Optimisation of sugar concentration and fermentation temperature to produce a low sugar green tea-flavoured water kefir beverage : a thesis submitted in partial fulfilment of the requirement for the degree of Master of Food Technology, Massey University, Albany, New Zealand

Abstract

Water kefir is a refreshing, self-carbonated, slightly sweet, low-alcoholic beverage characterised by a mildly acidic taste and yeasty aroma. Water kefir is prepared on a household scale or semi-industrial scale by inoculating sugar substrate with starter culture i.e., water kefir grains, consisting of a multispecies consortium of symbiotic lactic acid bacteria and yeast embedded in a polysaccharide matrix. Some of the microorganisms found in the kefir grains are recognized as probiotics.

The improved health awareness of consumers and popular trends such as healthy hydration, low added sugar, lactose intolerance and natural products are driving the research and development of non-dairy beverages which has created a niche market for fermented beverages such as water kefir. Previous research on water kefir was mainly focused on the identification and role of microorganisms present in the kefir grains. In recent years, growing interest in fermented lactose-free water kefir have led to the exploration of various sugar substrates (raw sugar, white sugar, brown sugar, molasses and honey), plant based substrates, dried fruits (apricot, dates, figs and raisin) and fermentation factors such as sugar concentration, temperature, kefir grain concentration and fermentation time. The aim of this study was to optimise the fermentation conditions (temperature and sugar concentration) to produce a novel, diary-free low added sugar water kefir beverage using green tea.

The production of green tea flavoured water kefir with low added sugar was optimised by investigating the effect of sugar concentration (3%, 4% w/v) and fermentation temperature (22℃ and 25℃) which were conducted in three phases. Phase Ⅰ enumerated the microflora in the water kefir grains starter culture (lactic acid bacteria and yeast). During Phase Ⅱ, physicochemical analysis (pH, titratable acidity, total soluble solids, and colour), microbiological analysis and consumer sensory analysis of four different formulations were evaluated to understand the effect of sugar concentration and fermentation temperature, with the aim of selecting an optimised green tea water kefir beverage. Phase Ⅲ analysed the stability of the selected water kefir beverage during storage (4±1℃) for two weeks. HPLC analysis was used to determine concentrations of sugar, ethanol, organic acids, and antioxidants present in the selected beverage (WK2) from Phase Ⅱ.

Lactic acid bacteria (7.80±0.14 log cfu/g) and S. cerevisiae (7.25±0.03 log cfu/g) were present in the water kefir grains starter culture. In phase Ⅱ, during fermentation the pH and total soluble solids decreased (p<0.05) with a concomitant increase (p<0.05) in titratable acidity. The pH of the four formulations ranged between 3.30±0.06 to 3.50±0.00, total soluble solids between 2.35±0.07-3.82±0.03°Brix and the titratable acidity was 0.178±0.01-0.228±0.03 %. Results from Phase Ⅱ showed that sugar concentration and fermentation temperature contributed to the physico-chemical and microbial properties of green tea water kefir. Increased metabolic activity of the microorganism was observed in samples prepared at high sugar concentrations and at the higher fermentation temperature. Water kefir sample (WK2) prepared at 22℃ using the 4% (w/v) sugar concentration received the highest consumer sensory scores (p<0.05), showed optimal physico-chemical results (p<0.05) (pH (3.50±0.00), T.A. (0.178±0.01 %), TSS (3.82±0.03 °B), colour L* (97.88±0.03), colour a* (0.99±0.09) and colour b* (3.15±0.03), and microbiological results (p<0.05) (viable cell count of LAB (8.50 ± 0.01 log cfu/mL) and S. cerevisiae (7.00±0.04 log cfu/mL). Therefore, water kefir beverage sample WK2 was selected for further studies in Phase Ⅲ. In Phase Ⅲ, the viable cell counts of lactic acid bacteria (8.42±0.00 log cfu/mL) and yeast (6.85±0.00 log cfu/mL) decreased (p<0.05) during refrigerated storage (4±1℃) for two weeks.

The overall sensory acceptability of green tea water kefir remained high during the two week cold storage period. Results showed it is possible to prepare a low sugar, low alcohol, probiotic green tea water kefir beverage containing no sucrose (0%, w/v), glucose (0.94±0.04%, w/v), fructose (1.45±0.04%, w/v), ethanol (0.67±0.06%, v/v) and lactic acid (0.626±0.00%, v/v). Higher concentrations of antioxidants such as gallic acid (3.88±0.13 µg/ml), epigallocatechin (89.03±4.45 µg/ml), epigallocatechin-3-gallate (122.81±2.79 µg/ml), epicatechin-3-gallate (25.67±1.10 µg/ml), caffeine (74.44±0.52 µg/ml), and theobromine (4.24±0.03 µg/ml) were found in green tea water kefir beverage (WK2) at the end of two weeks storage compared to 28 d cold storage black tea water kefir (except gallic acid 10 ug/ml, epigallocatechin 4.1 ug/ml, epigallocatechin-3-gallate 3 ug/ml, caffeine 7 ug/ml, epicatechin-3-gallate 2 ug/ml, theobromine 6.90 ug/ml) (Subardjo, 2017). Presence of probiotic microorganisms and antioxidants in green tea water kefir may confer health benefits when consumed regularly.

To the author’s knowledge, this is the first time that green tea (0.4%, w/v) has been used to prepare a novel, lactose-free, low added sugar (4%, w/v) green tea flavoured water kefir beverage. The fermentation process involved kefir grains (5%, w/v) at 22℃ for 144 h yielding green tea flavoured water kefir beverage with optimal physico-chemical, microbial, sensory properties that meets the stipulated regulations of non-alcoholic beverages and brewed soft drinks. Further studies should be conducted to investigate the antioxidant activity of the beverage produced. Additionally, isolation and identification of the strains of water kefir grains should be conducted which could help in better understanding the microbial interactions of the kefir grains.