The research team, led by Mengru Han and colleagues including senior author Baili Feng, selected two cultivars, Liuleng (black) and Ganqing10 (yellow), grown side by side in the same experimental plot of the Agricultural Science Research Institute of Gannan Tibetan Autonomous Prefecture. In previous work, the group had shown that black and yellow highland barley represent the extremes of compositional variation among colored cultivars, with black grains carrying roughly twice the flavonoids, phenolics, and anthocyanins of their yellow counterparts. The central question of the new study was whether those initial chemical differences would steer fermentation along fundamentally different trajectories, or whether the microbial communities driving the process would converge on a shared path regardless of raw material.

To find out, the researchers followed the traditional Tibetan brewing procedure. Barley grains were soaked, boiled, cooled, and inoculated with a starter combining indigenous Tibetan jiuqu—a traditional fermentation culture prepared from barley flour, plateau herbs, and aged starter—with high-activity dry yeast. The prepared grains then fermented in sealed earthenware jars at 29 degrees Celsius for 72 hours, with samples harvested at 0, 24, 48, and 72 hours. For each time point the team tracked antioxidant compound contents and radical-scavenging activities, sequenced bacterial 16S rRNA genes and fungal internal transcribed spacer regions to profile the microbiome, and used ultra-performance liquid chromatography coupled to tandem mass spectrometry to map the non-volatile metabolome at the final stage of fermentation.

The antioxidant measurements told a nuanced story. In both cultivars, fermentation pushed total phenolic content upward and anthocyanins peaked at 48 hours before easing slightly, while total flavonoid content declined progressively—suggesting that flavonoids were being transformed, deglycosylated, or converted into other phenolic derivatives rather than simply accumulating. Yellow barley showed the more dramatic relative gains, ultimately surpassing black barley in total phenolic content by the end of fermentation and posting a larger rise in total antioxidant capacity. Yet black barley never surrendered its lead in radical scavenging: it maintained consistently higher DPPH, ABTS, hydroxyl radical, and superoxide radical scavenging activities throughout all 72 hours, an advantage the authors attribute to its richer initial reserves of flavonoids, anthocyanins, and pigment-associated metabolites.

Microbial sequencing revealed a fermentation world that was simultaneously shared and distinct. In both jars, bacterial diversity fell steadily, and the fungal community collapsed toward near-total dominance by a single genus, Saccharomycopsis, whose competitive grip on carbohydrate utilization drove fungal diversity essentially to zero by the middle of fermentation. Among bacteria, Lactococcus flourished early, peaking at 24 hours, while Pediococcus rose steadily to dominate the late stage in both cultivars—an ecological succession that mirrors patterns seen in other cereal fermentations, where substrate consumption, acid accumulation, and falling pH progressively eliminate competitors and favor acid-tolerant lactic acid bacteria.

Beneath these broad similarities, however, the two cultivars hosted measurably different microbial worlds. Bacterial richness rose and then fell in black barley but followed the opposite pattern in yellow barley, and differences in alpha diversity were detectable even before fermentation began, hinting that the grains carried distinct resident microbiota shaped by their contrasting chemistry. LEfSe biomarker analysis identified cultivar- and stage-specific taxa: Gordonia and Frigoribacterium marked the 24-hour stage in black barley, Enterococcus marked 48 hours, and Levilactobacillus emerged at 72 hours, whereas yellow barley at 24 hours was characterized by Lactococcus and Flavobacterium. Principal coordinate analysis showed the overall bacterial community structures broadly overlapped, but the fungal communities differed significantly between cultivars, if modestly in magnitude.

Perhaps the most striking cultivar differences emerged from co-occurrence network analysis, which maps the statistical associations among microbial taxa. The yellow barley network was denser, more highly connected, and richer in positive correlations, suggesting a more cooperative, tightly interlinked community—the kind of structure that previous research has linked to efficient joint substrate utilization and metabolite exchange. The black barley network, by contrast, was more modular and compartmentalized, with taxa organized into relatively independent functional subgroups that may allow separate responses to acid stress, substrate availability, or phenolic inhibition. The putative keystone taxa also diverged sharply: Bacillus and Zoogloea anchored the black barley network, while Lactococcus, Enterococcus, and Aquabacterium held central positions in yellow barley. Predicted functional profiles from PICRUSt2 were broadly similar between cultivars—dominated by carbohydrate, amino acid, and energy metabolism—indicating a shared functional foundation, with differences concentrated in low-abundance pathways.

The metabolomics data sharpened the picture considerably. Principal component analysis and hierarchical clustering cleanly separated the fully fermented black and yellow samples, and while most detected metabolites were shared, black barley contained more cultivar-specific compounds. The metabolites enriched in black barley were overwhelmingly flavonoids and phenolic acids—derivatives of isoscoparin, tricin, chrysoeriol, caffeoyl compounds, and 1-acetyl-beta-carboline—and KEGG enrichment pointed squarely at flavonoid and anthocyanin biosynthesis pathways, including chrysoeriol aglycone, apigenin C-glycoside, and flavone and flavonol biosynthesis. Yellow barley, by contrast, accumulated more amino acids, small peptides, and their derivatives, suggesting its fermentation leaned more heavily toward primary nutrient transformation and the formation of taste-related compounds.

Correlation analyses then wove the three data layers together. Antioxidant indices were strongly and positively associated with specific flavonoid glycosides and phenolic derivatives: total flavonoid content tracked compounds such as chrysoeriol-7-O-rutinoside-5-O-glucoside and quercetin-5,4′-di-O-glucoside with correlation coefficients above 0.94, while anthocyanin content aligned with gallic acid and 1-acetyl-beta-carboline. Notably, the lactic acid bacterial genera Lactiplantibacillus and Levilactobacillus were the taxa most closely and positively correlated with antioxidant-related flavonoid and phenolic metabolites—a finding consistent with known β-glucosidase, esterase, and phenolic acid decarboxylase activities in some lactic acid bacteria that can liberate bound phenolics from grain matrices. Meanwhile, genera such as Aquabacterium, Bradyrhizobium, Lactococcus, and Microbacterium associated instead with peptide- and amino acid-derived metabolites, and Flavobacterium and Saccharomycopsis showed correlation patterns opposite to the antioxidant-linked lactic acid bacteria. The authors are careful to stress that these are statistical associations, not proof of causation, and that the functional predictions were inferred from taxonomy rather than measured directly by metagenomics or enzyme assays.

The study has clear limitations—pH, organic acids, and the free-versus-bound phenolic fractions were not fully characterized, and no cultivation or inoculation experiments were performed to test the candidate microorganisms directly. But the practical implications are compelling. For producers of highland barley wine and emerging functional foods, the work suggests that cultivar selection is a powerful lever: black barley delivers a fermented product with inherently stronger radical scavenging capacity, while yellow barley responds more dynamically to fermentation with larger relative gains in phenolics. Future experiments using sterile fermentations, defined co-cultures, and enzyme knockout studies could confirm whether Lactiplantibacillus and Levilactobacillus can be deployed as starter organisms to deliberately boost the phenolic content and antioxidant value of fermented cereal products. In an era when consumers increasingly seek foods with documented functional benefits, this plateau grain—and the microbes that transform it—may offer a scientifically grounded path from traditional brewing to evidence-based functional food design.

Subject of Research: Comparative multi-omics analysis of microbial succession, metabolite profiles, and antioxidant properties during fermentation of black and yellow highland barley.

Article Title: Comparative multi-omics analysis of microbial succession, metabolite profiles, and antioxidant properties in fermented black and yellow highland barley

Article References: Han, M., Hou, H., Wang, H., Zhang, M., Tang, X., Zhou, X., Liu, M., Ma, C., Yang, Q., & Feng, B. (2026). Comparative multi-omics analysis of microbial succession, metabolite profiles, and antioxidant properties in fermented black and yellow highland barley. Food Chemistry: X, 39, Article 104424. https://doi.org/10.1016/j.fochx.2026.104424

Image Credits: AI Generated

DOI: 10.1016/j.fochx.2026.104424

Keywords: highland barley, fermentation, antioxidant activity, flavonoids, anthocyanins, microbial succession, lactic acid bacteria, metabolomics, Pediococcus, Saccharomycopsis, Tibetan barley wine, functional foods