Technological quality and fungal community of Kombucha fermented with hemp leaves and milky mushroom flour (<i>Calocybe indica</i>)

Kombucha is traditionally a non-alcoholic beverage whose production is dependent on culture and the various ingredients used as substrates for fermentation. The goal of our study was to apply hemp leaf and milky mushroom (<i>Calocybe indica</i>) flour as functional ingredients to enhance phytonutrient quality, along with using a microbial consortium highly symbiotic with these ingredients. The study determined the content of phytonutrients (phenolic and flavonoids content), antioxidant activity through percentage inhibition of DPPH radical scavenging activity (%), and microbial communities changes during fermentation. The microbial changes were evaluated by cell viable count (total bacteria, Lactic Acid Bacteria, and Yeast & Mold) and ITS in prepared kombucha (using red tea leaves, pandan leaves, and sucrose) supplemented with functional ingredients: T1 (hemp leaves (control)) and T2 (hemp leaves with milky mushroom flour). The results indicated that microbial consortium changed during fermentation. In the first 7 days, the levels of yeast and mold increased to 6.17 and 6.18 log CFU/mL, respectively. By day 21, the levels of both T1 and T2 continued to rise, reaching 7.78 and 7.82 log CFU/mL, respectively. The viable count of lactic acid bacteria in T1 and T2 gradually increased to 6.79 and 6.70 log CFU/mL, respectively, by day 14. These changes resulted in a marked decrease in pH value, reaching 3.63 and 3.23 in T1 and T2, respectively, by the end of the process (21 days). The total bacterial viable count decreased with an increase in the fermentation time. During fermentation, unique genera of tea fungus observed in T1 and T2 were 64% and 19%, respectively. At the beginning (0 days), the top five genera found in T1 were: <i>g__Setophoma</i> (25.91%), <i>g__Macrocybe</i> (14.88%), <i>g__Cladosporium</i> (7.81%), <i>g__Phaeosphaeria</i> (7.12%), <i>g__Malassezia</i> (6.63%), while the top five genera in T2 were <i>g__Macrocybe</i> (94.55%), <i>g__Setophoma</i> (1.87%), <i>g__Cladosporium</i> (0.77%), <i>g__Phaeosphaeria</i> (0.40%), <i>g__Cordyceps</i> (0.38%). However, on day 21 (end of the process), it was found that <i>g__Dekkera</i> had the highest relative abundance in both T1 and T2. In addition, the supplementation of the two ingredients affected the total phenolic and total flavonoid content of the treatments. At the end of the process, T2 showed values of 155.91 mg GAE/mL for total phenolics and 1.01 mg CE/mL for total flavonoids, compared to T1, which had 129.52 mg GAE/mL and 0.69 mg CE/mL, respectively. Additionally, the DPPH inhibition was higher in T1 (91.95%) compared to T2 (91.03%). The findings suggest that kombucha fermented with these innovative ingredients exhibited enhanced phytonutrients, and served as substrate for LAB and tea fungus fermentation, while limiting the growth of fungal genera and diversity of microbial consortium.