MINISTRY OF EDUCATION & TRAINING
CAN THO UNIVERSITY
BIOTECHNOLOGY RESEARCH & DEVELOPMENT INSTITUTE
SUMMARY
BACHELOR OF SCIENCE THESIS THE
ADVANCED PROGRAM IN BIOTECHNOLOGY
FERMENTATION OF MANGO JUICE BY
LACTIC ACID BACTERIA
SUPERVISOR
STUDENT
MSc. HUYNH XUAN PHONG
NGUYEN LE VAN
Student code: 3082484
Session: 34
Can Tho, 2013
APPROVAL
SUPERVISOR
STUDENT
MSc. HUYNH XUAN PHONG NGUYEN LE VAN
Can Tho, May …, 2013
PRESIDENT OF EXAMINATION COMMITTEE
Abstract
In order to take advantage of available fruit and contribute to the
diversification of products from mango, this research was carried
out in term of added value of fruit juice fermented by lactic acid
bacteria (LAB). Four isolates of lactic acid bacteria from mango
were obtained. The results of research showed that all 10 strains
of lactic acid bacteria were able to develop in pH from 1.5 to 3.5
after 2 hours of incubation at 37°C with a density from 6.80 to
6.91 log cells/mL. After fermentation, the lactic acid content of 10
bacterial strains was in a range from 0.48 to 0.96% w/v, density
after 2 hours of incubation at 37°C from 8.85 to 9.3 log cells/mL.
Of which, strains isolated Lactobacillus acidophilus from Ybio
yeast powder gave the best results with the following levels of
lactic acid after fermentation was 0.96% w/v. The appropriate
ratio for fermentation mango juice was 40% for dilution and 9%
for blending sugar. The suitable conditions for mango juice
fermentation were as follow: at 37°C for 36 hours and at 5 log
cells/mL. In these conditions the sensory criteria and the density
of bacteria could reach requirements for probiotic products (> 6
log cells/mL). These levels could identically maintain during the
storage at temperature 4-6oC for 2 weeks.
Keyword: fermentation, L. acidophilus, lactic acid bacteria,
mango, probiotic.
i
CONTENTS
Abstract ................................................................................. i
Content ................................................................................... ii
1. Introduction ....................................................................... 1
2. Materials and methods ........................................................ 3
2.1 Material ............................................................................ 3
2.2 Methods ............................................................................ 3
2.2.1 Isolation and identification of LAB isolates at genus
level. ................................................................................ 3
2.2.2 Study on the tolerance at low pH of LAB isolates .... 3
2.2.3 Study on the ability to produce mango juice
fermented by LAB ............................................................ 4
2.2.4 Study on the ratio of dilution and blending sugar ...... 4
2.2.5 Study on the effects of LAB density, fermentation
temperature and time ........................................................ 5
2.2.6 Study on temperature and time for storage
conditions ......................................................................... 5
3. Results and discussion ........................................................ 6
3.1 Isolation of lactic acid bacteria .......................................... 6
3.2 The ability of LAB growing in low pH condition ............. 7
3.3 Applicability of mango juice fermented by LAB ............... 9
3.4 The ratio of dilution and blending sugar ............................ 11
3.5 Effect of inoculum density, temperature, fermentation
time ........................................................................................ 16
3.6 The effect of temperature and time for storage conditions .. 19
4. Conclusions and suggestions ............................................... 22
4.1 Conclusions ................................................................ 22
ii
4.2 Suggestions ................................................................ 22
References........................................................................ 23
iii
1. INTRODUCTION
Food is an indispensable necessity to humans. Growing
society, human life is more and more enhanced. So, eating is not a
main demand. The demand of enjoying and exploring the effect
of food on health are top priority. Since, the relationship between
food and dietary which have health benefits as well as help the
body fight the illness has become the trend research of the
nutritionist and scientists to create high-value products in terms of
biological products, is known as "probiotics". The probiotic
concept has been defined by Fuller (1989) to mean “a live
microbial feed supplement which beneficially affects the host
animal by improving its intestinal microbial balance”. Salminen
et al. (1999) proposed that probiotics are microbial cell
preparations or components of microbial cells that have a
beneficial effect on the health and well-being of the host. There
are many probiotic products use for human, livestock, plants. It is
produced and used of liquid or powder form.
Lactic acid are Gram-positive bacteria (Fooks et al., 1999),
ferment carbohydrates into energy and lactic acid (Jay, 2000).
Depending on the organism, metabolic pathways differ when
glucose is the main carbon source: homofermentative bacteria,
whereas the heterofermentative transform a glucose molecule into
lactate, ethanol and carbon dioxide (Caplice and Fitzgerald, 1999;
Jay, 2000; Kuipers et al., 2000). In addition, LAB produce small
organic compounds that give the aroma and flavor to the
fermented product (Caplice and Fitzgerald, 1999). LAB are
widely distributed in the nature. They could be isolated from
1
soils, water, plants, silages, waste products and also from the
intestinal tract of animals and humans (Axelsson, 1998). LAB are
widely used in probiotic products such as yogurt, nem chua,
pickle,... These products are not only used for eating but also used
to treat intestinal , stomach, due to lactic acid bacteria have the
ability to produce antibiotics to prevent and kill bacteria and
pathogenic microbes.
Juice is a good environment for the growth of bacteria and
probiotic products. Fruits and vegetables are foods with health
benefits because they contain antioxidants, vitamins, fiber and
minerals. Moreover, they do not contain any allergens for
consumers. In fruits, mango is a popular fruit and has high
nutritional value. Ripe mango has attractive yellow color, sweet
and sour, savoury aroma is more preferred. Mangoes contain
vitamin A, C, sugar, organic acids, so mango is widely used both
unripe and ripe fruit. Mangoes are eaten fresh, making juice, jam,
candy.
In order to take advantage of available fruit and contribute
to the diversification of products from mango, this research was
carried out in term for added value of fruit juice fermented by
lactic acid bacteria.
Objectives:
Study on the conditions of mango juice fermentation by
lactic acid bacteria.
2
2. MATERIALS AND METHODS
2.1 Materials
- Mango: Thanh Ca and Cat Chu.
- Six isolates of lactic acid bacteria from Luong Phuoc
Truong (2012).
- Medium: MRS broth, MRS agar.
- Chemicals, equipments in Food Biotechnology laboratory.
2.2 Methods
2.2.1 Isolation and identification of LAB isolates at genus level
Mango juice was contained in a flask, incubated at 37oC for
24 - 48 hours.
Transfered 1 mL mango juice fermented into a test tube
containing MRS broth environment.
After 24 hours, transfered bacteria from MRS broth to MRS
agar environment. Continue transfer bacteria on MRS agar
environment until pure.
Observe the bacteria under the microscope objective lens
X100.
Identification through the preliminary test: Gram stain,
catalase, oxidase test and dissolution of CaCO3.
2.2.2 Study on the tolerance at low pH of LAB isolates
Transferring LAB isolates from experiment 1 into tubes
containing MRS broth environment with 3 different levels of pH
(1.5, 2.5 and 3.5). This was the 2 experimental factors (strain and
pH) with 3 replications. Count the density of bacteria in
individual treatments at incubation time (T0) and after 2 hours of
incubation by plate counting method.
3
Monitor the indicators: density of the bacteria strains.
Analyze the data with statistics program STATGRAPHICS
Centurion XV.
2.2.3 Study on the ability to produce mango juice fermented
by LAB
Ripe mangoes were pressed (pasteurized with 140 mg/L of
NaHSO3 in 20 minutes) and put in the centrifuge tubes.
Inoculate each of LAB isolates into each tube. This was the 1
experimental factor (strains) with 3 replications.
Incubation at 37°C, analyzed after 48 hours.
Count density of lactic acid bacteria: plate counting method.
Monitor the indicators: Brix, pH, level of lactic acid after
fermentation and the density of lactic acid bacteria. Analyze the
data with statistics program STATGRAPHICS Centurion XV.
2.2.4 Study on the ratio of dilution and blending sugar
This was the 2 experimental factors with 3 replications.
Ratio of dilution (%): 30, 40, 50, 60
Ratio of blending surose (%): 6, 9, 12, 15
After pressing, 480 mL of mango juice was diluted with
ratios 30, 40, 50, 60 % and was blended sugar with rate 6, 9, 12,
15 %. After dilution and blending, these samples were pasteurized
by NaHSO3 (140 mg/L in 20 minutes). Then the best LAB isolate
in experiment 3 was inoculated in the samples and incubated at
37oC for 48 hours.
Monitor the indicators: Brix, pH before and after
fermentation, lactic acid content after fermentation and the
4
density of LAB, the sensory evaluation. Analyze the data with
statistics program STATGRAPHICS Centurion XV.
2.2.5 Study on the effects of LAB density, fermentation
temperature and time
This was the 3 experimental factors at 3 levels with 2
replications.
Density (log cells/mL): 3, 5, 7.
Incubation temperature (ºC): 30, 37, 28-32.
Time for fermentation (hours): 24, 36, 48.
After pressing, mango juice was diluted and blended sugar
by the appropriate rate in experiment 4, pasteurized by NaHSO3.
Then, LAB isolate was inoculated with density were 3, 5, 7 log
cells/mL, respectively. Incubated these samples at 30oC, 37oC,
28-32oC for 24, 36, 48 hours.
Monitor the indicators: Brix, pH and the density of LAB,
the sensory evaluation. Analyze the data with statistics program
STATGRAPHICS Centurion XV.
2.2.6 Study on temperature and time for storage conditions
This was the 2 experimental factors with 3 replications.
Temperature for storage: 4-6oC, 20-25oC, 28-32oC.
Time for storage: 1, 2 weeks.
Monitor the indicators: Brix, pH and the density of LAB, the
sensory evaluation. Analyze the data with statistics program
STATGRAPHICS Centurion XV.
5
3. RESULTS AND DISCUSSION
3.1 Isolation of lactic acid bacteria
After several transfers in MRS agar medium and visual
observations, 4 isolates of lactic acid were isolated from 2 kinds
of mango. Colonies of all isolates were round, smooth, grayish
white.
The cell morphology was shown in Table 2’
Table 2’. The characteristics of isolates
Cell
No
Sample
The isolate
morphology
1
Cat Chu
C213
Rod in pairs
2
Cat Chu
C111
Rod in pairs
3
Thanh Ca
T223
Rod in pairs
4
Thanh Ca
T133
Cocci in pairs
5*
Antibio
A
Short rod
6*
Probio
P
Short rod
7*
Ybio
Y
Short rod
8*
Lactomin plus
L
Cocci
9*
Biosubtyl
Bio
Long rod
10*
Papaya
ĐĐ
Rod in pairs
Note: The strains are marked * which were isolated from Truong (2012).
6
2 isolates from Cat Chu (C213, C111) were rod in pairsshaped, but the colony of C213 was smaller than C111. 2 isolates
from Thanh Ca (T223, T133) were rod in pairs-shaped and cocci
in pairs-shaped.
4 isolates were characterized in Gram positive, lack of
catalase and oxydase, produced clear zone around colonies in
medium containing CaCO3.
Figure 6: Representative
Figure 7: CaCO3 test
oxydase test of C213
of T223 isolate
isolate
From the above characteristics, it could be concluded that
4 isolates belong to lactic acid bacteria group.
3.2 The ability of LAB growing in low pH condition
The results in Table 3 showed that all bacterial strains at
different pH levels have increased density after 2 hours of
incubation at 37°C. At time T0, the density of bacteria in the MRS
medium at pH 1.5 and 2.5 decreased significantly (from 5 log
CFU/mL to 1.46-2.16 log CFU/mL). The reason was that the pH
1.5 and 2.5 are too low. So, most bacteria should be shocked. But
only after 2 hours of incubation at 37°C, they worked to restore
and increased the density up to 6.8-6.9 log CFU/mL.
7
At pH 3.5, most of bacteria could tolerate this pH. So, the
density of LAB at the time T0 decreased slightly from 4.66 to 4.77
log CFU/mL. After 2 hours of incubation at 37°C, the density of
bacteria increased up to 6.81-6.91 log CFU/mL.
Density of 10 isolates at pH 3.5 was higher than at pH 2.5
and density of 10 isolates at pH 1.5 was lowest. This showed that
the lower the pH affects the ability to survive and activity of
LAB. Through this experimental study showed that all isolate
were able to adapt and develop in a low pH medium, so they
could tolerate the environment in the stomach. Therefore, these
strains were able to apply in probiotic products.
Table 3. The density of bacteria
in MRS Broth environment at low pH.
pH
1,5
Isolates
logT0
Cv
logT2
Cv
(log CFU/mL)
(%)
(log CFU/mL)
(%)
j
A
1.48
P
j
1.46
Y
j
1.49
L
j
0.39
0.39
0.39
1.49
0.39
j
Bio
1.49
ĐĐ
j
0.67
1.48
0.00
C111
j
1.48
0.00
C213
1.47j
0.39
T223
j
1.46
T133
j
1.46
0.79
A
1.95h
2.56
0.39
8
e
0.15
de
0.31
cde
0.08
de
0.85
e
0.86
de
0.22
abcde
0.84
6.81
6.82
6.83
6.82
6.80
6.82
6.86
6.81e
0.90
abcde
0.29
e
6.81
0.34
6.84bcde
0.58
6.86
2,5
P
1.88i
Y
f
6.88abcd
0.80
2.45
bcde
0.34
bcde
0.30
L
i
1.89
0.53
Bio
2.13f
1.18
ĐĐ
g
C111
2.06
2.67
g
2.04
C213
g
2.07
T223
1.88i
1.98
6.84
6.84
6.82de
0.29
abcde
0.37
ab
0.65
6.86
6.9
1.21
de
6.82
0.29
0.81
6.82cde
0.37
T133
h
1.93
0.79
bcde
6.84
0.58
A
4.72c
0.53
6.84bcde
0.39
P
c
0.24
abc
0.73
abcde
1.27
a
0.88
de
0.15
bcde
0.22
cde
0.08
e
0.96
bcde
0.72
abcde
0.22
Y
L
Bio
3,5
2.16
0.53
ĐĐ
C111
C213
T223
T133
4.73
a
4.77
0.24
bc
4.74
0.24
cd
4.71
0.25
e
4.66
0.33
ab
4.77
0.21
c
4.73
0.32
cd
4.71
0.25
de
4.67
0.12
6.89
6.85
6.91
6.82
6.84
6.82
6.81
6.84
6.85
Note: The data in the table is the average of triplicates. The difference
was statistically significant mean only in columns. The same characters show no
difference statistically significant at 95%.
3.3 Applicability of mango juice fermented by LAB
Nowadays, probiotic products are commonly used as
fermented milk and yogurt. Besides, juice is offered as a good
9
environment for the fermented probiotic products (MattilaSandholm et al., 2002).
Table 4. Some indicators of mango juice after fermentation
Isolates
Brix
ab
pH
c
Acid
log T0
log T48
(% w/v)
(log CFU/mL)
(log CFU/mL)
a
a
Y
8.0
3.02
0.96
4.88
9.30a
A
7.67b
3.14bc
0.73c
4.82abc
9.0a
P
7.67b
3.42a
0.81bc
4.76c
8.89a
L
8.87a
3.2b
0.48d
4.85abc
9.15a
Bio
8.5ab
3.21b
0.90ab
4.78bc
8.85a
ĐĐ
8.0ab
3.07bc
0.69c
4.85ab
9.25a
C213
8.0ab
3.42a
0.92ab
4.83abc
9.03a
C111
8.0ab
3.39a
0.91ab
4.83abc
9.08a
T223
8.12ab
3.21b
0.75c
4.80abc
8.91a
T133
8.83a
3.11bc
0.75c
4.77bc
8.85a
Cv (%)
7.23
4.89
19.24
1.14
3.66
Note: The data in the table is the average of triplicates. The same
characters show no difference statistically significant at 95%.
The results in Table 4 showed that after 48 hours of mango
juice fermentation, all 10 isolates of lactic acid increased density,
produced lactic acid, reduced the amount of dissolved substances
(oBrix) and pH of the environment.
Brix of the treatments after fermentation was lower than the
original in a range 8.0-8.87, because LAB used sugar in the
mango juice to enrichment and generate lactic acid released to the
10
environment. After 48 hours of fermentation mango juice, pH of
the environment of the isolates decreased from 3.83 to 3.02-3.42.
As the results presented in the previous experiments, LAB
isolates were able to survive and grow at this pH. The cause of the
decrease in pH was due to the development of lactic acid bacteria
that produced lactic acid. Content of lactic acid produced from 4
isolates from Ybio powder, Biosubtyl powder and cat chu have no
significant differences with each other and higher than the other
isolates.
Density of lactic acid bacteria strains in time T0 were nearly
equal to the initial concentration of bacteria strains (4.76-4.88 log
CFU/mL) and after incubation all isolates had a significant
enrichment activity (8.85-9.30 log CFU/mL). Results showed
statistically processed, the density of lactic acid bacteria strains at
time T0 had no significant differences with each other and
significantly
different
from
3
strains
P,
Bio,T133.
After 48 hours of fermentation the density of bacterial strains
were > 6 log CFU/mL, consistent with standards of probiotic
products. Therefore, all strains were capable of application in the
production of fermented mango. However, lactic acid content of 4
isolates from Ybio powder, Biosubtyl powder and Cat Chu had no
significant differences with each other and higher than those of
isolates. Among those strains, the L. acidophilus isolate from
Ybio had the best content of lactic acid. So, L. acidophilus isolate
was selected for the next experiments.
3.4 The ratio of dilution and blending sugar
11
The results in Table 5’ showed that pH values before and
after fermentation was difference. After 48 hours of incubation,
pH was reduced from about 4.5-4.61 to 3.63-3.84. It caused by
bacterial growth, changed glucose into lactic acid. At the same
time, content of solution (oBrix) also decreased but not
significantly.
Table 5’. Some indicators of mango juice fermentation
Blending
Sucrose
(%w/v)
Dilution
(%)
pH after
fermentation
Brix after
fermentation
6
3.73bcde
15.33h
9
3.75abcde
17.43ef
12
3.77abcd
20.17b
15
3.83ab
21.8a
6
3.72cdef
14i
9
3.66ef
16g
12
3.76abcde
18.33d
15
3.68def
20.5b
6
3.84a
12.33j
9
3.71cdef
15.83g
12
3.79abc
17f
15
3.79abc
19.33c
6
3.74abcde
10.83k
9
3.67def
13.83i
30
40
50
12
3.7cdef
15.67gh
15
3.63f
17.5e
Cv (%)
2.09
17.83
60
12
Note: The data in the table is the average of triplicates. The same
characters
show
no
difference
statistically
significant
at
95%.
At the dilution rate of 30% of mango juice, the content of
lactic acid reduced while sugar content increased (Figure 8). The
high content of sugar inhibited the growth and development of
bacteria, acid content decreased. The lowest acid content when
the dilution rate was 60%.
Besides, at the same sugar content the samples which had
dilution ratio 30%, 40% had high content of lactic acid.
Therefore, at the dilution rate 30, 40% bacteria growth was better
than those of dilution ratios. In addition, when compared with the
other treatments the sample at the rate of 40% diluted and 9%
sugar content gave the highest acid content.
13
Figure 8. The change of lactic acid content according to sugar
content and dilution rate
In general, the density of LAB reduced when dilution rate
and the content of sugar increased. Density of LAB was highest in
30% dilution and 9% sugar. On the other hand the density of
bacteria at all dilution rates were above 6 (log CFU/mL). It was
suitable for the production of probiotic products.
Figure 9. The change of bacterial density according to
sugar content and dilution rate
14
Statistical results of sensory evaluation in Table 7 showed
that the samples with dilution ratio of 50% and 60% got lowest
score because these samples had a distinct aroma of mango. The
sample with 40% dilution and 9% content sugar had highest score
because this sample contained lactic acid, sweet taste and
aromatic smell.
Table 7. Sensory evaluation results of blending sugar content
and dilution rate
Dilution ratio of
juice
(% v/v)
30
40
50
60
Sugar content
(% w/v)
Score of sensory
evaluation
6
9
12
15
6
9
12
15
6
3.6cd
4.0b
3.75c
3.3fg
3.4ef
4.65a
3.65cd
3.5de
2.6i
9
12
15
6
9
12
15
2.8h
2.95h
3.15g
2.2j
2.5i
2.8h
2.9h
15.49
Cv (%)
Note: The maximum score is 5. The data were the average of 5 sensory
evaluators.
In short, from the results of pH, Brix, lactic acid content, the
density of bacteria and the results of the sensory evaluation
15
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