Multi-Strain Probiotics Enhance the Bioactivity of Cascara Kombucha during Microbial Composition-Controlled Fermentation

Abstract

Kombucha is a widely consumed fermented tea beverage with diverse health benefits. In a previous study, we demonstrated that the use of cascara as a substrate results in a special kombucha beverage with high bioactivity. Traditional kombucha fermentation using a symbiotic culture of bacteria and yeast (SCOBY) can lead to inconsistent product quality because of the lack of control over microbial composition. We successfully isolated and identified yeast and bacteria, including Saccharomyces cerevisiae, Komagataeibacter rhaeticus, and Lactobacillus brevis that are appropriate starter cultures for cascara kombucha fermentation. We also demonstrated that a supplementation with lactic acid bacteria (LAB) and a mixture of S. cerevisiae and K. rhaeticus resulted in higher total polyphenol and flavonoid content of cascara kombucha compared with the traditionally fermented product using SCOBY as the inoculum. The free radical scavenging activity, inhibitory effects on α-amylase, tyrosinase activity, and antibacterial properties of cascara kombucha were also enhanced as a result of LAB supplement. These findings provide valuable insights into the controlled microbiological composition required for the fermentation of cascara kombucha, thereby ensuring consistent quality and enhanced bioactivity of the product. Further, the use of cascara as a substrate for kombucha production not only offers various health benefits and biological effects, but also repurposes by-products from the coffee industry, which contributes to sustainable development and is eco-friendly.

Keywords: antibacterial; antioxidant; fermentation; probiotics; sustainable development.

Fig. 1

Characterization of the microbial strain responsible for kombucha fermentation. Gram stain microscopic morphology of yeast (A), acetic acid bacteria (AAB) (B), and lactic acid bacteria (LAB) (C) isolated from symbiotic culture of bacteria and yeast under a microscope at 1,000× magnification. A phylogenetic tree based on internal transcribed spacer gene sequences of yeast (D), AAB (E), and LAB (F) isolates and reference strains in the Gene Bank database.

Fig. 2

(A) The growth of acetic acid bacteria (AAB) on Carr agar supplemented with ethanol and bromocresol green resulted in a color change from green to yellow. (B) Bacterial cellulose production of AAB in Hestrin-Schramm medium.

Fig. 3

Effects of the inoculum on the growth of nascent pellicles symbiotic culture of bacteria and yeasts (SCOBYs) using a cascara substrate (A), pH values (B), total acid accumulation (C), and total polyphenol content (TPC)/total flavonoid content (TFC) (D) in cascara kombucha. Data are presented as the means of triplicate analysis±SD. *Indicate the significant difference from the SCOBY group (P<0.05). Lowercase letters (a-e) indicate significant differences between groups according to Duncan’s test (P<0.05). SK, Saccharomyces cerevisiae and Komagataeibacter rhaeticus; SK Multi-Lab, S. cerevisiae, K. rhaeticus and mixture of Lactobacillus rhamnosus, Weizmannia coagulans, and Lactobacillus brevis; SKLR, S. cerevisiae, K. rhaeticus, and L. rhamnosus; SKW, S. cerevisiae, K. rhaeticus, and W. coagulans; SKLB, S. cerevisiae, K. rhaeticus, and L. brevis.

Fig. 4

Effects of the inoculum on the free radical scavenging activities of cascara kombucha expressed by DPPH (A), and ABTS (B). Data are presented as the means of triplicate analysis±SD. Lowercase letters (a-e) indicate significant differences between groups according to Duncan’s test (P<0.05). CFS, cell-free supernatants; SCOBY, symbiotic culture of bacteria and yeast; SK, Saccharomyces cerevisiae and Komagataeibacter rhaeticus; SK Multi-Lab, S. cerevisiae, K. rhaeticus and mixture of Lactobacillus rhamnosus, Weizmannia coagulans, and Lactobacillus brevis; SKLR, S. cerevisiae, K. rhaeticus, and L. rhamnosus; SKW, S. cerevisiae, K. rhaeticus, and W. coagulans; SKLB, S. cerevisiae, K. rhaeticus, and L. brevis.

Fig. 5

Effects of the inoculum on α-amylase inhibition activity (A) and tyrosinase inhibition activity (B) of cascara kombucha. Data are presented as the means of triplicate analysis±SD. Lowercase letters (a-f) indicate significant differences between groups according to Duncan’s test (P<0.05). CFS, cell-free supernatants; SCOBY, symbiotic culture of bacteria and yeast; SK, Saccharomyces cerevisiae and Komagataeibacter rhaeticus; SK Multi-Lab, S. cerevisiae, K. rhaeticus and mixture of Lactobacillus rhamnosus, Weizmannia coagulans, and Lactobacillus brevis; SKLR, S. cerevisiae, K. rhaeticus, and L. rhamnosus; SKW, S. cerevisiae, K. rhaeticus, and W. coagulans; SKLB, S. cerevisiae, K. rhaeticus, and L. brevis.