DSpace at VNU: Screening the optimal ratio of symbiosis between isolated yeast and acetic acid bacteria strain from traditional kombucha for high-level production of glucuronic acid

Accepted Manuscript
Screening the optimal ratio of symbiosis between isolated yeast and acetic acid
bacteria strain from traditional kombucha for high-level production of glucuronic acid
Nguyen Khoi Nguyen, Phuong Bang Nguyen, Huong Thuy Nguyen, Phu Hong Le
PII:

S0023-6438(15)30032-3

DOI:

10.1016/j.lwt.2015.07.018

Reference:

YFSTL 4812

To appear in:

LWT - Food Science and Technology

Received Date: 11 March 2015
Revised Date:

26 May 2015

Accepted Date: 9 July 2015

Please cite this article as: Nguyen, N.K., Nguyen, P.B., Nguyen, H.T., Le, P.H., Screening the optimal
ratio of symbiosis between isolated yeast and acetic acid bacteria strain from traditional kombucha for
high-level production of glucuronic acid, LWT - Food Science and Technology (2015), doi: 10.1016/
j.lwt.2015.07.018.

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ACCEPTED MANUSCRIPT

Screening the optimal ratio of symbiosis between isolated yeast and acetic acid bacteria strain from traditional

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kombucha for high-level production of glucuronic acid

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Nguyen Khoi Nguyen a,*

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Email:

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Phuong Bang Nguyena

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Email:

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Huong Thuy Nguyen c

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Email:

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Phu Hong Le a,b

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Email:

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Vietnam.

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City, 70000, Vietnam.

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c

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University, Ho Chi Minh City, 70000, Vietnam.

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*Corresponding author

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Tel: +84 08 37244270; Fax: +84 08 37244271; Mobile: +84 938 105157.

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Abbreviations:

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AAB: acetic acid bacteria

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GlcUA: glucuronic acid

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KBC: kombucha

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LAB: lactic acid bacteria

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SBT: Sweetened black tea

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School of Biotechnology, International University, Vietnam National University, Ho Chi Minh City, 70000,

Center of Research and Technology Transfer, International University, Vietnam National University, Ho Chi Minh

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Department of Biotechnology, Faculty of Chemical Engineering, University of Technology, Vietnam National

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ABSTRACT

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Glucuronic acid- a human detoxifying drug can be found in traditional kombucha which is sweetened-black tea

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fermented by symbiotic microflora between yeast and acetic acid bacteria embedded within a microbial cellulose

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membrane. The main purpose of the study is to obtain the new designed symbiosis from the isolated yeasts and

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bacterial strains which can produce the high-level glucuronic acid kombucha and avoid unexpected microbial

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contaminants. The isolation, selection and identification showed the best initial combination ratio between Dekkera

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bruxellensis KN89 and Gluconacetobacter intermedius KN89 is 4Y (yeast):6A (acetic acid -bacteria) in number of

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living cell per milliliter which produced 175.8 mg L-1 glucuronic acid in 7-day fermentation (P<0.05). This study

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also provides a basic understanding about fermentation kinetics of this symbiosis in order to control and enhance the

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final products at the critical time point (after 54 hours of process). The findings of this study are practically relevant

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in producing a safe and glucuronic acid enriched kombucha

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Keywords: combination ratios, fermented tea, glucuronic acid, kombucha, sweetened-black tea.

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Chemical compounds studied in this article

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Glucuronic acid (PubChem CID: 444791)

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1. Introduction

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Kombucha or “tea fungus” is a healthy sugared tea, which is fermented by a symbiosis of acidophilic yeast, and

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acetic acid bacteria (AAB) embedded in a microbial cellulose layer (Greenwalt, Steinkraus, & Ledford, 2000). One of

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the most significant organic compounds found in this drink is glucuronic acid (GlcUA) (Jayabalan, Malbasa, Loncar,

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Vitas, & Sathishkumar, 2014). This acid has been a topic of interest in recent years, because of its detoxifying

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properties. It can eliminate many types of toxicants such as pollutants, exogenous chemicals, excess steroid

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hormones, and bilirubin from the human body via the urinary systems (Vīna, Linde, Patetko, & Semjonovs, 2013;

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Vīna, Semjonovs, Linde, & Patetko, 2013). Moreover, GlcUA can be converted into glucosamine, a beneficial

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substance associated with of cartilage, collagen, and fluids related to the treatment of osteoarthritis (Yavari, Assadi,

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Moghadam, & Larijani, 2011). GlcUA is also a precursor for the biosynthesis of vitamin C (Merchie, Lavens, &

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Sorgeloos, 1997). In kombucha, AAB assimilated the monosaccharide (glucose and fructose) products of yeast

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metabolism. These monosaccharides are the main substrates for the production of various organic compounds

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including acetic acid, gluconic acid, some micronutrients, and glucuronic acid (Vijayaraghavan et al., 2000).

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In many previous studies, the optimization of GlcUA production during the fermentative process was limited by the

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use of the whole traditional “mother-tea fungus” layer as the starter culture. This layer contains various types of

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microorganisms, and the particular yeasts or glucuronic acid producing bacterial strains had not been fully

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characterized (Vīna, Semjonovs, et al., 2013; Yavari et al., 2011). Also, kombucha may contain many contaminant

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microorganisms such as Penicillium spp. and Candida albicans, these contaminating strains can compete with the

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essential kombucha microorganism for nutrition and reduce the efficiency of fermentation and glucuronic acid

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production. Moreover, Penicillium spp., a hyalohyphomycosis mold and C. albicans are infectious and opportunistic

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human pathogens (Kumamoto & Vinces, 2005; Schinabeck & Ghannoum, 2003). Home- cultured kombucha has a

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high risk of contamination due to non-aseptic operation and careless transfer among households. Although kombucha

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is good for health, its conventional culturing method, which uses the whole kombucha layer as the starter culture,

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make it difficult to control the unwanted microorganisms including pathogenic bacteria, yeasts and mold during

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fermentation (Dufresne & Farnworth, 2000; Eric & Jessica, 2013). To produce clean and safe GlcUA-rich kombucha,

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our first and foremost priority is to develop a simple and effective microbial symbiosis model. Since yeast and AAB

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play crucial roles in kombucha, it is important to isolate and select the best strains and characterize their fermentative

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features within the symbiosis before production on an industrial scale (Achi, 2005). These objectives were addressed

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in our study.

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To manage the beneficial microbial strains in the production of GlcUA-rich kombucha, we isolated and screened for

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the most suitable yeast and bacterial strain and evaluated their ratios for GlcUA production. Another vital objective is

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to reduce the number of contaminant microorganisms in kombucha. Appropriate microbial control and biotechnology

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methods can manage the growth of microorganisms during the large-scale process and improve the quality of the

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traditional fermented tea. We believe that the results of this study will enable the beverage industry to produce higher

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quantities and qualities of healthy kombucha.

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2. Materials and Methods

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2.1 Reagents, apparatus and medium preparations.

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Traditional Kombucha with the whole cellulose membrane was kindly provided by biotechnology department,

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Faculty of Chemistry, University of Technology, Vietnam National University, Ho Chi Minh City. Black tea (Lipton,

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Unilever, London) One Liter of autoclaved sweetened black tea (SBT) contains 100 g of sucrose and extract of 1 g of

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Lipton black tea in boiling water. The new kombucha was cultured in the sweetened black tea medium by adding 5 g

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of the wet previous KBC layer to 100 mL of the tea volume. The eight-day fermented tea served as the source for

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microbial isolation.

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2.2 Isolation, phenotypic characterization and identification of Acetic acid bacteria

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Pretreatment: 5g of wet KBC layer was tore into small pieces, mixed with 5 mL solution of 10 (ml L-1) cellulase

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enzymes (Sigma-Aldrich) then incubated for 2 hours at 370C. The liquid sample was diluted then streaked onto MYP

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(D-mannitol 25 g L-1, yeast extract 5g L-1, and bacteriological peptone 3 g L-1, pH 5) agar plate and incubated for 5

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days at 28-300C (Vuyst et al., 2008). Gram-negative bacterial strains which showed the positive results according to

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AAB’s morphological, physiological and cultural characteristics were carried out in further biochemical tests.

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H2O2 3% (ml L-1) was used in catalase test. Single strain was spread on WL (Himedia, Mumbai, India) agar plates at

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pH 6.8 with the presence of 0.2 (g L-1) bromolthymol blue (pH indicator) (Sigma-Aldrich) to determine acid

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production ability (Franke et al., 1999). The yellow zone-surrounding colonies indicate the low pH area. Water-

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soluble brown pigment determination in GYP broth culture (Kadere, Miyamoto, Oniang, Kutima, & Njoroge, 2008).

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The cellulose floating layers were stained with 2-3 drops of Schulze’s reagent resulted in dark blue or black

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color(Amelio & Frank, 2002). Finally, the filtrates from 5-day fermented tea broth produced by the isolated strains

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were used for GlcUA detection. The selected AAB strains which were conferred the positive GlcUA production

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ability were selected to analyze in further experiments.

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2.3 Isolation, selection and identification of yeast strain for symbiosis combination

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Each 10 mL kombucha broth from top, middle, and bottom of fermented tea jar was collected to isolate yeast strains.

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The samples were diluted and then streaked onto 10 mg L-1 cycloheximide containing WL agar medium (Curtin,

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Bellon, Henschke, Godden, & Lopes, 2007). The incubation carried out in total 3-4 days at (29±1) 0C, the

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distinguished colonies were picked up based on the yeast morphology (Yarrow, 1998). Different isolated yeast strains

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were tested for the acid tolerant ability in pH-3 SBT medium, at (29±1) 0C for 24 hours. pH of the cultures was

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adjusted by the filtrate of 7-day fermented tea broth produce by the isolated AAB strains in previous section. Yeast’s

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densities were determined by measuring OD value (600 nm) using UV-VIS Spectrophotometer (UV-2700, Shimazu,

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Japan). The highest density yeast strain is the most highly adapted one which would be carried out in further

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experiments.

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2.4 Screening for the optimal ratio between the two isolated strains for glucuronic acid production.

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Yeast and bacteria were cultured separately in SBT medium and were determined their densities by counting colonies

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on ML agar plates at every six hours. The results were presented in form of log CFU. The inoculums of yeast and

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AAB were prepared separately in SBT medium for 72 hours at 29±10C. Then, their densities (number of living cell

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per milliliter) were determined as 23 x 108 (Yeast), 2 x 108 (AAB) (CFU mL-1) before combining two strains for

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fermentation. The ratios of living cells between these two microbial strains were obtained by different transferring

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volume from each inoculum into one final 50 mL-SBT medium containing glass. Different combinations of the two

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isolated strains were designed by increasing one’s population while decreasing the other. The central point

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experiment was set at (5:5) ratio (table 1).

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All experimental samples were fermented at 29±10C for 5 days before filtering through 0.22 µm spore size membrane

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and measuring GlcUA concentration by HPLC-MS. The unfermented tea and traditional kombucha served as the

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controls.

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2.5 Study on the kinetics fermentation of the new designed symbiosis.

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The best ratio of microbial symbiosis was used to ferment SBT medium with 100 g L-1 sucrose at 300C, pH 4.5 and for

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7 days to assess GlcUA producing, pH level, consumed sucrose, specific growth rate (µ), and velocity of glucuronic

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acid formation (ρ). GlcUA concentration was determined by HPLC-MS, pH values were checked by electronic pH

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meter (Seven CompactTM S220-K, Mettler-Toledo AG, Switzeland), consumed sucrose was obtained by the

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difference of the initial sucrose concentration and the sucrose concentration at a particular period that were measured

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using refractometer (RX-SOOOX, Atago, Japan) for every 24 hours. Specific growth rate (µ) and the velocity of

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glucuronic acid formation (ρ) were calculated by equation (1) and (2) , respectively (Doran, 2013).

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ࣆ=

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Wherein:

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xt and x0 are the microbial population (CFU mL-1) at t and initial time (obtained every six hours in this study).

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t and t0 are the t and initial time when the sample is measured.

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µ: is specific growth rate (1 h-1)

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࢚࣋ = μ࢚ . ࢅࡼ/࢙

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Wherein:

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(1)

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࢒࢔(࢚࢞ ି࢞࢕ )

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࢚ି࢚૙

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ρt: velocity of glucuronic acid formation at particular t period

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µ t: specific growth rate at specific t time.

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YP/S: yield of glucuronic acid biosynthesis. P: product (g L-1). S: substrate (g L-1)

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2.6 Quantification of glucuronic acid by HPLC-MS

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Glucuronic acid detection was performed exactly according to the method described by Nguyen, Dong, Le, and

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Nguyen (2014). Briefly, before the fermented tea samples were injected into HPLC vials, they were loaded onto an

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SPE C18 column and passed through a millipore filter (0.45 µm). Then, 20 µL of the filtrate was pumped to an HPLC

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system (Agilent 1200) equipped with a mass spectrometer (micrOTOF-Qll, Bruker) and ACE3 C-18 column (4.6 x

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150 mm) with the standard Glucuronic acid (G5269-10G, Sigma-Aldrich) for analysis. The resolution peaks recorded

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on the HPLC chromatogram report were relative to the retention time of the GlcUA standard. The concentrations

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were quantified from standard curves and multiplied by the dilution factors. The column (150 x 4.6 mm) in stationary

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phase is composed of silica particle (3.5 µm). Formic acid (1g L-1) in deionized water (A) and formic acid (1g L-1) in

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methanol (B) was used as the mobile phase. The flow rate of isocratic elution was adjusted to 0.5 mL min-1 at 40°C.

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Mass spectrometer source of electrospray ionization settings were as follows: dry gas temperature 200 0C, dry gas

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flow 10.0 L min-1, nebulizer gas pressure 1.2 bar, capillary voltage 4500 V, end plate offset -500 V. Mass spectra

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were detected from 100 to 300 m z-1 at 1 Hz acquisition speed. (Fig.2c.)

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2.7 Molecular identification of Yeast and AAB strains

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In order to avoid bias in research, we employed NK-Biotek Company (Ho Chi Minh City, Vietnam) to perform the

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sequencing analysis of 28s rRNA and 16s rRNA for yeast and AAB strain, respectively. The whole cultures of yeast

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and AAB strain on the agar plates were sent to this company and the obtained sequences were sent back to us as the

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results. Finally, we analyzed and compared the results using BLAST software />
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to figure out the specific name of each strain.

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2.8 Triangle Sensory Evaluation

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Traditional kombucha and the new designed kombucha (4Y:6A) were fermented in the same conditions 10% sucrose

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concentration, initial pH 5, 5 days, at 300C and used for sensitive test. 25 customers were required to identify the

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different sample from three kombucha samples which have two identical one (Rousseau, Meyer, & O'mahony, 2007).

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The records were analyzed by excel and SPSS 19.00 software which are available on http://www-

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01.ibm.com/software/analytics/spss/

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Results and Discussion

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3.1 Isolation, phenotypic characterization and identification of acetic acid bacteria

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One of the most abundant prokaryotes cells in traditional kombucha is AAB family which responsible for GlcUA and

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microbial cellulose membrane producing. Thereby, our experiments aim to obtain AAB with the ability of GlcUA

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production for further functional food applications. Four bacterial strains which showed gram-negative, rod shape,

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transparent and round colonies were screened for their behaviors in different culture mediums (Table 2). Only two

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strains (AAB2, AAB3) showed both abilities of microbial cellulose and glucuronic acid production then they were

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sequenced. The comparisons of sequenced nucleotide of these bacteria gave in the same name of Gluconacetobacter

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intermedius then was coded as Gluconacetobacter intermedius KN89. Our results agree with those have been

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reported in other studies.

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In many kombucha samples from Canada, UK, and US, Gluconacetobacter family was found to be predominant over

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85% in population of acetic acid bacteria (Marsh, Sullivan, Hill, Ross, & Cotter, 2014). G. intermedius has been used

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in a wide range of food, industrial and pharmaceutical applications since it is not only known as high ethanol and

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acetic acid tolerance but also as an exopolysaccharide, nanofibrillated cellulose, and glucuronic acid producer (Kose,

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Sunagawa, Yoshida, & Tajima, 2013).Therefore, G. intermedius KN89 gathers enough useful characteristics to be

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combined with yeast strain for glucuronic acid production in our further designed experiment.

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3.2 Isolation, selection and identification yeast strain for symbiosis combination

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Fig.1. shows the highest OD value of Y2 strains that reflects its high adaptation in low pH medium. The sequenced

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Y2 strain shows the result of Dekkera bruxellensis and was coded as Dekkera bruxellensis KN89. This species is

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familiar with kombucha microbial association as it also has been identified in other studied. D. bruxellensis showed

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the advantage in high-ethanol and acid tolerance (Schifferdecker, Dashko, Ishchuk, & Piškur, 2014) and even higher

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in biomass yield, ethanol production compared to other industrial strains such as Saccharomyces cerevisiae due to its

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higher efficiency in energy metabolite (Blomqvist, Nogué, Gorwa-Grauslund, & Passoth, 2012). In addition, D.

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bruxellensis is able to consume nitrogenous compounds, and metabolize phenolic acids to vinylphenol and ethyl

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derivatives. These organic compounds are regarded as the contribution to the flavor of some fermented food like

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Lambic beer, sourdough and kombucha (Pita, Silva, Simoes, Passoth, & Morais, 2013; Schifferdecker et al., 2014). In

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general, this yeast strain is qualified enough to be combined with G. intermedius KN89 for further kombucha

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fermentation. Yeast plays an important role in kombucha not only as ethanol producer but also as a component in the

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microbial symbiosis that supports AAB in fermentation (Jayabalan et al., 2014). Hence, the selection and

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enhancement of yeast fermentative characteristics for the industrial process have drawn much scientific attention, i.e.

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ability of acid tolerance (Mitsumasu et al., 2014). Prolonging fermentative process in kombucha always reduces the

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pH of the tea broth thus in order to maintain the equilibrium of yeast and bacteria in their symbiosis, yeast strain

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should has high acid-tolerance.

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3.3 Screening for the optimal ratio between Dekkera bruxellensis KN89 and Gluconacetobacter intermedius

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KN89 for glucuronic acid production

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The original ratio between yeast and AAB in kombucha brewing is vital for its metabolites production. Since both

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strains can consume sucrose in the medium, the increasing of one’s initial population size may rapidly colonize the

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niches, dominate the other strain and cause the influences on the composition of the final metabolites compounds as

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the consequence of microbial competition (Hibbing, Fuqua, Parsek, & Peterson, 2010). Thus, screening of different

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initial amount of living cell between these two components is essential, particularly, in reformation of the symbiosis

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for specific fermentative purpose. Fig.2a. demonstrates the concentration of glucuronic acid produced by different

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combination ratios between D. bruxellensis KN89 and G. intermedius KN89. The unfermented sample (only tea

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broth) showed only 1.06e ± 0.8 mg L-1 glucuronic acid while the combination 4Y6A gave the highest one of 102.56a ±

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3.55 mg L-1. The traditional culture and the 3Y7A symbiosis produced the similar level of glucuronic acid (30.3d ±

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2.3 and 28.7d ± 1.45 mg L-1). Other ratios of symbiosis presented the moderated levels within a range from 32.3c±3.5

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to 44.76b ± 1.75 mg L-1 (Fig.2a.)

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The new symbiosis of single yeast and a bacterial strain showed more effective in glucuronic acid production

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compared to traditional kombucha. The unwanted microbial strains were removed; hence their competitions no longer

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exist. This improvement not only supports for the growths of D. bruxellensis KN89, G. intermedius KN89 but also

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helps control fewer microbial strains in large scale process and avoid the risks of health problems from unexpected

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microorganisms such as Candida albicans and Penicillium spp. (Dufresne & Farnworth, 2000). However, the

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glucuronic acid amount of 4Y6A ratio (102.56a ± 3.55mg L-1) increased dramatically and fell down immediately at

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3Y7A ratio (28.7d ± 1.45 mg L-1), additional ratio (4Y7A; 3Y6A) were carried out to have a closer evaluation of the

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glucuronic acid production.

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Yeast not only contributes to special flavor of kombucha but also stimulates AAB in GlcUA production. Most

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combinations (7Y3A: 44.26b ± 3.2; 6Y4A: 42.36b ± 1.5; 5Y5A: 44.76b ± 1.75; 4Y6A: 102.56a ± 3.55; 4Y7A: 40.26b ±

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4.5) (mg L-1) gave the higher GlcUA amount compared to those produced by single culture of AAB (36.1c±5 mg L-1).

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This result emphasized the advantaged GlcUA production of designed symbiosis which reflects the microbial

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association through their metabolisms. Yeast breakdowns sucrose into glucose and fructose and then uses fructose for

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ethanol production rather than glucose. AAB consumes glucose and ethanol to produce organic acid and microbial

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cellulose (Dufresne & Farnworth, 2000) . In addition, some other characteristics of D. bruxellensis KN89 could bring

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advantages in GlcUA excretion by G. intermedius KN89. It can produce and raise the level of acetic acid in the

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environment that triggers the feedback inhibition in glycolysis metabolism of G. intermedius KN89, results in

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stimulation of glucuronate pathway in bacteria (Freer, Dien, & Matsuda, 2003). Moreover, the metabolization of

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cinnamic acid to vinylphenol derivatives by D. bruxellensis KN89 not only contribute to special taste in its original

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fermented food habitats but also support for the growth of bacteria. Since cinnamic acid has negative effects to

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microbial propagation, it is reduced to less toxic compounds by the yeast metabolism (Schifferdecker et al., 2014).

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Glucuronic acid concentration has therefore can be increased by these mechanisms.

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Role of yeast in this combination can be determined by its population when their interactions directly effect on the

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glucuronic acid production of symbiosis.

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In Fig.2b, the yeast started exponential and stationary phase (12 – 30 hrs) earlier than bacteria did (18 – 60 hrs). This

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result is vital to explain the effects of different ratios in GlcUA production. When the initial amount of yeast was

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equal or higher than amount of AAB (7Y3A; 6Y4A; 5Y5A), the yeast could get over advantaged and compete with

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the bacterial population, hence, the GlcUA concentrations were average (42 - 45 mg L-1). In other cases, when initial

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AAB population was higher than yeast or even there was no yeast in the last sample (4Y7A; 3Y6A; 3Y7A; A), AAB

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predominated over yeast strain. Thus, supports from yeast for AAB to produce GlcUA were reduced either. With

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4Y6A ratio, when AAB strain gathered appropriate population and supports from yeast, this symbiosis generated the

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highest GlcUA concentration (102.56a ± 3.55mg L-1). The new designed symbiosis allowed the more flexibility and

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less complication in microbial control as well as significant productivity of GlcUA.

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3.4 Study on the kinetic fermentation of the new designed symbiosis.

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Since the kinetic fermentation plays a vital role in large-scale operation even in modification of fermentation types

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which affect to the productivity, more research into fermentative behaviors of the new designed symbiosis (4Y:6A) is

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essential such as the specific growth rate (µ) and velocity of product formation (ρ). (Doran, 2013).

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In this study, we accessed the growth phase of the bacteria to obtain µ and measured the consumed sucrose, pH, and

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GlcUA concentration every 24 hours to evaluate ρ of GlcUA formation. In Fig.3. G. intermedius KN89’s log phase

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ends after 54 hours when µ change its direction. At this time, ρ continues increasing significantly. It is indicated that

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GlcUA is the primary metabolite that was highly produced at the beginning of stationary phase of G. intermedius.

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More illustrations have been shown in Fig.4. When GlcUA amounts went up slowly in the first two day, and

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dramatically increased after the 3rd day of fermentation (93.6c – 131.8b mg L-1) due to the increases of ρ. The 54th

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hours of fermentation was an important critical time point that can be intervened by changing batch fermentation to

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continuous fermentation in order to prolong the highest µ value status to obtain the highest yield of GlcUA

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production. In addition, evaluations of µ can determine the possible explanation in pH level and the consumed

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sucrose. The consumed sucrose level reflects the growth phase of the microorganism; the higher population the larger

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sucrose amount was utilized (Fig.5). The pH level dropped down quickly as the beginning of process but it was slow

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down after 72 hours due to the feedback inhibition in acetic acid production, when the microorganism is on the

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stationary phase (Fig. 5.).

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3.5 Triangle Sensory Evaluation

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In table 3, among 24 customers participated in the sensitive test, there were only one can distinguish the different

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sample from three kombucha samples. With the high reliability of 95%, we can conclude that there is no difference in

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organoleptic perception to consumers between the new designed kombucha and traditional one.

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bruxellensis KN89 and G. intermedius KN89 which were isolated from the traditional kombucha are very common

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and can occupy more than 85% of microorganism population of conventional tea culture (Marsh et al., 2014). In

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addition, the new fermented tea sample and the traditional one were cultured in the same conditions such as substrate,

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time, pH and temperature. Therefore, regarding the organoleptic perception, these two products are the same.

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Although kombucha can be contaminated by some pathogenic microorganisms, the new designed kombucha which

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was fermented by selected yeast and AAB; carried out in aseptic conditions can avoid the risk. Besides, another

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advantage of the new kombucha is its high GlcUA concentration. These are very promising results for developing

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new product not only due to its high acceptance flavor and taste but also its improved bioactivities and safety

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compared to traditional kombucha. However, due to the high acidity during the long fermentation, people with

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gastropathy problems may get susceptibility when drinking kombucha (Dufresne & Farnworth, 2000). Hence, further

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experiment should focus on reducing the acidity while maintaining the bioactivities.

In fact, D.

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Conclusion

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Since the microorganism plays a significant role in final fermented products, our findings have contributed a further

282

step in improvement of traditional fermented tea. The new designed symbiosis 4Y6A between the isolated yeast (D.

283

bruxellensis KN89) and AAB (G. intermedius KN89) which showed the highest glucuronic acid production up to

284

175.8 ±7(mg L-1) in 7-day fermentation is ready for further applications in pilot scale of production. Moreover,

285

identification of specific growth rate of G. intermedius KN89 and the velocity of GlcUA formation have provide a

286

critical time point for intervention of fermentative technology. After 54 hours of the process, G. intermedius had

287

reached its stationary phase and increased the GlcUA production velocity. It was the most suitable time to modify the

288

process by supplying more nutrition, growth factors, or switching from batch fermentation to continue fermentation

289

in order to prolong the high potential stage and obtain large amount of GlcUA. However, optimization of

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fermentative conditions for this new designed symbiosis is required before industrial application. These preliminary

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findings are essential for a high quantity and quality of GlcUA-rich kombucha production.

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Conflict of interest statement

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The authors declare that there is no conflict of interest

295

Role of the funding source

296

This study was not supported by grants. The corresponding author and his beloved family provided the finance for the

297

research. HNT and PLH are the sponsors of this study who contribute in raw material pretreatment, and in the

298

decision to submit the article for publication.

299

Acknowledgements

300

The authors would like to give special thanks to Manh Khac Nguyen (Mr) for his helps in performance glucuronic

301

acid detection.

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Reference (Endnote)

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Table 1
The combination ratios between yeast and AAB living cells in reformation of kombucha symbiosis for glucuronic
production
AAB
3
4
5
6
7
Yeast
7
7Y:3A
6
6Y:4A
5
5Y:5A
4
4Y:6A
3
3Y:7A
0
A(no yeast)

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A: acetic acid bacteria, Y: yeast. Records of viable cells counting in the inoculums of yeast and AAB are 23 x 108 and 2 x 108
(CFU mL-1), respectively. Different ratios reflect the different transferring volumes from the inoculum into the total 50-mL SBT
medium containing glass for the symbiotic fermentation.


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Strain
AAB1
AAB2
AAB3
AAB4

Catalase
Activity

Acid
producing

+
+
+
+

+
+
+
+

Watersoluble
brown
pigment
+
+
-

Bacterial
floating
layer

Glucuronic
acid

+
+
+

NA

+
+
-

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Table 2
Phenotypic characterization of isolated acetic acid bacteria from kombucha

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AAB1, AAB2, AAB3, AAB4 are the coded names of distinguished gram-negative bacteria strains which showed the positive
results according to the AAB’s morphological, physiological, and culture characteristics. These strains were tested in their
biochemical experiments; the glucuronic acid producing strains were collected for molecular identification.
+: positive results, - : negative results, NA: no assessment.

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Table 3
The triangle sensory test to evaluation the customer’s perception of traditional kombucha and new designed
kombucha
T

Customer’s
number
7
8
9
10
11
12

F
x
x
x
x
x
x

T

F
x

X

x
x
x
x

Customer’s
number
13
14
15
16
17
18

T

F
x
x
x
x
x
x

Customer’s
number
19
20
21
22

23
24

T

F
x
x
x
x
x
x

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Customer’s
number
1
2
3
4
5
6

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The customer has to find out 1 different sample among three kombucha tea samples which have two identical one.
T: True (the customer can distinguish the different sample); F: False (the customer can NOT distinguish the different sample)
Total number of customer: 24. ( p<0.05)


0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0

a

b

b

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OD value
at 600 nm

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Sweetened black
tea

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Fig.1. The optical density (OD) of different isolated yeast cultures ( Y1, Y2, Y3) measured at 600nm wave
length. The highest OD value reflects the most highly adapted yeast strain in pH 3 SBT medium. Mean with various
letter are significantly different (P< 0.05) (N=3)

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a

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80

b

b

b

b

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60

c

c

40

d

d

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Glucuronic acid production in
different combination ratio (mg L-1)

100

20
e
0

TE
D

U.Tea T.Tea 7Y3A 6Y4A 5Y5A 4Y6A 4Y7A 3Y6A 3Y7A
A
Different combination ratios of new designed microbial symbiosis

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Fig.2a. Glucuronic acid production by different new designs in microbial symbiosis of kombucha
Y: yeast; A: acetic acid bacteria; U.Tea: unfermented tea (control); T. Tea (Traditional fermented tea-traditional
kombucha fermented by a “mother” starter kombucha layer). ( ) glucuronic acid production by different ratios of the
designed symbiosis between yeast and AAB; Since the glucuronic acid amount of 4Y6A ratio increased dramatically

and fell down immediately at 3Y7A ratio, additional ratio ( ) (4Y7A; 3Y6A) were carried out to have a closer
evaluation of the glucuronic acid production. Mean with various letter are significantly different (P< 0.05) (N= 3).


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Gluconacetobacter
intermedius

6

Dekkra bruxellensis

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4

2

0
0

6

12

18

24

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Growth phase (log CFU mL-1)

10

30

36

42

48

54

60

66

72

Time of fermentation (hours)

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Fig.2b. Growth phases of ( ) Dekkera bruxellensis & ( ) Gluconacetobacter intermedius in SBT medium were
indicated by the number of counted viable cells in every six hours which presented in form of logCFU mL-1. (P<
0.05) (N= 3).


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Fig.2c. The Glucuronic acid chromatogram report of HPLC-MS performance. The molecular mass of Glucuronate
anion is 193.0342 g/mol. Approximately 3 minutes of the glucuronic acid retention time was recorded.


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Time of fermentation (days)
2

1

3

a

0.024

0.25

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b
bc

cd
de

0.2

a

de

0.02

cd

ef

0.018

SC

f
0.15

b

0.1

c

0.05

g

h
gh
0
12

18

24

30

36

42

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6

0.016

54

60

66

0.014
0.012

Velocity of glucuronic acid synthesis (ρ)

0.022

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Specific growth rate of Gluconacetobacter intermedius (µ)

0.3

0.01
0.008

72

Time of fermentation (hrs)

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Fig.3. Specific growth rate µ (1 h-1) of G. intermedius and velocity of GlcUA formation ρ were checked at different

period of time.
Means in the same line that do not share a letter are significantly different (P <0.05)
(N=3)


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200

a

180

ab
b

140
120
100
80

b

c
d

d

1

2

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Glucuronic acid
concentration (mg L-1)

160

60
40
20

SC

0

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3
4
5
Time of fermentation (days)

6

7

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Fig.4. Glucuronic acid production of the symbiosis designed by 4Y6A ratio between yeast and AAB were checked
every 24 hours by HPLC-MS. Means that do not share a letter are significantly different (P <0.05) (N=3).


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4.5

a

a

0.8
b

3.5

0.4

c
cd

3

d
e

e

d

3
4
5
Time of fermentation (days)

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e

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1

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0.6

SC

pH level

c

6

Consumed sucrose (%) (g L-1)

b

4

b

e
0
7

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Fig.5. pH levels and the consumed sucrose (g L-1) of the symbiosis designed by 4Y6A ratio between yeast and AAB
were checked every 24 hours
.
. Means that do not share a letter are significantly different
(P <0.05) (N=3).


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Screening the optimal ratio of symbiosis between isolated yeast and acetic acid bacteria strain from

2

traditional kombucha for high-level production of glucuronic acid

3

Nguyen Khoi Nguyen a,*

4

Email:

5

Phuong Bang Nguyena

6

Email:

7

Huong Thuy Nguyen c

8

Email:

9

Phu Hong Le a,b

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Email:

11

a

12

Vietnam.

13

b

14

City, 70000, Vietnam.

15

c

16

University, Ho Chi Minh City, 70000, Vietnam.

17

*Corresponding author

18

Tel: +84 08 37244270; Fax: +84 08 37244271; Mobile: +84 938 105157.

19

Highlights

20

► Glucuronic acid has a detoxifying property against drug, bilirubin and chemicals

21

► Traditional kombucha is a rich source of glucuronic acid

22

► D. bruxellensis KN89 and G. intermedius KN89 were isolated from kombucha

23

► New designed microbial symbiosis improved significantly glucuronic acid production

24

► Using fermentation kinetics to increase final products.

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School of Biotechnology, International University, Vietnam National University, Ho Chi Minh City, 70000,

Center of Research and Technology Transfer, International University, Vietnam National University, Ho Chi Minh

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Department of Biotechnology, Faculty of Chemical Engineering, University of Technology, Vietnam National