Food Biotechnology

ISSN: 0890-5436 (Print) 1532-4249 (Online) Journal homepage: http://www.tandfonline.com/loi/lfbt20

Effects of Lactobacillus casei and Alterations

in Fermentation Conditions on Biosynthesis
of Glucuronic Acid by a Dekkera bruxellensisGluconacetobacter intermedius Kombucha
Symbiosis Model System
Nguyen Khoi Nguyen, Huong Thuy Nguyen & Phu Hong Le
To cite this article: Nguyen Khoi Nguyen, Huong Thuy Nguyen & Phu Hong Le (2015) Effects of
Lactobacillus casei and Alterations in Fermentation Conditions on Biosynthesis of Glucuronic
Acid by a Dekkera bruxellensis-Gluconacetobacter intermedius Kombucha Symbiosis Model
System, Food Biotechnology, 29:4, 356-370, DOI: 10.1080/08905436.2015.1092446
To link to this article: http://dx.doi.org/10.1080/08905436.2015.1092446

Published online: 11 Dec 2015.

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Food Biotechnology, 29:356370, 2015

Copyright Taylor & Francis Group, LLC
ISSN: 0890-5436 print / 1532-4249 online
DOI: 10.1080/08905436.2015.1092446

Effects of Lactobacillus casei

and Alterations in Fermentation
Conditions on Biosynthesis of
Glucuronic Acid by a Dekkera
bruxellensisGluconacetobacter
intermedius Kombucha
Symbiosis Model System
Nguyen Khoi Nguyen1 , Huong Thuy Nguyen3,
and Phu Hong Le1,2
1

School of Biotechnology, International University, Vietnam National University, Ho

Chi Minh City, Vietnam
2
Center of Research and Technology Transfer, International University, Vietnam
National University, Ho Chi Minh City, Vietnam
3
Department of Biotechnology, Faculty of Chemical Engineering, University of
Technology, Vietnam National University, Ho Chi Minh City, Vietnam
A combination of Dekkera bruxellensis and Gluconacetobacter intermedius is used as a
model system to emulate the microbial symbiosis between yeast and acetic acid bacteria in kombucha, and to study glucuronic acid (GlcUA) production in this system. In the
current study, the addition of Lactobacillus casei and variations in the fermentative
conditions, such as sucrose concentration, temperature and duration, were examined
to evaluate their effects on GlcUA formation. Results showed that L. casei acts as a
supporter species and stimulated GlcUA production in the kombucha symbiosis model
to levels 54.1% greater than in the control. Varying the fermentation parameters also
affected GlcUA production. Therefore, the range of each factor value was screened
and restricted to achieve a higher amount of target acid, as it was 515%, 2531 C,
37 days, and 57 units in sucrose concentration, temperature, fermentation time and

Address correspondence to Nguyen Khoi Nguyen, School of Biotechnology, International

University, Vietnam National University, Ho Chi Minh City, 70000, Vietnam; E-mail:
nguyenkhoibiotech@gmail.com
Color versions of one or more of the figures in the article can be found online at www.
tandfonline.com/lfbt.

Effects of Lactobacillus casei

initial pH value, respectively. These findings are an important step for optimizing GlcUA
production by this model system.

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Key Words:
Dekkera bruxellensis; Gluconacetobacter intermedius; glucuronic acid;
kombucha; Lactobacillus casei; microbial symbiosis

INTRODUCTION
Traditional kombucha, or mushroom tea, is a sweetened black tea that
is fermented by a complex symbiosis between yeast and acetic acid bacteria (AAB) covered by a microbial cellulose membrane (Dufresne and
Farnworth, 2000). The AAB in kombucha belong to the genera Acetobacter,
Gluconobacter, Bacterium, and Gluconacetobacter, with the yeast genera
including Brettanomyces, Saccharomyces, Zygosaccharomyces, and Pichia (Liu
et al., 1996; Teoh et al., 2004). The novel yeast strain Dekkera bruxellensis,
also known as anamorphic Brettanomyces bruxellensis, which shows asexual
reproduction, has been isolated from kombucha. This strain has many helpful
fermentative characteristics, including a natural ability to produce bio-ethanol
from lignocellulosic biomass, potential to ferment sucrose from sugarcane juice,
and the ability to digest nitrate and other nitrogenous compounds (Pita et al.,
2013; Reis et al., 2014). In kombucha, D. bruxellensis is usually a co-fermenting
synergistic pair with Gluconacetobacter intermedius, which is a member of
the Acetobacteraceae family previously known as Acetobacter intermedius. The
Acetobacteraceae are a group of Gram-negative, obligate-aerobic AAB that perform several incomplete oxidation reactions to allow them to colonize both fruit
and flowers (Hommel and Ahnert, 2000). Because Gluconacetobacter species
can produce microbial cellulose, oxidize ethanol/sugar to organic acid, and tolerate an environment high in ethanol/acetic acid, they are widely used in the
production of nata de coco, vinegar, acetic acid, and kombucha, among others
(Iida et al., 2008).
In recent years, kombucha has drawn much scientific attention because
it is a rich source of glucuronic acid (GlcUA), which is synthesized by AAB
during fermentation. GlcUA is a carboxylic acid, and acts as a detoxifying
agent to remove exogenous chemicals from the human body (Vina et al., 2013).
Hence, many studies have focused on enhancing the production of this food and
pharmaceutical substance in kombucha by modifying the substrate, enhancing co-fermentation with the culture with Lactobacillus species (Nguyen et al.,
2015a), or optimizing the fermentation conditions (Vna et al., 2013). However,
to improve the production of GlcUA in kombucha, the interactions and the
roles of the microorganisms must first be understood. In our previous study,
Lactobacillus casei showed potential for enhancing GlcUA production in traditional fermented tea (Nguyen et al., 2015a). However, the exact role of L.
casei and its effect on GlcUA production remain unclear because traditional
kombucha contains a variety of bacterial and yeast strains whose interactions

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N.-K. Nguyen et al.

are unpredictable. Additionally, different microorganisms react differently to

environmental conditions; hence, identification of specific bacteria or yeasts in
kombucha is vital for studies on production of metabolites under distinct fermentation conditions. In this study, we examined the effects of L. casei synergy
on GlcUA production by a model kombucha culture system. D. bruxellensis and
G. intermedius were selected as representative yeast and AAB species, respectively, and were combined to form a simple model of kombucha symbiosis that
allowed us to study metabolite production, and to more accurately predict the
behavior of the strains following alteration of the environmental conditions.
We first assessed the effects by treating with different amounts of L. casei on
GlcUA production, and then used biochemical analyses and high performance
liquid chromatography-mass spectrometry (HPLC-MS) to predict the role of L.
casei in the fermentation reaction. Variations of the fermentation conditions
(sucrose concentration, temperature, and time) were then evaluated in combination with L. casei treatment in the kombucha symbiosis model to determine
their effects on GlcUA production. This study helps to understand the role of
L. casei in GlcUA production, and further optimization of this pharmaceutical
acid in large-scale production.

MATERIALS AND METHODS

Chemicals, Raw Materials and Medium Preparations
Freeze-dried Dekkera bruxellensis and Gluconacetobacter intermedius
strains, which had their origins from conventional kombucha, were selected
from the microbial collections of Biotechnology Department, Faculty of
Chemical Engineering, University of Technology, Vietnam National University,
Ho Chi Minh City (Nguyen et al., 2015b). Lactobacillus casei was isolated
from kefir milk in our previous study (Nguyen et al., 2015a). Carbazole
(C5132, Sigma-Aldrich), Cetylpyridinium chloride (C0732, Sigma-Aldrich) and
Disodium Tetraborate (221732, Sigma-Aldrich, St. Louis, MO, USA) were used
to detect GlcUA and other glucuronic part-containing acid. Sweetened black
tea (SBT) medium used to carry out the fermentation, were prepared by method
described by (Nguyen et al., 2015a). Briefly, one liter of autoclaved medium contains 100 g of sucrose and extract of 1 g of Lipton black tea (Unilever, London)
in boiling water.

Microbial Culture and Preparation for the Fermentation

MRS medium (11066, Merck Millipore, Germany), Yeast extract peptone
dextrose broth medium (YPD broth, Y1375, Sigma-Aldrich) and Wallerstein
Laboratory Nutrient Broth (WL) medium (17215, Sigma-Aldrich) were used to
activate L. casei, D. bruxellensis, and G. intermedius for 24 h at 30 C. A volume

Effects of Lactobacillus casei

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of 100 L of each culture was transferred to 50-mL SBT medium and incubated
for 24, 48, and 72 h at 30 C, respectively. Numbers of living cell per milliliter
of each inoculum were counted on agar plate and recorded as 23 x 108 , 2 x 108 ,
and 38 x 108 (CFU/mL).

Effects of Different Ratios of L. casei on Glucuronic Acid

Production
The experiment was carried out by a combination of three microbial strains.
The ratio of living cells between three strains were obtained by transferring
specific volume of each microbial inoculum to a new 50-mL SBT medium in
total (Table 1).

Prediction of the Role of L. casei in GlcUA Production

GlcUA and hyaluronic acid were assessed in different types of filtrate
form SBT cultures of L. casei with and without combination of yeast and
AAB (D. bruxellensis & G. intermedius) using HPLC-MS and Cetylpyridinium
chloride (CPC), respectively.
Precipitation of hyaluronic acid was performed by adding 2 mL of CPC 1%
into the filtrate from fermented tea broth and the result was compared with
Table 1: Summary of experimental designs.
Other conditions
No

Examined
conditions

>1

Ratios of
Lactobacillus
casei

Sucrose % (g/L)

Time (days)

4
5

pH
Temperature
( C)

Value

Ferment

0.5; 1; 1.5; 2; Ratio between

2.5
yeast and
AAB
(kombucha
model
symbiosis) (1:
1.5)
5; 7.5; 10;
Ratio between
12.5; 15;
yeast, AAB
17.5
and LAB
(1:1.5;1)
3; 5; 7; 9; 11;
13
4; 5; 6; 7; 8
22; 27; 32;
37; 42

Sucrose Time Temperature

% (g/L) (days)
( C)

10

37

37

10

37

10
10

5
5

37

The ratios between each component in the microbial symbiosis were obtained by transferring specific living cell-containing volume (mL) from each individual inoculum to one new
50-mL SBT medium for fermentation based on CFU/mL of each strain. All of experiments are
independent.

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N.-K. Nguyen et al.

a positive control of sodium hyaluronate (H1876, Sigma-Aldrich) (Selyanin

et al., 2015).

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Verification of the GlcUA Enhancement by L. casei

Effects of three potential ratios of L. casei selected from previous
experiment on GlcUA production were verified by HPLC system (Agilent
1200) equipped with a mass spectrometer (micrOTOF-Qll, Bruker) with a positive control of glucuronic acid (G5269-10G,Sigma-Aldrich). The control sample
contained no L. casei but a microbial symbiosis of yeast (D. bruxellensis) and
AAB (G. intermedius) with (1;1.5) ratio, respectively. Traditional kombucha and
SBT medium fermented only by G. intermedius were also examined.

Quantification of Glucuronic Acid by HPLC-MS

Glucuronic acid determination followed the procedure described by Nguyen
et al. (2015b). Briefly, before the fermented tea samples were injected into
HPLC vials, they were loaded onto an SPE C18 column and passed through
a millipore filter (0.45 m). Then, 20 L of the filtrate was pumped to an
HPLC system equipped with a mass spectrometer and ACE3 C-18 column for
analysis. The column (150 x 4.6 mm) in stationary phase is composed of silica
particle (3.5 m). Formic acid (1g/L) in deionized water (A) and formic acid
(1g/L) in methanol (B) was used as the mobile phase. The flow rate of isocratic
elution was adjusted to 0.5 mL/min at 40 C.The resolution peaks recorded
on the HPLC chromatogram report were relative to the retention time of the
GlcUA standard.

Glucuronic Acid Production under Different Fermentative

Conditions: Sucrose Concentration, Time, pH and Temperature
GlcUA production was studied in different fermentative conditions. Four
independent experiments were described in Table 1.

Detection of Glucuronic Acid by Carbazole Reaction

Detection of GlcUA was processed as the method using carbazole
and sodium borate under sulfuric acid environment described in British
Pharmacopoeia 2007 (British Pharmacopoeia Commission, 2006). The sample
changed from colorless to purple red color, showed the positive result with
GlcUA.

Statistical Analysis
All the experiments were performed in triplicate for each sample, and the
analysis of variance (ANOVA) was performed using Minitab software version

Effects of Lactobacillus casei

16.00 available at www.minitab.com. A difference was considered statistically

significant if p0.05.

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RESULTS AND DISCUSSION

Effects of Different Ratios of L. casei on GlcUA Production
Our previous study showed that L. casei isolated from kefir was superior
to other Lactobacillus strains in improving GlcUA production in traditional
kombucha (Nguyen et al., 2015a). However, traditional kombucha contains
multiple different microorganisms and it is difficult to interpret the many
microbial interactions, let alone elucidate the role of one specific microorganism. Therefore, D. bruxellensis and G. intermedius were selected as representative strains to generate a kombucha microbial symbiosis model for closer examination of the effects of L. casei treatment and synergy on GlcUA production.
Fig. 1 shows GlcUA production by the model system when treated with different amounts of L. casei. Although the graph shows fluctuations, the highest
concentration of GlcUA was recorded at a ratio of 1:1.5:0.5 (D. bruxellensis:
G. intermedius: L. casei). This microbial combination produced 20.4% more
GlcUA than the control culture (674.5a 12 mg/L vs. 560c 26 mg/L). At a
ratio of 1:1.5:1, GlcUA production decreased, but increased again at a ratio of
1:1.5:1.5 (D. bruxellensis: G. intermedius: L. casei)
Our previous study showed that treatment of a traditional kombucha culture with L. casei enhanced GlcUA production by up to 39.6% compared with
the control culture (without L. casei) (Nguyen et al., 2014; Nguyen et al.,
2015a). While it is too early to compare the effect of L. casei on GlcUA production in traditional kombucha culture based on the effect in the model symbiosis
system, our results clearly show that L. casei treatment has a positive effect on
GlcUA production in both cases. Further investigation should be carried out to
understand the interaction between L. casei and the two other strains in the
model system, and how this effects GlcUA production.

Prediction of the Role of L. casei in GlcUA Production

The role of Lactobacillus strains in combination with AAB in the production of GlcUA and D-saccharic acid 1,4 lactone (DSL) in kombucha has
recently been the focus of several studies. Lactobacilli stimulated DSL production by Gluconacetobacter sp. A4 (Yang et al., 2010), and improved GlcUA
concentrations in traditional kombucha (Nguyen et al., 2014; Nguyen et al.,
2015a). In our previous study, GlcUA was detected by carbazole reaction.
However, the role of L. casei remained unclear because we could not know
whether this increase in GlcUA concentration was caused by the GlcUA
excreted by L. casei itself, or whether it induced an increase in expression by AAB present in the kombucha culture. In addition, some L. casei

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N.-K. Nguyen et al.

Lactobacillus casei ratio in number of living cell (CFU/mL)
Control

0.5

1.5

2.5

Detection of GlcUA by HPLC-MS (mg/L)

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450
a
400
350

ab

700

bc

bc

600

300

a
500

250
b

200

c
400

150
100
d

300

50
0

Traditional
kombucha

Control

0.5

1.5

G.I

Determination of GlcUA by carbazole (mg/L)

800

500

200

Figure 1: Glucuronic acid production from different combinations between Lactobacillus casei

and the former kombucha symbiosis (Dekkera bruxellensis & Gluconacetobacter intermedius).
( ) Effects of different ratios of L. casei on the glucuronic acid production were firstly screened
by carbazole reaction. ( ) Three most effected ratios of L. casei were checked for confirmation of its positive enhancement in glucuronic acid production by HPLC-MS. The ratio of
microorganisms were obtained by transferring specific living cell-containing volume (mL) from
its individual inoculum to the new 50-mL SBT medium for fermentation based on its CFU/mL.
The control sample contained no L. casei but kombucha symbiosis of yeast (D. bruxellensis)
and AAB (G. intermedius) with (1;1.5) ratio. G.I: contained 100% of G. intermedius. Traditional
kombucha fermented by the mother kombucha layer (starter culture). Means do not share
the letter (a-d) are significantly different (N = 3).

strains produce hyaluronic acid, which can react with carbazole and give
false-positive results (Hor et al., 2014; Selyanin et al., 2015) (Pham &
Tu, 2010) (Fig. 2). Therefore in the current study, cetylpyridinium chloride
(CPC) was used to detect hyaluronic acid, and HPLC-MS was used to identify GlcUA in the filtrates of L. casei cultured alone, and in co-culture with the
kombucha symbiosis model species (Table 2). Results from each sample were
compared with a positive control of sodium hyaluronate and D-glucuronic acid.
Negative results were obtained for all samples, regardless of the presence of
D. bruxellensis and G. intermedius, indicating that the L. casei strain used in
this study does not excrete either hyaluronic acid or GlcUA in SBT medium.
This suggests that the L. casei strain only plays a supporting co-culture role
in the biotransformation towards production of GlcUA by G. intermedius. Our
results agree with a previous report by Yang et al. (2010), which suggested that
Lactobacilli excrete other metabolites that assist the growth and metabolism

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Effects of Lactobacillus casei

Figure 2: Mechanism of glucuronic acid in the reaction with carbazole. Since hyaluronic acid
composed by glucuronic acid, it still can react with carbazole in acid environment to form a
purple-red complex.

Table 2: Screening for hyaluronic acid/glucuronic acid production of Lactobacillus

casei in different culture medium.

No

1
2
3
4

Sample

Filtrate from Lc individual culture

in SBT medium
Filtrate from the co-culture of Lc
with Db & Gi in SBT medium
Sodium hyaluronate
D-GlcUA

Precipitation by
Cetylpyidinium
chloride

Glucuronic
acid detection
by HPLC-MS

NE

+
NE

NE
+

Lc: Lactobacillus casei, Db: Dekkera bruxellensis, Gi: Gluconacetobacter intermedius (+) positive results; (-) negative results; (NE) No experiment. Sodium hyaluronate and D-GlcUA served
as the positive control in precipitation and HPLC-MS test.

of AAB. This evidence emphasizes the importance of fundamental studies of

microbial interaction for industrial application. Further work should therefore
investigate the metabolites produced by L. casei during fermentation.

Verification of the GlcUA Enhancement by L. casei

The carbazole reaction confined the range and pointed out the three possible ratios (0.5; 1; 1.5) of L. casei treatment in combination with kombucha
symbiosis model for GlcUA enhancement. These ratios and their effects then
were verified using highly accurate method (HPLC-MS). The two highest
concentrations of GlcUA were recorded at (1:1.5:0.5) and (1:1.5:1) ratio of
3-component symbiosis (D. bruxellensis: G. intermedius: L. casei) (238.6a
6.45 and 224.9a 15.3 mg/L). They showed higher GlcUA amount, up to 160%
compared to the control sample (without L. casei, 154.8c 9.56 mg/L) and

363

N.-K. Nguyen et al.

6 times higher than that of the individual fermentation of G. intermedius (36.8d

1.75 mg/L) (Fig. 1). This suggests that L.casei treatment can significantly
improve GlcUA production of kombucha symbiosis model.
Moreover, this L. casei strain which was isolated from kefir has been
reported to increase the antimicrobial, antioxidant activities of traditional
kombucha (Nguyen et al., 2014; Nguyen et al., 2015a). Therefore, the evidence highlights a potential in biotransformation improvement of kombucha
by treatment with L.casei.

Glucuronic Acid Production under Different Fermentative

Conditions
To our knowledge, this is the first time L. casei isolated from kefir was combined with D. bruxellensis and G. intermedius in tea fermentation to obtain a
high-level GlcUA concentration. The pre-assessment of these microbial behaviors under distinct environmental circumstances is therefore significantly
important step before performing GlcUA optimization of the whole tea culture.

Effects of Sucrose Concentrations on GlcUA Production

In the present study, the effects of various sugar concentrations on the
GlcUA excreted by the designed microbial symbiosis are shown in the Fig. 3.
The GlcUA concentration increased when the initial sugar percentage in
medium rose from 5% to 12.5%. In contrast, when sugar continuously rose from
12.5% to 17.5%, the GlcUA amount dropped down dramatically.
Carbon source is essential for living microorganisms particularly for their
growth and metabolites production (Caldwell, 1999). It is the same in microbial
350
GlcUA concentrations (mg/L)

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364

300
b

250

200
150
100

c
c

50
0

7.5
10
12.5
15
Sucrose concentration g/L (%)

17.5

Figure 3: The glucuronic acid production of the microbial symbiosis based on different initial

sucrose-containing SBT mediums. The ratio of symbiosis between D. bruxellensis, G. intermedius

and L. casei used in this experiment are 1; 1.5; 1 respectively. Means do not share the letter
(a-d) are significantly different (N= 3).

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Effects of Lactobacillus casei

association of kombucha, glucose must be sufficiently provided in the medium

to meet the requirements for cellulose and GlcUA production, since these products share the same sugar source in the culture (Frank, 1995; Vna et al., 2013).
As shown in Fig. 3, the GlcUA concentration decreased while the sugar amount
decreased from 12.5% to 17.5% due to the imbalance of nutritional uptake
and utilization inside microbial cells. In addition, the high sugar concentration may cause the osmotic stress that suppress the growth of microorganisms.
Hence, our preliminary results emphasize the impact of sucrose concentration
on GlcUA production and suggest 12.5% (g/L) for an appropriate level of sugar.

Effects of Temperature on GlcUA Production

In our research, effects of GlcUA produced by the designed microbial symbiosis were evaluated under various levels of temperature form 22 to 42 C.
The GlcUA amount was recorded as a fluctuating line in Fig. 3 which shows
a high level at 27 o C and 37. At the moment, research on similar kombucha
symbiosis is limited, and thus, mechanisms of alterative optimum temperature
on GlcUA production remain unclear. However, there are some possible explanations that need to be taken into account. Overall, increasing temperature
speeds the microbial sucrose utilization higher and results in the faster GlcUA
production in kombucha fermented juice (Yavari et al., 2011). It also stimulates
D. bruxellensis generate much more acetic acid at 30 C compared to that produced at 24 C (Freer et al., 2003). According to Yang et al. (2010), acetic acid
can cause inhibition of glycolysis pathway. So, when its concentration is high
enough in the medium, it can reduce the activities of glycolysis and enhance
the flux of glucuronate pathway of G. intermedius. These could be a possible
explanation for the increasing of GlcUA production in our study, when temperature rises from 22 C to 27 C. Nevertheless, further investigations on the
optimal temperature and excreted metabolites are required.
With regard to AAB, they can survive in a wide range of temperature
(845 C) and their optimum is 2530 C (Hommel and Ahnert, 2000). However,
in our experiment, G. intermedius strain which combined with D. bruxellensis & L. casei, can produce GlcUA in large amount at 37 C but a smaller
amount at 42 C. The differences between single strain and symbiosis experiment could be a main reason. In some early reports, published by other studies
(Malbasa et al., 2014), the microbial flora of kombucha can ferment milk and
give a successful process at 37 C but failed at 43 C. In another study mentioned by Moghadami et al. (2013), their Acetobacter sp. strain 53 was selected
from 39 AAB species produced the highest acetic acid amount at 33 C but
reduced at 40 C. The behavior of our bacterial strains is somehow similar
to those mentioned in above studies. In short, our findings provided helpful
information to narrow down the temperature range from 2737 C for further
optimization.

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Effects of pH on Glucuronic Acid Production

As can be seen from Fig. 5, glucuronic acid increased gradually as the pH
increased from 4 to 6 and suddenly reduced at level 7 whose glucuronic acid
concentration was about one-third of the highest value (123.5 d 24.7 vs 367a
45.3 mg/L). The lowest concentration was recorded at level 8 of pH which
account for only 33.5e 17.5 (mg/L).
Regarding Acetobacter/Gluconacetobacter metabolism, their oxidation abilities based on membrane-bound enzymes involving glucose, alcohol, aldehyde
and sorbitol dehydrogenases, which have acidic optima. The optimal pH level
is also determined as a range of 5.46.3 (Hommel and Ahnert, 2000). In addition, our results agree with those that have been reported in other studies
that pH 6 was the most favorable condition for glucuronic acid production(Beigmohammadi et al., 2010). In general, pH range 5-7 is suitable for
further optimization.

Effects of Fermentation Time on GlcUA Production

The results are presented in single line graph of Fig. 4. The GlcUA concentration increased to the highest point (270.56 13.03 mg/L) at the fifth
day of fermentation and slightly declined when the duration was extended.
Correlation between fermentative duration and temperature in kombucha
brewing on certain valuable substances such as GlcUA has been recently documented (Vina et al., 2013). Since microorganisms are sensitive to temperature
and GlcUA production depends on microbial growths and interactions, the
temperature conditions can be used to interpret the fermentation time.
The optimum temperature for Lactic acid bacteria (LAB) to grow was
3040 C (Hommel and Ahnert, 2000), and it is higher than those of AAB
as mentioned before. At the beginning of the process, L. casei may assist G.
intermedius to produce GlcUA until its population is large enough after 5 d
fermentation and then it can compete with G. intermedius.
In addition, heterofermentative lactic acid bacteria can ferment GlcUA
(member of uronic acid family) as a sole carbon source for energy (Stamer and
Stoyla, 1968) and L. casei belongs to group II Lactobacilli that are referred
to as facultative heterofermentative specie (Hutkins, 2006). This L. casei can
convert GlcUA to other organic acids such as lactic and acetic acid; hence,
potentially reduce the GlcUA concentration. For this reason, we suppose that,
a smaller initial L.casei supplementation is more appropriate for higher GlcUA
in a long-term process. In order to test this hypothesis, we repeated the experiment using the ratio (1: 1.5: 0.5) for the microbial combination. Interestingly,
with the longer fermentation time the higher GlcUA amount was achieved
(Fig. 4). GlcUA concentration increased strongly from the third day (161.67c
14.57 mg/L) to the fifth day (237b 9.85 mg/L) of fermentation and continued to increase to reach 285a 17.09 (mg/L) at the 13th day. This result is

Effects of Lactobacillus casei

27

32

37

42

500

280

450

260

400

ab

ab

350

220

300

240

bc
ab

ab

200

250
180

200

150

bc

160

100

140

50

120

11

100

13

Glucuronic acid production at different temperatures

(mg/L)

GlcUA production at different fermentation times (mg/L)

Fermentation time (days)

Figure 4: The effect of temperature (

) and time ( ) on glucuronic acid production in fermentation. The ratio of symbiosis between D. bruxellensis, G. intermedius and L. casei used
in this experiment are 1; 1.5; 1 respectively. ( ) glucuronic acid production using the ratio
(1;1.5;0.5) of the microbial symbiosis. Means do not share the letter (a-d) are significantly
different (N = 3).

Glucuronic acid concentrations (mg/L)

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Temperature (C)
22

450

400
350
300

b
c

250
200

150
100

50
0

6
Initial pH level

Figure 5: Effects of different initial pH on glucuronic acid production of symbiosis model in SBT

medium. The ratio of symbiosis between D. bruxellensis, G. intermedius and L. casei used in
this experiment are 1; 1.5; 1 respectively. Means do not share the letter (a-d) are significantly
different (N= 3).

important enough to contribute to one criterion for choosing a suitable amount

of L. casei at the beginning of fermentation. It depends on the purpose and
technology in fermentation when certain amount of L. casei is more likely to
play a vital role in products quality.

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N.-K. Nguyen et al.

In short, the findings once again emphasized the correlation between the
fermentative conditions. The temperature seems to be a vital key need since it
controls most of the microbial behavior. In addition, the different amount of L.
casei relates to the fermentation time because the long-term process with the
high concentration of L. casei may cause a reduction in GlcUA amount with L.
casei utilizing it as a carbon source.
Thus, one parameter can have both positive and negative effect to certain
metabolites and products of microorganisms. This depends on its level and
other fermentative conditions under various circumstances. It is necessary to
design a further method such as Plackett-Burman design and Response surface methodology to optimize GlcUA production with multiple parameters at
the same time.

CONCLUSION
This is the first study which focuses on glucuronic acid production using a
microbial symbiosis model. As the result, it is certainly clear that treatment
of L. casei has the ability to enhance GlcUA production in kombucha without
producing any GlcUA or glucuronic-containing by products. The new symbiosis
model consisted of D. bruxellensis, G. intermedius and L. casei with a corresponding ratio (1: 1.5: 0.5) producing the highest GlcUA (238.60a 6.45 mg/L)
and allowed us to investigate the microbial interactions easily and more accurately compared to traditional kombucha. Besides, the evaluation of GlcUA
production in this study has provided a vital transitional step to optimize
the range of four fermentative parameters: sucrose concentration (515%),
time (37 d), pH (57), and temperature (2737 C) for further improvement of
biotransformation potential. These preliminary findings would be helpful steps
toward producing GlcUA rich kombucha in a large scale fermentation.

ABBREVIATIONS
AAB:
GlcUA:
KBC:
LAB:
SBT:

acetic acid bacteria

glucuronic acid
kombucha
lactic acid bacteria
sweetened black tea

ACKNOWLEDGMENTS
The authors would like to give special thanks to Mr. Manh Khac Nguyen
and Mr. Phuong Ngoc Nguyen for their help in performance this study.

Effects of Lactobacillus casei

ORCID

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Nguyen Khoi Nguyen

http://orcid.org/0000-0001-8919-3977

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