Fermented Soybean Peptides Show Antioxidant Effects in Immunosuppressed Mice

A team of Chinese researchers has isolated and identified ten antioxidant peptides from Tongchuan Douchi, a traditional fermented soybean product from Sichuan Province, and shown in a mouse study that the purified peptide fraction can counteract some of the biochemical signs of oxidative stress in liver and kidney tissue. The work, published in February 2026 in the Springer journal Agricultural Products Processing and Storage, combines classical protein chemistry with mass spectrometry and an animal model of chemically induced immunosuppression, offering one of the more systematic characterizations to date of the bioactive peptides in this heritage food.

Douchi is produced by steaming black or yellow soybeans and then fermenting them in solid state with filamentous fungi such as Mucor or Aspergillus species, or with bacterial proteases. Tongchuan Douchi, made in Santai County, relies on a distinctive mucor-based koji-making fermentation and carries more than three centuries of production history; it was listed as National-level Intangible Cultural Heritage in 2008. Fermented soy products are rich in oligopeptides, free amino acids and flavonoids, and peptides from such foods have previously been associated with antihypertensive, antioxidant, antimicrobial, antidiabetic and anticancer activities in earlier research. The authors note, however, that local specialty foods like Douchi have received relatively little systematic evaluation compared with other fermented soybean products such as the Indian alkaline-fermented Kinema, whose peptide content and gastrointestinal stability were characterized in prior work.

The extraction pipeline began with dual-frequency ultrasound-assisted extraction. Ten grams of Douchi were ground with phosphate buffer at pH 7.9, the liquid-to-material ratio was adjusted to 37 milliliters per gram, and the slurry was treated in a dual-frequency ultrasonic reactor operating at a 25 to 40 kilohertz working-time ratio of 30:20 seconds, at 40 degrees Celsius, a power density of 297 watts per liter, for 30 minutes. After centrifugation, proteins were precipitated with ice-cold 15 percent trichloroacetic acid, and the remaining supernatant served as the crude polypeptide extract.

The crude extract was then passed through a 0.22-micrometer membrane and fractionated with ultrafiltration cartridges cut off at 10 and 3 kilodaltons. The fraction between 3 and 10 kilodaltons made up the largest share of the material at 52.4 percent, peptides above 10 kilodaltons accounted for 18.6 percent, and the smallest peptides, below 3 kilodaltons, represented 29.1 percent. Despite their lower abundance, the sub-3-kilodalton peptides showed the strongest DPPH radical scavenging rate, at 91 percent, consistent with the wider observation that low-molecular-weight antioxidant peptides tend to have greater bioavailability and cellular absorption. This fraction was therefore carried forward.

Next, the sub-3-kilodalton material was resolved on a Sephadex G-25 size-exclusion column, producing four peaks designated F1 through F4, eluted in descending order of molecular weight. When each fraction was tested at a concentration of 1 milligram per milliliter, F3 achieved the highest DPPH radical scavenging rate, 93.2 percent. Interestingly, F4, although it contained the smallest peptides, scored lowest at 29.1 percent. The authors suggest that very small peptides may lack sufficient active groups such as hydroxyl or thiol moieties, may be less stable to pH and temperature, and may include free amino acids with weak electron-transfer capacity. Tricine-SDS-PAGE confirmed the purification trajectory: crude extracts showed broad bands near 6.5 and 3.3 kilodaltons, the ultrafiltered fraction concentrated near 3.3 kilodaltons, and F3 migrated around 1 kilodalton, indicating that the most active antioxidant peptides were smaller than 1 kilodalton.

Amino acid analysis of F3, performed by acid hydrolysis and an automated amino acid analyzer, revealed a composition consistent with antioxidant function. Glutamic and aspartic acids accounted for 20.3 percent and 11.6 percent of total amino acids respectively, hydrophobic amino acids made up 27.4 percent, branched-chain amino acids 12.58 percent, and the aromatic residues tyrosine and phenylalanine 7.09 percent. Hydrophobic residues can promote peptide solubility at the lipid-water interface and act as hydrogen donors, while aromatic and sulfur-containing residues are known contributors to radical scavenging in plant-derived peptides.

Using LC-MS/MS on an Orbitrap Fusion Lumos instrument and searching against a soybean protein database, the team identified ten peptides in F3 with high confidence scores: LLLPH, KDFPPR, DDFFL, ADFYNPK, IIGGI, DTPMIG, SVVDM, LVSPP, C(+57.02)ADLF and VIVEL. Most are short chains with molecular weights below 1 kilodalton, in line with reports that peptides of two to nine residues show stronger antioxidant activity than longer chains. Hydrophobic amino acids exceeded 50 percent of the composition in eight of the ten peptides, a feature that favors interaction with lipid peroxides. In KDFPPR, the positively charged lysine and arginine may stabilize radicals electrostatically, while proline’s rigid structure may enhance peptide stability, and the aromatic phenylalanine and tyrosine in ADFYNPK are efficient radical scavengers.

The in vivo phase of the study used 60 male Kunming mice randomly assigned to six groups of ten: a normal group, a cyclophosphamide-induced immunosuppressive model group, a positive control receiving carnosine at 100 milligrams per kilogram per day, and low-, medium- and high-dose peptide groups receiving 100, 300 and 500 milligrams per kilogram per day by gavage for 15 days. Immunosuppression was induced with intraperitoneal cyclophosphamide at 70 milligrams per kilogram daily for three consecutive days. Compared with the normal group, model mice showed significantly reduced liver, kidney and spleen organ coefficients, confirming the model. Medium and high doses of F3 significantly raised these coefficients relative to the model group, although only the high dose differed significantly from the positive control in renal coefficient.

The oxidative stress markers told a consistent story. Liver T-SOD activity rose significantly, by 32.0 percent at the medium dose and 28.8 percent at the high dose, versus the model group, while the positive control rose 40.1 percent. Kidney T-SOD increased by 23.38 percent at the high dose, reaching significance, whereas low- and medium-dose increases of 8.36 and 17.11 percent did not. Hepatic GSH-Px rose significantly at the medium and high doses, by 34.4 and 29.5 percent, and renal GSH-Px increased significantly at all three peptide doses, by 63.7, 77.0 and 112.4 percent. Malondialdehyde, a marker of lipid peroxidation, fell significantly in the liver at medium and high doses, by 21.2 and 18.8 percent, and fell significantly in the kidney at all doses, by 29.1, 39.4 and 35.8 percent. Together, the authors conclude, the enzyme activation and lipid peroxide reduction indicate that Douchi peptides act both by scavenging free radicals directly and by stimulating endogenous antioxidant defenses.

The researchers are careful about the limits of their findings. The peptide mixture administered to the mice contained all ten sequences together, so the contribution of any single peptide remains unknown, and the authors call for future work isolating individual peptides and studying them through cell culture, metabolomics and genomics. As an animal experiment in a chemically induced immunosuppression model, the study demonstrates mechanistic antioxidant effects in mice rather than clinical benefits in humans, and no human efficacy can be inferred from these results. Even so, the combination of a well-characterized heritage food, a fully specified extraction and purification chain, and quantified in vivo endpoints gives the work unusual technical completeness for a food-derived peptide study, and supports the authors’ suggestion that Douchi-derived peptides may hold potential as ingredients for functional foods pending further research.

Subject of Research: Isolation, structural identification and in vivo antioxidant activity of peptides from the fermented soybean food Tongchuan Douchi

Article Title: Purification, structural identification and in vivo antioxidant activity of Douchi antioxidant peptides

Article References: Jin, J., Deng, Y., Jian, X., Luo, Y., Zhao, W., Li, L., Zeng, F., Liang, L., & Muhammed, A. (2026). Purification, structural identification and in vivo antioxidant activity of Douchi antioxidant peptides. Agricultural Products Processing and Storage, 2(1), Article 32. https://doi.org/10.1007/s44462-026-00066-4

Image Credits: AI Generated

DOI: 10.1007/s44462-026-00066-4

Keywords: Douchi, antioxidant peptides, fermented soybeans, LC-MS/MS, oxidative stress, mice study, cyclophosphamide, SOD, GSH-Px, malondialdehyde, functional foods, Sichuan