was also susceptible to inactivation of microbes,
peptidoglycan synthesis, and amino acid metabolism . Therefore, metabolism was simultaneously related
[28]
to growth and occurred in the inactivation process of microbes.

Differences in metabolites based on multiple samples comparison
There were 21 (MF-B), 19 (MF-C), 25 (MF-D), 24 (MF-E), 23 (MLS-A), 25 (MF-A), and 22 (MR-A)
metabolites whose relative content was higher than 1% in each sample. Queuine, cyclopentanol, pc (16:0/
0:0), palmitic acid, isorhamnetin, fisetin, and kaempferol 3-o-sophoroside were universally predominant
metabolites (the relative content > 3%) in non-salt Suancai. Among them, queuine was highlighted in MF-B
(5.24%) and MF-C (13.30%), which was derived from a de novo synthesized metabolite in bacteria .
[29]
Moreover, the relative content of cyclopentanol, isorhamnetin, pc (16:0/0:0), palmitic acid, and
pyroglutamic acid was higher in MF-D, MF-E, MLS-A, MF-A, and MR-A, respectively, among which
cyclopentanol endows pleasant flavors and isorhamnetin possess antioxidant properties . Palmitic acid and
[30]
pyroglutamic acid were involved in fatty acid metabolism and glutathione metabolism, respectively.

The overall patterns of metabolite differences in non-salt Suancai fermented by different substrates/
suppliers of ingredients are shown in PLS-DA score plots. As shown in Figure 1A, there was an obvious
difference in metabolites between samples. The response permutation test confirmed the absence of
overfitting and misinformation in the data. It can be concluded that the metabolic preference and metabolic
diversity varied remarkably with the substrates/suppliers. Heatmap and cluster analysis illustrate the
composition of top 50 metabolites [Figure 1B]. The parallel assays of each sample were clustered together,
indicating good assay performance. Compared to different substrates, MR-A was clustered into a single
cluster because of the high abundance of indole-3-carboxylic acid, 3-hydroxybenzoic acid, (3Z,6Z)-3,6-
nonadien-1-ol, and Pc (17:0/0:0). Among them, indole-3-carboxylic acid was involved in the biogenesis of
ascorbigen in Brassica oleracea L. , the bulb of which was similar to Manjing rhizome. 3-Hydroxybenzoic
[31]
acid is an antioxidant phytochemical . Interestingly, (3Z,6Z)-3,6-nonadien-1-ol endows a strong and waxy
[32]
flavor to fresh vegetables with a creamy taste, which is probably the reason for an overall flavor difference of