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

[12]
level . For example, metabolomic analysis revealed both targeted and untargeted metabolites of fermented
[13]
soybean foods, which suggested their role in flavor development and therapeutic properties of them . The
shelf life of green tea beverage could be predicted by identifying markers determined by metabolomic
[14]
analysis . In addition, the variation of metabolites during cold storage of Torreya yunnanensis seeds was
investigated through metabolomic analysis, which provided insights into conservation efforts and habitat
restoration .
[15]
To reveal the microbial and metabolite characteristics of non-salt Suancai, the simulated fermentation
system was constructed. Furthermore, the effects of different substrates and suppliers of ingredients on
microbial counts, physicochemical properties, organic acids, metabolites, and microbial community were
also evaluated. This is the first study investigating the non-salt Suancai and its microbial and metabolite
characteristics. Therefore, the present study contributes to the scientific inheritance of traditional
technology and provides the basis for quality control.

MATERIALS AND METHODS
Sample preparation
Fresh vegetables were purchased from a local supermarket and used to produce non-salt Suancai. The
preserved dried Suancai was purchased from different local suppliers in Xichang City, Sichuan Province,
China (101.97° E, 27.93° N), which was used as a starter for fermentation. There were five Manjing leaves
(MF) (Brassica rapa L.), one Manjing rhizome (MR), and one mustard leaf stem (MLS) (Brassica juncea var.
latipa). Fresh vegetables were boiled for over 5 min and then transferred to a bottle containing different
preserved dried Suancai. The fermentation was done at room temperature (10 ± 2 °C). After 15 days of
fermentation, the fermented vegetables and liquids were evaluated. To differentiate the sample information,
samples collected from the same suppliers were numbered MF-A, MR-A, and MLS-A according to their
substrate ingredients. The other four purchase channels (suppliers) of samples were labeled as MF-B, MF-C,
MF-D, and MF-E, respectively, which were all made by Manjing leaf (Brassica rapa L.).

Microbial counts and physicochemical properties assessment
The fermented liquid of each sample was serially diluted in normal saline (NaCl, 0.85%, w/v), and the
diluent was plated on de Man, Rogosa and Sharpe (MRS; Oxoid Ltd, Hampshire, UK) and Rose Bengal agar
(RBA; Qingdao Hope Bio-Technology Co., Ltd, China) to enumerate lactic acid bacteria and yeast,
respectively . MRS plates were incubated at 37 °C for 48 h, and Rose Bengal agar plates were incubated at
[16]
30 °C for 48 h. Each microbial count was recorded as log colony-forming units per gram of sample (log
CFU/mL). The total acid (TA) and reducing sugar (RS) contents of the fermented vegetable samples were
measured. Briefly, 2 g of each sample was subjected to ultrasonic (40 kHz, room temperature) extraction
with ultrapure water, and the mixture was filtrated (qualitative grade filter circles) to obtain supernatant for
further analysis. TA and RS were determined based on Chinese national standards (GB 12456-2021 and GB
5009.7-2016), which refer to the acid-base titration method and the 3,5-dinitrosalicylic acid (DNS) method,
[17]
respectively. Total sugar (TS) was analyzed by a modified procedure of a previous publication : a 5 g
sample was mixed with 100 mL HCl (2%, v/v) and treated at 121 °C for 30 min. Then, the pH of the
extraction solution was adjusted to neutrality with NaOH (20%, w/v) after cooling down. The solution was
then diluted to 250 mL and filtered using filter paper.

Analysis of organic acids
High-performance liquid chromatography (Agilent G1260 HPLC, Agilent Technologies Co. Ltd., USA) was
used to analyze organic acids (OAs) based on a previous publication but with some optimizations. For
[18]
fermented mustard, a 3 g sample was mixed and homogenized with 0.004 M H SO at a ratio of 2:7 (w/v),
4
2
followed by extraction for 30 min. Prior to analysis, the treated sample was centrifuged at 12,000 r/min for