VIETNAM NATIONAL UNIVERSITY HOCHIMINH CITY

INTERNATIONAL UNIVERSITY

FERMENTATION OF GRAPE JUICE

DRINKING BY KOMBUCHA LAYER

A thesis submitted to
The School of Biotechnology, International University
In partial fulfillment of the requirements for the degree of
B.S. in Biotechnology

Student name: Dng Hong Bo Khnh ID: BT070117

Supervisor:

Dr. L Hng Ph

February, 2013

VIETNAM NATIONAL UNIVERSITY HOCHIMINH CITY

INTERNATIONAL UNIVERSITY

FERMENTATION OF GRAPE JUICE

DRINKING BY KOMBUCHA LAYER

A thesis submitted to
The School of Biotechnology, International University
In partial fulfillment of the requirements for the degree of
B.S. in Biotechnology

Student name: Dng Hong Bo Khnh ID: BT070117

(dhbk18889@gmail.com)
Supervisor:

Dr. L Hng Ph

February, 2013

ACKNOWLEDGEMENT
A deeply appreciation is addressed to all those people who have made a
significant contribution to my completion of this thesis.
I would like to express my special thanks to my supervisor, Dr. Le Hong Phu,
who helped and supported me throughout my project. I am thankful to him for
many valuable discussions that helped me understand my research area better,
as well as for his advice on doing a lot of research. I have gained so much useful
knowledge from this wonderful project.
I am also grateful to MSc. Tran Thi Quynh Dao, MSc. Nguyen Hong Long and
BSc. Nguyen Khac Manh for their assistance and guidance on laboratory
technique and collecting data. Thanks for their enthusiasm and patience in
helping me to get familiar with various laboratory instruments. I have gained a
wider experience on doing cell culture.
Last but not the least, I wish to thank my mommy, my grandfather and my
close friend Le Thi Ngoc Anh who always supported and encouraged me to
complete this thesis. I warmly appreciate their concern about my health and
their support to help me overcome this stressful time. More important, thanks
for their belief on me.

FERMENTATION OF GRAPE JUICE DRINING BY KOMBUCHA

LAYER
Khanh B.H Duong, Phu H. Le*
School of Biotechnology, International University Vietnam National University in
HCMC
*

Corresponding authors email address: lhphu@hcmiu.edu.vn

ABSTRACT
Kombucha is a refreshing beverage obtained through the fermentation of
sugared grape juice with a symbiotic culture of acetic bacteria and fungi
consumed for its distinct antibiotic effects against the number of disease
organisms and several therapeutic purposes in human medicine. Grape juice
phytochemicals such as resveratrol and polyphenol antioxidants have been
positively linked to inhibit cancer heart disease degenerative nerve disease viral
infections and mechanisms of Alzheimer's disease. Glucuronic acid is the key
component in human health due to its detoxifying action through conjugation to
the xenobiotic metabolisms in liver and associated with cartilage, shown
substantial benefit in the treatment of osteoarthritis. Here I report first analysis
of glucuronic acid production (g/L) as well as monitored changes in pH,
remained sucrose (g/L), reducing sugar (g/L) and total acidity (g/L) by sing
Kombucha layer on grape juice. High Performance Liquid Chromatography (HPLC)

is one mode of chromatography, one of the most used analytical techniques used
to separate a mixture of compounds in analytical chemistry and biochemistry with
the purpose of identifying, quantifying and purifying the individual components of
the mixture. This method showed that glucuronic acid production on grape juice
Kombucha had a significant higer than that with black tea, which was reached
nearly 160 g/L on the 7th day.

Key words: healthy drink, fermentation, Kombucha, glucuronic acid, grape

juice

1. Introduction
1.1

Kombucha

KOMBU-CHA: Kombu= Japanese name for an edible species of seaweed.

Cha= Japanese for tea = Seaweed-tea.
Kombucha is a popular beverage among many traditional fermented foods across
the world. It originated in northeast China (Manchuria) and later spread to Russia
and the rest of the world. Kombucha has several health benefits have been reported.
Consumption of Kombucha has been shown to have beneficial effects on gastric,
intestinal, and glandular activities and to overcome on arteriosclerosis, toxin
excretion, diabetes, nervousness, and aging problems (Teoh et al., 2004).
Additionally, it can also act as a laxative and are known to relieve joint rheumatism,
gout, and hemorrhoids (Reiss, 1994; Dufresne and Farnworth, 2000; Bhattacharya
et al., 2011). The therapeutic benefits of consuming kombucha have been reported to
range from weight loss up to curing of cancer and AIDS (Teoh et al., 2004). The
free radical scavenging and antioxidant activities of Kombucha tea has been reported
recently (Jayabalan et al., 2008; Malbaa et al., 2011).
Kombucha is a traditional beverage prepared by fermenting sweetened black tea
with the tea fungus which is a symbiosis of Acetobacter, including Acetobacter
xylinum as a characteristic spieces, and various yeasts, such as the genera of
Brettanomyces, Zygosaccharomyces, Saccharomyces, and Pichia depending on the
source (P. Mayser, et al., 1995). It is fermented using something called a SCOBY
(Symbiotic Colony of Bacteria and Yeast), or a symbiotic colony of bacteria and
yeast (Chen and Liu, 2000). The scoby digests the sugar in the tea, creating acetic
acid (vinegar) and glucuronic acid, which is one of the components of kombucha
that may be responsible for its purported health benefits (Teoh et al., 2004). Once
fermented, kombucha contains multiple species of yeast and bacteria along with
active enzymes, amino acids, organic acids, and polyphenols produced by the yeast
and bacteria. It can contain a small percentage of alcohol. Kombucha layer has been
claimed to be a prophylactic and therapeutic agent to human health from weight loss
to metabolic diseases, arthritis, indigestion, curing cancer, and AIDS (C. Dufresne
and E. Farnworth, 2000).

Kombucha beverage is composed of two portions: a floating cellulose pellicle

layer and the sour liquid broth (Chen and Liu, 2000). Typical of such fermentation is
the activity of Acetobacter xylinum which enables synthesis of a floating cellulose
pellicle and converts glucose to glucuronic acid and fructose to acetic acid, in which
the embedded cells benefit from the close contact with the atmospheric oxygen
(Siever et al., 1995) and yeast cells hydrolyze sucrose into glucose and fructose,
producing ethanol (Reiss, 1994). During the fermentation process, bacteria and
yeasts metabolize sucrose into a number of organic acids such as acetic acid and
glucuronic acid (Chu and Chen, 2006), amino acids, antibiotics and a variety of
micronutrients produced during fermentation (Vijayaraghavaity et al., 2000). In
addition to ethanol and acetic acid, a great number of other compounds emerge as a
result of numerous reactions (Balention et al., 1997; Pasha and Reddy, 2005).
Important metabolites are organic acidsactive ingredients of kombucha tea that
may exert beneficial effects (Jayabalan et al., 2007). The US Food and Drug
Administration has evaluated the practices of several commercial producers of the
kombucha and found no pathogenic organisms or other hygiene violations (CDC,
1996). This beverage has been reported to have medicinal effects against metabolic
diseases, arthritis, indigestion and various types of cancer (Sreeramulu et al., 2000).
1.2

Glucuronic acid

One of the main metabolites identified in the kombucha beverage is a glucuronic

acid (C.H. Liu, et al., 1996). Glucuronic acid is a highly water-soluble carboxylic
acid, its structure is similar to that of glucose. However, glucuronic acid's sixth
carbon is oxidized to a carboxylic acid. Its formula is C6H10O7 (Fig.1) and the
molecular weight is 194.14 g mol1. Glucuronic acid normally produced by a
healthy liver, that can be converted into glucosamine and related chondroitin-sulfate
are associated with cartilage, collagen and the fluid which lubricate the joints (Frank
and Gnther, 1991). Produced by the bacteria (Acetobacter), it can break down to
caprylic acid is of great benefit to sufferers of candidiasis and other yeast infections
such as thrush. (D. Cvetkovic, et al., 2008). Kombucha layer researchers believe
that, its detoxifying property is presumably due to the capacity of glucuronic acid
binding to toxin molecules and increasing their excretion from the organism by the
kidneys or the intestines (R. Jayabalan, et al., 2007). Butyric acid, also found in
kombucha beverage protects human cellular membranes and combined with
2

glucuronic acid strengthens the walls of the gut and also protects against parasites as
a result of its bond to glucuronic acid (U. Mann, 1988).

Figure 1: Glucuronic acid structure

1.3

Grape juice

Grape juice is a kind of food and is therefore primarily consumed for its
nutritional characteristics (e.g. energy value, nutrients). Red grape juice contains
several vitamins (the most important: vitamin C), minerals, as well as flavonoids
(quercetin, myricetin and anthocyanins). Flavonoids are phenolic compounds that
are widespread in commonly consumed fruits and vegetables such as apples and
onions and beverages derived from plants like tea. Thousands of flavonoids are
distributed throughout the plant world and many have antioxidant functions. Recent
research reports a range of health-beneficial effects from antioxidants in the diet.
Due to the protection they confer against oxidative damage caused by free radicals
and general physiological activities, they may play a significant role in preventing
diseases such as cancer, and cardiovascular and neurological diseases.
It is obviously true that Kombucha beverage supplied hundreds of benefits for
human health with the high concentration of glucuronic acid (C.H. Liu, et al, 1996).
In this study I would like to increase its health benefits by supplant the tea material
by grape juice which has more vitamins and nutrients for human body. In addition,
Although the Kombucha beverage was found for a long time (J BLANC, et al, 1996)
the Kombuchas researches just appear in Vietnam several years ago, it is a quite
new object for scientist to study. There are also some unclear ideas about the health
benefits of this drinking but no any project has research about it in Vietnam before.

Therefore, I would like to indentify the advantages of this beverage and give a new
method to make this drinking become more healthy. However because of the limited
time and budget, the project was conducted with red grape which is raised in Phan
Thiet and is the most popular grape of Vietnam.
2. Materials and methods
2.1 Preparation and cultivation of Kombucha layer

Black tea (yellow label tea by Lipton, which made from 100% tender tea leaves
was bought in Co.op Mart) was used as the substrate for the fermentation of
Kombucha and the SCOBY was given by Lab A101. The sucrose used as the carbon
source was by Granulate Sugar which was produced from Thanh Thanh Cong
Company. 250 gram of sucrose were added to three litters of distilled water that had
been just boiled for 15 min in a big tea pot (Sreeramulu et al, 2000). Subsequently,
three black tea bags were added and allowed to steep for 15 min and then was taken
out. The tea was then cooled to 25C, and 1000 mL of tea was aliquoted into three
5L plastic bottles that had been previously sterilized ( all the plastic equipment in
this research will be pasteurised at 80C for 20 min by the standard for plastic
material (Balentine, 1997)). The samples was cultivated by adding 200 g of SCOBY
in each bottle which had been cultured in the same medium for 14 days, and the
bottles were covered with sterile gauze towels (size 25 x 30 cm) secured with a glue
tap to allow aeration ( Shukla et al, 2000).
Finally, the fermentation was carried out in lab 101 with room temperature at
25C. After two weeks, the culture were done by preparing another three plastic
bottles of 1 litter black tea with 83.33 gram sucrose for the next batch of kombucha
(Sreeramulu et al, 2000). With clean hands, the kombucha layers were gently lifted
out of the solutions and set one by one on each prepared bottles. As well as , it was
checked over and removed the bottom layer if the SCOBY was getting very thick.
The new SCOBYs was used to inoculate grape juice fermentation.

2.2 Preparation the grape juice

The CJ-26A CORNELL (Fig. 2) which is the most common type of juicer that
can easily find in any electrical super market was used in this research. It revolution
can be up to 44000rpm and the blending capacity is 800 millilitres (Cornell
appliances). It is upright and cylindrical in shape and can extract juice from grapes
by grating them into tiny pieces, then used a sieve to spin the juice out of the pulp at
high speeds. This centrifugal juicer is much faster for making juice so it is more
convenient. It usually has larger mouths so can eat a lot of grapes at the same
time. It is also easier to clean than other types of juicers. Moreover, there is a large
variety of centrifugal juicers on the market that everyone can lay his hand on.

Washing and pasteurizing all part of the machine was considered as the first
especially the sieve, the cover, the pusher and the juice groove. All of them was
pasteurized by steeping in hot water in 15 min then washed again by mild detergent,
the other parts were wiped by a wet soft cloth (Cornell appliances). The cleaning
steps will be repeated whenever finishing the grape juice extraction.

Red grape was bought from the supermartket (Co.op Mart). First of all, grapes
were removed from the sterms. Stems, leaves, bugs and any unsuitable grapes were
thrown away (eg green, dried up, or with black spots on them). The grapes were
washed gently and carefully to remove any unwanted stuff from the skins, firstly
with clean water, then steeped in the salty water, finally rinsed with distilled water
(Fig.3). The cleaned grapes were placed in large pots and then were put in the juice
extractor which already prepared. The grape juice extraction was come out to the
juice groove. After finishing extraction, a lot of pulp still among the grape juice
(Fig.4a), so the pulp should be separated by another step.

Figure 2: Juice extractor

Figure 3: Cleaned grapes

Fig 4: The grape before and after removed pulp. (a)before (b)after

To do with the grape juice, three 15 litter plastic bottles, ten 20 cm plastic
colanders with hole size is 0.2 x 0.2 cm2 (America's Test Kitchen, 332 Ly Thuong
Kiet street, ward 14, distric 10) and ten 20 cm plastic bowls were pasteurized, each
colander was set inside each bowl, then ten sterile gauze towels (size 25 x 30 cm,
4ply, can find in any pharmacy store) which were lined on each colander and allow
for at least two centimetres of overhang on all sides (Fig.5). The grape juice was
poured into the the colander on the cheesecloth and allow it to strain into the bowl.
Once most of the juice has passed through, gather the sides of the cheese cloth
together and squeeze all of the juice out. Remove the colanders from the bowls, the
juice was then poured into two 15 litter plastic bottles and put in the refrigerator
overnight for precipitation. On the next day, the juice had two layers are the
precipitation and the supernatant. The supernatant was pour out into another 15 litter
plastic bottle which is the juice without any pulp, this juice was kept in refrigerator
for the next fermentation. The precipitation were centrifuged at 8000 rpm 8C for 10
minute (Oliveira, et al, 2006), now the precipitant was completely tight, we could
take the pure grape juice from the upper layer and remove the lower layer (Fig.4b).
This pure grape juice was poured together with the first one to make 17 litters of
grape juice without pulps. The grape juice was kept in refrigerator at 8C which
used for the fermentation during the next ten days (Rodrigues, Teixeira, & Oliveira,
2006). This process would be repeated several times during the research after
finishing a shift of grape juice fermentation with Kombucha and followed an
approximate proportion: every 2 kilograms of red grape will give 1 liter of pure
grape juice roughly.

2.3 Ferment with Grape Juice

6

Use a 1000 mL cylinder to contain 200 mL of clear grape juice, then pour this
into twelve sterilized 500 mL plastic bottles (Fig.6). SCOBYs was prepared in the
procedure mentioned; except for the weight of kombucha layers used: 25, 50, and
75g. These sizes of SCOBY were chosen based on the results of preliminary
experiments and was described in the Food Research Journal (E. Basehoar-Powers,
2001). For conducting the experiments, three grape juice bottles were in turn added
with 25 g, 50 g and 75 g of SCOBYs. The grape juice in the other three bottle were
held up 100 mL tea broth on the top (Malbasa et al., 2008). All the bottle with glue
taps were covered with sterile gauze towels (size 25 x 30 cm) to allow aeration as
the tea broth (Fig.12).

Figure
Figure 5: Tools for filtering the juice

6: Sample preparation

Fermentation was then carried out in Lab 101 at room temperature. This process
were repeated continually in the next 6 days. After 7 days, the process was done by
pouring the liquid medium out into a small cup, these samples were used to check
the sensory evaluation daily by a form attached in the appendix 1.

2.4 Sensory evaluation

Sensory evaluation is an analytical method in which the human senses serve as a

measurement tool to determine the quality and to describe the condition of the food
product. In this research sensory testing, method was used is hedonistic testing
7

which is used within the scope of consumer tests and serve to characterize consumer
behavior. The main objective of this sensory evaluation is the measurement of
sensory attributes and the quantification of the influence of these attributes on
consumer acceptance. Sensory attributes are directly linked to the concept of quality
and thereby ultimately contribute to the success or failure of the fermentation in a
period of time.

The sensory evaluation was carried out at HCMC University of Science,

Vietnam National University, 227 Nguyen Van Cu Distric 5, HCMC, from 11 to
2 pm, which time the students and teachers from the Universities and people
living around the area go out for lunch, as well as they go to the afternoon class.
The scale of this survey was 100 people. Each one joining the test tasted twenty
eight samples such as M25-1, M25-2, M25-3, M25-4, M25-5, M25-6, M25-7.
These samples were from grape juice with 25 g of SCOBY for seven days.
Similarly, M50-1, M50-2, M50-3, M50-4, M50-5, M50-6, M50-7 with 50 g
kombucha layers and M75-1, M75-2, M75-3, M75-4, M75-5, M75-6, M75-7 for
75 g kombucha layers. The 100 mL tea broth and grape juice was marked D1,
D2, D3, D4, D5, D6, D7.
Each sample was evaluated by each group. The average and standard
deviation of the results was computed by combining the results of evaluations
from the other groups in the session. The taste, color, odor, overall appearance
and overall acceptability were evaluated according to the scale provided by
Elizabeth L. 1977 with a slight moderation as below:
a) Odor - not acceptable-1; moderately acceptable-2; very acceptable-3
b) Color - not acceptable-1; moderately acceptable-2; very acceptable3
c) Taste - not acceptable-1; moderately acceptable-2; very acceptable-3
d) Overall appearance- not acceptable-1; moderately acceptable-2;
very acceptable-3
e) Overall acceptability- not acceptable-1; moderately acceptable2; very acceptable-3

The measurement of chemical changes were then carried out after the process
had an acceptable time by sensory evaluation.

2.5 Determination of pH and concentration of sucrose

The pH of the sample was measured with an electronic pH meter (PHM 82,
Standard pH Meter, Radiometer Copenhagen) available in lab 101 and the sucrose
changes was also measured by a refractometer available in lab 702 in a fermentation
period which was chosen by sensory evaluation.

2.6 Determination of total acidity

In this process, titration technique is used to determine the concentration Total

Acidity which present in the product. The procedure was follow by Chris Kraemer,
2004 with briefly described as below
2.6.1 Preparation of NaOH solution:
Dilute approximately 10 mL of 6N NaOH with approximately 450 mL of
distilled water in a 600 mL beaker. Rinse the graduated cylinder with distilled water
and add to the beaker to make sure that all of the NaOH were gotten, Stir the
solution thoroughly.
2.6.2 Titration of NaOH solution:
1. Obtain a 50 mL burrett, close the stopcock and fill it to the top with distilled
water. Open the stopcock and allow all of the water to drain.
2. Close the stopcock and fill your burrett with 50 mL of your NaOH solution
(from above) so that the solution comes in contact with the entire inner
surface of the burrett.
3. Open the stopcock and allow all of the NaOH to drain through the tip.
4. Fill the burrett to the top with the NaOH. Open the stopcock all the way to
flush all bubbles out of the tip. When all bubbles have been flushed out (it
may take several tries), close the stopcock and refill the burrett.

5. Read the bottom of the meniscus and record the initial reading to the nearest
0.01 mL. The Teflon stopcock should turn smoothly with a little resistance.
If the stopcock is too loose, tighten it a little, otherwise the solution will leak
around the stopcock and the titration will be for naught.
6. Use a repipetter to deliver 10 mL of the standard HCl into a clean 150 mL
beaker. Wash down the sides of the beaker with the wash bottle. The
addition of water at this stage has no effect on the total amount of acid
already present in the beaker. Be sure to record the concentration of the
standard HCl!
7. Add 2 drops of phenolphthalein indicator to the sample ( which was diluted
10 times). The solution should remain colorless.
8. Add a magnetic stir bar to the beaker and place on the heating stir plate.
Adjust the stirring rate to obtain a vortex without any of the solution
splattering on the sides of the beaker. Avoid spilling any of the beaker
contents. Any loss of sample would render the titration worthless.
9. Rinse down the inside of the beaker occasionally and continue, slowly
adding NaOH until the first permanent, faint pink color persists for at least
30 seconds. At this point the titration is complete (the endpoint).
10. Read the final volume of NaOH and record to the nearest 0.01 mL.
11. Repeat Steps number 7 to 12 with the rest samples.

2.7 Determination of reducing sugar

The reducing sugar was determined according to the colour intensity of the
analytical sample and the standard curve. The procedure was followed by
Process Biotechnology lab manual with briefly described as below.
The chemicals used for this experiment include DNS solution: dinitro 3,5
salicylic acid (10g/L), NaOH (16g/L), Na-K tartarate (300g/L) and Sugar
standard: glucose. The procedure was processed by using a pipette to transfer
1mL diluted samples (1:100) into a tube and then added 1mL DNS solution. The
tubes were heated up by putting in a water-thermostat at 100oC for 5min. After
10

the tubes were cooled down to the ambient temperature, then added 10mL
distilled water and mixed. The spectrophotometric absorbance were read at
540nm. A control sample was made by replacing 1mL analytical sample by 1mL
distilled water. This control sample was used to regulate the spectrophotometric
absorbance to 0. The standard curve (quantitative relationship between reducing
sugar concentration and spectrophotometric absorbance) can be formed by
preparing 4 samples with sugar concentrations 0.5, 1.0, 1.5 and 2.0g/L.

2.8 High performance liquid chromatography analysis of glucuronic

acid:

High Performance Liquid Chromatography (HPLC) is one mode of

chromatography, one of the most used analytical techniques. Chromatographic
process can be defined as separation technique involving mass-transfer between
stationary and mobile phase. HPLC utilises a liquid mobile phase to separate the
components of a mixture. The stationary phase can be a liquid or a solid phase.
These components are first dissolved in a solvent, and then forced to flow through a
chromatographic column under a high pressure. In the column, the mixture separates
into its components. The amount of resolution is important, and is dependent upon
the extent of interaction between the solute components and the stationary phase.
The stationary phase is defined as the immobile packing material in the column. The
interaction of the solute with mobile and stationary phases can be manipulated
through different choices of both solvents and stationary phases. As a result, HPLC
acquires a high degree of versatility not found in other chromatographic systems and
it has the ability to easily separate a wide variety of chemical mixtures.

The procedure was conducted at HCMC University of Science, Vietnam

National University, Laboratory analysis center, 227 Nguyen Van Cu Distric 5,
HCMC with the guidance of Mr. Nguyen Khac Manh.
Firstly, 5 mL of sample was mixed and well shaked with 200 L of formic
acid in one minute then the solution were centrifuged 4000 rpm at 8 C for 2
minute. Secondly, the SPE C-18 column was activated by using 10 mL MeOH
and 5 mL H2O containing 0.1% formic acid. Thirdly, 5 mL of centrifugal sample
11

was poured to the SPE C-18 column to get solution 1. Next, solution 2 was
gotten by washing the SPE C-18 with 5 mL MeOH and 5 mL H2O containing
0.1% formic acid. Finally, solution 1 and solution 2 were combined and shacked
regularly then passed through Millipore filter (0.45 ) into HPLC vials. A 10 mL
sample of filtrate was injected to a HPLC system equipped with a MS detector.
The mobile phase A was H2O containing 0.1 % formic acid and the mobile
phase B was MeOH containing 0.1% formic acid. The flow rate was maintained
as 0.5ml/min and column was at room temperature. Detection was carried out at
0.5 m. The resolution peaks were recorded on the HPLC chart according to the
retention time of glucuronic acid.

2.9 Statistical analyses

All analyses were carried out on each treatment. The sensory evaluation were
analysed by two-way ANOVA 16.0. Results were presented as means and SED.
Statistical significance was considered when P < 0.05.

3. Results and discussion:

3.1 Sensory evaluation
As the ANOVA test result (appendix 2), there are no any significant different
between four samples in one day. The sensory evaluation result were determined by
the range 3, which sample has the average score under 1.5 mean failings (Elizabeth
L., 1977). Therefore I can conclude the result as below:

12

Figure 7: Odour of the Grape juice Kombucha samples

Kombucha has its own special smell that will be immediately recognize long
time brewer. The sweet-sour smell of Kombucha wafting from the brewer is a
unique delight. It may take a couple of days for the smell to appear. Its a sharp odor
akin to cider vinegar which is strong enough to prickle consumers sense of smel.l
That is reason why after 5 - day fermentation the smell appearance makes the
consumers able to be unacceptable. Even there were some consumers like that smell
but it was not enough to pass the test. In general after the 5th day fermentation all the
samples had the score under 1.5 (Fig.7)

13

Figure 8: Colour of the Grape juice Kombucha samples

As the result all the colour of the samples passed the test. The mediums have the
colour of the red grape juice, which was changed lighter slightly during the
fermentation (Fig.12), as well as the kombucha tea is dependent upon the type of tea
used as well as how long the tea was allowed to brew ( Wong, Crystal. July 12,
2007).

Figure 9: Taste of the Grape juice Kombucha samples

The taste of fermented grape juice varies greatly depending on the amount of
time it was allowed to ferment. The growth of acetic acid bacteria and yeast during
the fermentation made the batch had a strong vinegar taste (Ferson, MJ,1998). This
is right with the survey which most of consumers did not want to taste any samples
after the 5 th day fermentation. As the result, all the samples which were fermented
after 5 days were fail because they was too sour and sharp. This fermentation time
was rather low when compared with the earlier research on Kombucha tea which is
able to use after a week (R. Jayabalan, et al., 2007)

14

Figure 10: Overall appearance of the Grape juice Kombucha samples

Figure 11: Overall acceptability of the Grape juice Kombucha samples

The overall appearance and the overall acceptability were the conclusion of
consumers after smelling and tasting. Its right that all the samples were good
appearance and high acceptability in the first 4th day fermentation because it still
have the taste of the grape juice and not too much vinegar which consumers cannot
accepted.

15

As a consequence, the grape juice fermented with Kombucha layer should be use
withing 5 days fermentation. Even a longer fermentation process will allow the
grape juice to fully culture, the taste and the odour made the consumer unaccepted
the product. Therefore, in my next experiments for chemical changes, I just choose
the fermentation period within 7 days, and the sample is the grape juice fermented
with 50g of Kombucha layer. It is because all types of Kombucha layer give a
similar trend for sensory evaluation result, and with the 50g Kombucha layer the

sample grew gradually in 200ml grape juice (Fig.12)

Figure 12: The grape juice femented by Kombucha layers

3.2

Chemical Changes during Kombucha on grape juice

Fermentation

The microorganisms utilized the carbon source and started producing

cellulose, which appeared as a thin layer on top of the grape juice. In present
study, the chemical changes was investigated on grape juice fermented mediums
with the Kombucha mother size was 50 g. The duration of the fermentation was
7 days. The values are means of triplicate measurements.

PH:
16

Figure 13: Ph value of M50 during 7-day fermentation

Generally, the changes in pH for the grape juice fermented dropped gradually as
the fermentation process. Initially the pH value of the grape juice medium was
approximately 3.57, and it dropped to about 3.1 0.2 (Fig.13). These results are
consistent with some of the earlier findings (Hwang et al., 1999, Chen and Liu,
2000). The increase in acidity just a consequence of the physiological activity of the
Kombucha layer and synthesis of organic acids and glucuronic acid. The highest pH
values measured at the end of fermentation was 3.18, whereas the lowest was 2.92.
This appeared to be rather low in comparison with any results of other authors after
the sucrose fermentation on tea (R. Jayabalan, et al., 2007). During the fermentation
process, yeasts and bacteria metabolize sucrose into a number of organic acids, such
as acetic acid and glucuronic acid. These observations are in agreement with the
findings of other studies (Steinkrauset al., 1994; Greenwalt et al., 1998).

Remained Sucrose:

Figure 14: The remain sucrose of M50 during 7-day fermentation

17

The sucrose used since it is a traditional carbon source for kombucha

fermentation. It is used by the yeasts to produce ethanol, which is initially oxides to
acetaldehyde and then oxidized by acetic acid bacteria . It is used by the yeasts to
produce ethanol, which is initially oxides to acetaldehyde and then oxidized by
acetic acid bacteria (Brown et al., 1976; Greenwalt et al., 1998).Therefore,
throughout the fermentation, the used carbon source in the cultivation medium is
hydrolyzed by the enzyme inverted from tea fungus yeasts in to glucose and
fructose. After the first day, the sucrose consumption began at 8.1 g/L and then
dropped gradually until 7.3 0.2 on the 7th day (Fig.14). The reduction of sucrose
concentration occurred significantly faster than Kombucha fermented with black tea
which just reached 6.9 0.2 on the 12th day (Jayabalan, et al., 2007).
Reducing Sugars:

Figure15: The reducing sugar of M50 during 7 days fermentation

Glucose is used by the yeasts to yield ethanol, which is initially oxides to
acetaldehyde then oxidized by acetic acid bacteria and carbon dioxide. Fructose
remains part of the ferment broth and is utilized by the microorganisms to a lesser
degree. Glucose was not produced in parallel with fructose but was produced with a
lower initial rate (Chen and Liu, 2000). The reducing sugars content 12.35 g/L in
grape juice was decreased to 10.99 0.4 during 7-day fermentation due to the
increase in total acidity by beverage (Fig.15). This appeared to be rather high in
comparison with any the results of other authors after the fermentation on tea (R.

18

Jayabalan, et al., 2007) or previous findings for the fermentation on molasses (R.V.
Malbasa, et al., 2007)
Total Acidity:

Figure 16: The total acid content of M50 during 7-day fermentation
The content of total acid as a function of fermentation time is presented in Figure
16. It was changed from 41 g/L in grape juice to 170 2.5 g/L as preparation
condition. That was much higher than the Kombucha fermented with black tea when
just reach about 26.63 3.4 g/L on the 12th day (Jayabalan, et al., 2007). The
differences between the total acid and the concentrations of glucuronic acid in the
different substrates can be attributed to the presence of other acid metabolites such
as gluconic, lactic, acetic.

Glucuronic Acid:

19

Fig. 17: The glucuronic acid of M50 during 7-day fermentation

Using grape juice, as a medium for the fermentation process of kombucha layer,
the layers bacteria and yeasts metabolize sucrose into a number of organic acids
such as acetic acid and glucuronic acid by different and complementary ways that
one of the possible ways of glucose transformation is also its oxidation at C-6
position into glucuronic acid this is what appeared in the results investigated (Chu
and Chen, 2006). Although there were reported that green and black tea were found
to be the best substrate for glucuronic acid production (17.3 1.4 g/L and 23.3 2.4
g/L) respectively on the 12th day by kombucha culture (Jayabalan, et al., 2007) and
until now there have been no reports about the influence of Kombucha layer activity
on the component changes in grape juice. In this study, the Glucuronic acid yields
ranged from 35.8 g/L to 159.23 9.5 g/L in 7 days (Fig.17) was unbelievably very
high, it synthesized faster than on black tea or green tea with sucrose substrate as a
source of energy.

20

4. Conclusion
The results of the sensory evaluation demonstrated that the acceptability of
consumers for grape juice fermented by Kombucha layer is within a period of 5
days. There were some differences among the samples during the fermentation, and
on the 7th day the glucuronic acid increased more than five times as much in
comparison with the grape juice on the first day. At the same time the pH degree, the
remain sucrose and the reducing sugar decreased during the fermentation, while the
total acid was increased. Consequently, the fermentation with grape juice was faster
in chemical changes than that with tea.

The study contributed to the research a new process of fermentation on grape

juice especially on red grape juice, in addition to the increasing of Glucuronic acid.
The further study is highly recommended on gluconic acid with the different
variable such as microbial change, anitmicrobial activity, carbon source content,
etc...
21

I hope that, the research about grape juice fermented by Kombucha layer could
contribute to the industrial nutritious beverage production in our country as a
premise of the economic development.

22

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APPENDICES
APPENDIX 1: Attributes of grape juice fermented by 4 types of Kombucha
layers with their associated significance level as analyzed by ANOVA.

Attribute Acceptance

P-value

Odor
Color
Taste
Overall appearance
Overall acceptability

P=0.415
P=0.885
P=0.162
P=0.801
P=0.323

APPENDIX 2: Raw data of sensory evaluation of 28 samples.

Day Odor
Colour Taste
Overall
Overall acceptability
appearance
M25

M50

M75

1
2
3
4
5
6
7
1
2
3
4
5
6
7
1
2
3
4
5
6
7
1
2
3
4

2.1
2.3
2.3
2
1.9
1.4
1.2
2.1
2.5
2.6
2.4
2.1
1.2
1.1
2
2.3
2.3
2.1
1.5
1.5
1.3
2
2.1
2.2
1.9

2.2
2.3
2.3
2.3
2.2
2.1
2
2.2
2.3
2.1
2.3
2.1
2.2
2.1
2.1
2.3
2.4
2.1
2.2
2.2
2.3
2.1
2
2.1
2

3
2.5
2.5
2.4
1.9
1.6
1.1
2.9
2.8
2.8
2.1
1.5
1.2
1.2
2.7
2.4
2.4
1.7
1.5
1.1
1.1
2.5
2.6
2.7
2.5

2.7
2.7
2.8
2.1
1.5
1.1
1.2
2.7
2.8
2.9
2.2
1.4
1.1
1.1
2.8
2.4
2.5
1.8
1.7
1.3
1
2.9
2.9
2.8
2.9

2.7
2.7
2.7
2.2
1.4
1.3
1.1
2.9
2.8
2.9
2.3
1.3
1.2
1.1
2.8
2.4
2.4
1.8
1.5
1.2
1.1
2.9
2.8
2.8
2.7

5
6
7

1.5
1.3
1.2

2.3
2.2
2.3

2.4
2.1
1.9

2.7
2.1
1.5

2.7
2.1
1.4

APPENDIX 3: Glucose standard for reducing sugar

APPENDIX 4: ANOVA for Glucuronic acid of grape juice fermented on

Kombucha layer among M25, M50, M75, D

Source

Sum of
squares

Df

Mean
value

F value

P value

Significant

Day 1

619.84

206.613

1.72E+00

0.000

Significant

Day 2

4198.465

1399.488

147.562

0.000

Significant

Day 3

442.877

147.626

15.02

0.001

Significant

Day 4

7283.008

2427.669

101.176

0.000

Significant

Day 5

1226.301

408.767

21.403

0.000

Significant

Day 6

579.344

193.115

17.036

0.001

Significant

Day 7

531.976

177.325

28.083

0.000

Significant

APPENDIX 5: The result for each samples during 7 days fermentation

Sampl
e
M25

M50

M75

Time

pH

Sucrose

Day
1
2
3
4
5
6
7
1
2
3
4
5
6
7
1
2
3
4
5
6
7
1
2
3
4
5
6
7

3.65
3.22
3.19
3.17
3.15
3.14
3.11
3.57
3.33
3.24
3.21
3.16
3.12
3.1
3.4
3.29
3.2
3.17
3.15
3.13
3.11
3.78
3.46
3.34
3.32
3.29
3.27
3.17

g/L
8.35
7.27
7.43
7.54
7.39
7.35
7.3
8.1
7.78
7.7
7.71
7.63
7.49
7.23
8.8
7.86
7.79
7.8
7.81
7.41
7.31
8.74
8.51
8.48
8.39
8.31
8.24
8.25

Reducing
Sugar
g/L
12.19
12.37
12.3
12.24
11.68
11.28
10.93
12.35
12.51
11.95
12.22
11.43
11.27
10.99
12.21
12.29
12.23
11.9
11.81
11.4
10.73
12.25
12.29
12.35
11.84
11.76
11.34
11

Total
Acid
g/L
42
48
79
83
125
139
146
41
78
90
149
154
165
170
57
83
103
147
157
168
180
38
47
86
118
145
152
157

Glucuroni
c Acid
g/L
3.167
4.59
6.847
7.953
12.306
13.684
14.567
3.58
6.76
7.856
13.506
14.967
15.511
15.923
4.791
7.828
8.296
14.112
14.632
15.007
15.585
2.897
3.038
7.927
10.928
13.958
14.227
14.345

APPENDIX 6: Sensory evaluation consumer form.

VIETNAM NATIONAL UNIVERSITY HOCHIMINH CITY
INTERNATIONAL UNIVERSITY

ti lun vn tt nghip
Sinh vin: Dng Hong Bo Khnh ( BT070117)
Gio vin hng dn: Tin s L Hng Ph

PHIU NH GI CM QUAN SN PHM

NC P NHO KOMBUCHA
Tn ngi th:.
Ngy th:.
Mu..

BNG CU HI

Cu hi

Kt qu
Xin vui lng cho bit anh ch nh gi nh th no v hng thm ca sn phm?
1
Nng mi qu nh so vi thch ca ti
Nng mi va phi vi thch ca ti
2
Nng mi qu nng so vi thch ca ti
3
Xin vui lng cho bit anh ch nh gi nh th no v mu sc ca sn phm?
1
Mu sc qu lt lt so vi thch ca ti
Mu sc va phi vi thch ca ti
Mu sc qu m vi thch ca ti
Xin vui lng cho bit anh ch nh gi nh th no v mi v ca sn phm?

2
3

1
Chua so vi thch ca ti
Va phi, chp nhn c so vi thch ca ti
2
Ngt so vi thch ca ti
3
Xin vui lng cho bit mc thch ca anh ch v sn phm sau khi cm quan?
1
Hon ton khng thch
Va phi, chp nhn c

Rt thch
3
Xin vui lng cho bit anh ch nh gi nh th no v v hi ha ca sn phm?
1
V khng ngon
V bnh thng
V ngon

2
3

RT CM N NHNG KIN NG GP QU BU CA ANH/CH