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INTERNATIONAL UNIVERSITY
A thesis submitted to
The School of Biotechnology, International University
In partial fulfillment of the requirements for the degree of
B.S. in Biotechnology
Dr. L Hng Ph
February, 2013
A thesis submitted to
The School of Biotechnology, International University
In partial fulfillment of the requirements for the degree of
B.S. in Biotechnology
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.
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.
1. Introduction
1.1
Kombucha
Glucuronic acid
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).
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.
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.
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.
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.
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 measurement of chemical changes were then carried out after the process
had an acceptable time by sensory evaluation.
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.
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.
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.
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.
12
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
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
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
3.2
PH:
16
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:
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
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.
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
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
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
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
ti lun vn tt nghip
Sinh vin: Dng Hong Bo Khnh ( BT070117)
Gio vin hng dn: Tin s L Hng Ph
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