Characterisation of acetic acid bacteria and yeast isolated from kombucha produced in New Zealand : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Auckland, New Zealand

Abstract

Background: Kombucha is a popular functional tea beverage commonly fermented by a complex symbiotic culture of acetic acid bacteria (AAB) and yeast (SCOBY) in a base of sugared tea infusion at ambient temperature for 7-14 days. Regular consumption of Kombucha confers potential health benefits due to the presence of live cultures and high concentrations of bioactive components such as vitamins, polyphenols, and organic acids. However, industrial production of Kombucha faces challenges due to the limited information on the dynamic changes in its microbial community composition and the lack of knowledge regarding their health-promoting characteristics. The impact of fermentation conditions and added substrates on starter cultures, physicochemical characteristics and functional activities is also not well understood.

Objectives: This study aimed to determine the microbiological characteristics of New Zealand Kombucha starter cultures and evaluate the probiotic potential of AAB and yeast isolated from commercial Kombucha products. The bioactive components, antioxidant activity, and antimicrobial activities of Kombucha fermented using a New Zealand starter culture under different fermentation conditions were also determined.

Methods: In phase I, the dominant AAB and yeast were isolated from Kombucha (tea broth and cellulosic pellicle) and screened for their biochemical characteristics. Dominant microorganisms were identified by sequencing of the ribosomal RNA gene (16S rRNA for AAB and ITS for yeast). In phase II, 62 yeast strains and 36 AAB strains were isolated from the Kombucha starter culture and commercial Kombucha. Representative yeast (n=15) and AAB (n=9) isolates were selected for evaluation using various techniques comprising tolerance to bile salt, NaCl, different temperatures, acidity, cell surface characteristics (auto-aggregation, co-aggregation with pathogens, and hydrophobicity with chloroform), and functional activities (antioxidant, antimicrobial and bile salt hydrolase activities). The safety of the strains was evaluated by testing their susceptibility to antibiotics and enzymatic activities. In phase III, the Plackett-Burman design was applied to evaluate the impact of different fermentation conditions on the physicochemical and functional activities of black tea Kombucha. The following factors were tested: fermentation temperature (20-30℃), fermentation time (7-14 days), sugar levels (20-100 g/L), black tea levels (1.5-12 g/L), tea infusion time (5-15 min), inoculum volume (100-200 mL/L) and SCOBY (10-40 g/L). The total phenolic content (TPC) of black tea Kombucha prepared under different conditions was determined by the Folin-Ciocalteu assay, and the total flavonoid content (TFC) was measured using the modified aluminium chloride colorimetric assay. Seven individual phenolic components in the Kombucha broth were analysed by high performance liquid chromatography (HPLC). The 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ABTS+ assays were used to determine the in vitro antioxidant activity of Kombucha tea broth. Ethanol was determined by enzymatic assay and antimicrobial activity of the Kombucha was screened by agar well-diffusion assay.

Results: The dominant AAB species in the Kombucha starter culture were identified as Komagataebacter rhaeticus, which contributes to the cellulosic pellicle formation. The isolated yeast belonged to Debaryomyces prosopidis and Zygosacchromyces lentus. During fermentation, the acidity increased, while the total soluble solids decreased. Of the fifteen yeast strains tested, two strains yeast of Pichia kudraivzevii GBY1, Saccharomyces cerevisiae GBY2, three AAB strains of Acetobacter musti LOAAB1, and Gluconobacter potus (LOAAB2 and GBAAB3) isolated from New Zealand Kombucha showed promising probiotic characteristics.

Kombucha fermented under 17 different conditions generated by the Plackett-Burman design were all acidic (pH 2.55 - 3.16) with visible colour differences. Three phenolic components (gallic acid, theobromine, caffeine) were detected from all seventeen fermented Kombucha samples. All the Kombucha samples (seventeen) exhibited antioxidant activity for both DPPH (20.78 - 91.28%) and ABTS (22.63 - 551.10 µg TE/mL) relative to their total phenolic and flavonoid content. High phenolic content and strong antioxidant capacity were detected in Kombucha samples prepared with high levels of tea (12 g/L). Kombucha samples with the highest titratable acidity (1.03%) showed the strongest antimicrobial activities against Staphylococcus aureus, Bacillus cereus and Escherichia coli. The ethanol concentration of Kombucha prepared under the different conditions varied from 0.03% to 0.38% ABV and were all below the levels stipulated by Australia-New Zealand regulations for non-alcoholic beverages (1.15 % ABV).

Conclusion: The New Zealand Kombucha starter culture was composed of K. rhaeticus, D. prosopidis, and Z. lentus. Results indicated that the yeast and AAB strains isolated from commercial Kombucha produced in New Zealand may be probiotic. The antioxidant activity of Kombucha prepared under different fermentation conditions may be attributed to the phenolic components of the tea. Additionally, acidity and phenolic content may have contributed to the antimicrobial activity of the Kombucha tea against the three pathogens. Kombucha produced in this study may be considered as a functional product due to the presence of phenolic contents, potential probiotics, and antimicrobial activities against three pathogens. The results of this study showed the probiotic potential of AAB and yeast strains isolated from Kombucha based on some in vitro tests.