fermentation of mango juice by lactic acid bacteria

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


showed that the samples with dilution ratio of 40% and 9% sugar
was appropriate to ferment mango. These ratios were chosen for
subsequence experiments.
3.5 Effect of inoculum density, temperature and time for
fermentation
After fermentation, the values of pH and Brix decreased.
The results of Figure 10 showed that, acid content reached
the highest value at 37°C. Moreover, lactic acid content of the
samples with bacterial density 7 log cells/mL were higher than the
samples with bacterial density 3-5 log cells/mL. When the
bacterial density was higher the ability converting sugar into
lactic acid of bacteria was stronger.

Figure 10. The change of acid content according to
inoculum density, temperature and time of fermentation
In general, the acid content increased when incubation time
increased. Acid content of the samples incubated in 24 hours were
lower than the samples incubated in 36-48 hours. Lactic acid
content reached the highest value (1.02% w/v) in the sample of
bacterial density 7 log cells/mL at 37°C for 48 hours incubation.
16

Next, the sample was 5 log cells/mL, incubated for 48 hours at
37°C (0.95% w/v).

Figure 11. The change of bacterial density according to
inoculum density, temperature and time of fermentation
The results of Figure 11 showed that density of bacteria
reached the highest value at 37oC and the lowest at 30oC. Besides,
the density at 48 hours of incubation was higher than the other
incubation time.
In the same temperature and incubation time, the samples
with 7 log cells/mL of bacterial density had the higher density
than the other. Highest density bacterial in samples with density
of 7 log cells/mL, incubated at 37°C for 48 hours (9.74 log
CFU/mL). After incubation, bacterial density of all samples were
reached over 6 log CFU/mL, so these samples had the standard of
probiotic products.
The results of the sensory evaluation showed that the sample
which was inoculated 5 log cells/mL, incubated at 37°C for 36
hours was appreciated (4.6) because of smell of mango, the
harmony of sweet and sour. Sample with bacterial density 7 log
17


cells/mL, incubated at 37°C for 48 hours was the worst (1.6).
Although the results of lactic acid content, bacterial density
of the samples which were inoculated 7 log cells/mL, incubated at
37oC for 48 hours were higher than the other samples. However,
these are the products for humans. Therefore, these samples were
not selected because the less aroma of mango in product, sour and
sweet was not harmony. While the samples were inoculated 5 log

cells/mL and incubated at 37oC for 36 hours were still reached the
standard of probiotic products (>6 log CFU/mL) and had the
highest score in the sensory evaluation.
In short, the samples were inoculated 5 log cells/mL and
incubated at 370C for 36 hours were suitable for the production of
mango juice fermentation.
Table 9. Results sensory evaluation of mango juice fermented
by inoculum density, inoculation time and fermentation time
Time
(h)

Inoculum density
log cells/mL

3

24

5

7

18

Temperature
(oC)

Score of
sensory
evaluation

30

3,35g

P

4,0b

37

3,5ef

30

3,5ef

P

3,7cd

37

4,0b

30

4,05b

P

3,05h

37

2,9i


3

36

5

7

3

48

5

7

Cv (%)

30

3,5ef

P

3,6de

37

4,1b

30

3,75c

P

4,05b

37

4,6a

30

4,1b

P

3,1h

37

2,65j

30

3,65cd

P

3,6de

37

3,05h

30

3,65cd

P

3,4fg

37

2,35k

30

1,9l

P

1,95l

37

1,6m
21.9

Note: The maximum score is 5. The data were the average of 5 sensory
evaluators.

3.6 The effect of temperature and time for storage conditions
The samples which storage at 4-6oC, cool room (20-25oC)
and ambient temperature (28-32oC) had higher density more than
19


6 log CFU/mL. These samples were consistent with the
requirements of probiotic products. In the first week, the density
of bacteria at 28-32oC was higher than samples stored at 4-6oC
and 20-25oC. The reason was that, this was the appropriate

Time (weeks)

temperature for bacterial growth.
Table 10. Results of the survey time and temperature for
storage products

1

2
Cv
(%)

Acid content
(%w/v)

pH

Bacterial density
(log CFU/mL)

4˚C
–
6˚C

20˚C
–
25˚C

28˚C
–
32˚C

4˚C
–
6˚C

20˚C

–
25˚C

28˚C
–
32˚C

4˚C
–
6˚C

20˚C
–
25˚C

28˚C
–
32˚C

3,81a

3,71b

3,43d

1,07c

1,10bc

1,37a

8,51a

8,52a

8,71a

3,84a

3,71b

3,65c

1,10bc

1,14b

1,4a

8,16b

7,34c

6,15d

3.72

11.81

11.88

Note: The data in the table was the average of triplicates. The same
characters show no difference statistically significant at 95%.

In the second week, the bacterial density at 3 storage
temperature was reduced. However, these samples were still
reached the standard of probiotic products (>6 log CFU/mL). The
bacterial density of the sample stored at room temperature 28320C decreased from 8.71 to 6.15 log CFU/mL.
In short, products stored at 4 - 60C and content of lactic acid
bacterial density almost stable after 2 weeks of storage. Content
of lactic acid from 1.07 to 1.1 %w/v and bacterial density 8.51 20