Mung bean seed coat (MBC) was purchased from Kittitat Co., Ltd. (Thailand). The raw material was kept at 0 °C before use. For the preparation of MBC extract, polyphenols were extracted from MBC with 50% ethanol using pressurized liquid extraction [20 (link)]. The conditions for extraction were 1300 PSI at 160 °C. The extract was filtered and concentrated using a rotary evaporator, and then centrifuged at 7000 rpm for 10 min at 25 °C. The concentrated extract was dried with a pilot spray dryer using maltodextrin and gum arabic as carriers. The obtained polyphenol extract was stored at −20 °C in an aluminum bag before use.
For the preparation of another fermented vegetable formula, polyphenol extract was dissolved in the liquid from the fermented vegetables, and after thorough mixing, the solution was added back to the fermented vegetables. The fermented vegetables supplemented with polyphenol extract were stored at 4 °C before analysis.
Vitexin quantification was conducted as described below. First, polyphenol was extracted from kimchi samples according to the previously reported method by Kim, Lee et al. (2018) [21 (link)]. Briefly, kimchi was ground with a blender. Then, 5 g of ground kimchi was mixed with 25 mL of 60% acetonitrile and vortexed for 1 min. The mixture was sonicated at 37 °C for 10 min. Then, the mixture was centrifuged at 3000 rpm for 10 min. The supernatant was collected and filtered through a syringe filter (0.45 µM) before the determination of the vitexin content using HPLC according to Pavasutti, Sinthuvanich et al. (2023) [22 (link)]. Samples were subjected to HPLC equipped with a diode array detector (DAD) (Waters 600, Spectralab Scientific Inc., Milford, MA, USA). The absorption spectra were recorded from 210–600 nm for all peaks. UV absorbance at 280 nm and 337 nm was used to monitor phenolic compounds and flavonoids, respectively. Samples were passed through an analytical column C18 (4.6 × 250 mm, 5 µm, Waters Symmetry Column, Agilent Technologies Inc., Dublin, Ireland) and were stored at 30 °C. The injection volume of the sample was 10 µL. Elution was performed using two solvent gradients: solvent A (1% acetic acid in deionized water, v/v) and solvent B (1% acetic acid in methanol, v/v). Elution was carried out at a flow rate of 1 mL/min. The gradient program was as follows: 10–35% B (10 min), 35–42% B (15 min), 42–75% B (10 min), 75% B (5 min), 10–75% B (5 min), and 10% B (5 min). We obtained the preliminary results of the vitexin content and chromatographic analysis of the standard formula (N) and fermented vegetables with vitexin (V), shown as follows.Figure 1 a shows that the vitexin content in fermented vegetables with vitexin (V sample) was significantly higher than that in standard fermented vegetables (N sample). The vitexin contents of the standard fermented vegetables and fermented vegetables with vitexin were stable during a 15-day storage time. Figure 1 b,c show chromatograms of the standard fermented vegetables and fermented vegetables with vitexin, respectively. The chromatograms show the peak of vitexin at the retention time of 15.8 min, which agrees with the standard vitexin. The second peak in Figure 1 c is isovitexin. This agrees with a previous study that extracted polyphenols from mung bean seed coat extract [20 (link)], and the present study used the same method.
All fermented vegetable samples were stored for 15 days at 4 °C and sampled on days 0, 3, 6, 9, 12, and 15. The fermented vegetables samples were analyzed according to quality indexes (pH, LAB, and L. rhamnosus GG count). Moreover, sampling at 4 °C from days 0 to 15 was performed to investigate the microbial diversity and antioxidant activity.
For the preparation of another fermented vegetable formula, polyphenol extract was dissolved in the liquid from the fermented vegetables, and after thorough mixing, the solution was added back to the fermented vegetables. The fermented vegetables supplemented with polyphenol extract were stored at 4 °C before analysis.
Vitexin quantification was conducted as described below. First, polyphenol was extracted from kimchi samples according to the previously reported method by Kim, Lee et al. (2018) [21 (link)]. Briefly, kimchi was ground with a blender. Then, 5 g of ground kimchi was mixed with 25 mL of 60% acetonitrile and vortexed for 1 min. The mixture was sonicated at 37 °C for 10 min. Then, the mixture was centrifuged at 3000 rpm for 10 min. The supernatant was collected and filtered through a syringe filter (0.45 µM) before the determination of the vitexin content using HPLC according to Pavasutti, Sinthuvanich et al. (2023) [22 (link)]. Samples were subjected to HPLC equipped with a diode array detector (DAD) (Waters 600, Spectralab Scientific Inc., Milford, MA, USA). The absorption spectra were recorded from 210–600 nm for all peaks. UV absorbance at 280 nm and 337 nm was used to monitor phenolic compounds and flavonoids, respectively. Samples were passed through an analytical column C18 (4.6 × 250 mm, 5 µm, Waters Symmetry Column, Agilent Technologies Inc., Dublin, Ireland) and were stored at 30 °C. The injection volume of the sample was 10 µL. Elution was performed using two solvent gradients: solvent A (1% acetic acid in deionized water, v/v) and solvent B (1% acetic acid in methanol, v/v). Elution was carried out at a flow rate of 1 mL/min. The gradient program was as follows: 10–35% B (10 min), 35–42% B (15 min), 42–75% B (10 min), 75% B (5 min), 10–75% B (5 min), and 10% B (5 min). We obtained the preliminary results of the vitexin content and chromatographic analysis of the standard formula (N) and fermented vegetables with vitexin (V), shown as follows.
All fermented vegetable samples were stored for 15 days at 4 °C and sampled on days 0, 3, 6, 9, 12, and 15. The fermented vegetables samples were analyzed according to quality indexes (pH, LAB, and L. rhamnosus GG count). Moreover, sampling at 4 °C from days 0 to 15 was performed to investigate the microbial diversity and antioxidant activity.
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