fermentation of dragon fruit juice by lactic acid bacteria

MINISTRY OF EDUCATION & TRAINING
CAN THO UNIVERSITY
BIOTECHNOLOGY RESEARCH & DEVELOPMENT INSTITUTE

SUMMARY
BACHELOR OF SCIENT THESIS
THE ADVANCED PROGRAM IN BIOTECHNOLOGY

FERMENTATION OF
DRAGON FRUIT JUICE
BY LACTIC ACID BACTERIA

SUPERVISOR

STUDENT

MSc. HUYNH XUAN PHONG

NGUYEN THUY VI
Student code: 3082567
Session: 34

Can Tho, 2013


APPROVAL

SUPERVISOR

STUDENT

MSc. HUYNH XUAN PHONG

NGUYEN THUY VI

Can Tho, 2013
PRESIDENT OF EXAMINATION COMMITTEE


Abstract
This study was carried out to isolate and select the isolates
of lactic acid bacteria applied for the fermentation of dragon fruit
juice. Experiments were done including dilution rates, sugar
content, inoculum level, incubation temperature, fermentation
time, and storage conditions. Six strains of lactic acid bacteria
isolated from dragon fruit juice and three strains of probiotic
powder were tested. All 6 tested bacterial strains could grow in
low pH conditions (1.5 – 3.5) and could be used for fermentation
of dragon fruit juice, with the bacterial level was at 6 log
CFU/mL. A strain Lactobacillus acidophilus isolated from
Antibio powder gave the best capable of dragon fruit juice
fermentation (acid content was 1.38% w/v and bacteria
concentration was 8.44 log CFU/mL). The favourable conditions
of fermentation were determined as follow: pure dragon fruit
juice, 9% of supplemented sugar, 37ºC of fermentation
temperature, 48 hours of fermentation, and 6 log CFU/mL of
inoculum level. The appropriate temperature and time storage for
fermented juice were found at 4 – 6ºC for 3 weeks.
Keywords: dragon fruit juice, fermentation, lactic acid bacteria,
Lactobacillus acidophilus, probiotic

i


Contents
Abstract ...................................................................................... i
Contents ...................................................................................... ii
1. INTRODUCTION .................................................................. 1
2. MATERIALS AND METHODS ............................................ 3
2.1 Materials ............................................................................ 3
2.2 Methods ............................................................................. 3
2.2.1. Isolation and identification of LAB isolates at genus
level .................................................................................... 3
2.2.2. Study on the acidity tolerant ability of LAB isolates... 4
2.2.3. Study on application in producing of fermented
dragon fruit juice by LAB ................................................... 4
2.2.4. Study on dilution rates of dragon fruit juice and
sucrose rates ........................................................................ 5
2.2.5. Study on inoculum levels, incubation temperature
and fermentation time ......................................................... 5
2.2.6. Study on storage temperature and storage time ........... 5
3. Results and discussion ............................................................. 6
3.1. Isolation and identification of LAB isolates at genus level . 6
3.2. Acidity tolerant ability of LAB isolates.............................. 7
3.3. Application in producing of fermented dragon fruit juice
by LAB .................................................................................... 9
3.4. Dilution rates of dragon fruit juice and sucrose rates .......... 10
3.5. Inoculum levels, incubation temperature and fermentation
time ......................................................................................... 15
3.6. Storage temperature and storage time ................................ 18
4. Conclusions and suggestions ................................................... 21

4.1 Conclusions........................................................................ 21
ii


4.2 Suggestions ........................................................................ 21
References ................................................................................... 22

iii


1. INTRODUCTION
Probiotics

are

defined

as

the

non-pathogenic

microorganisms capable of survive in the digestive process,
having positive impact on the health and physiology of the hosts.
There are many probiotic products for human and livestock.
Lactic acid bacteria (LAB) are able to produce probiotics
(Temmerman et al., 2002). Thus, LAB are used in probiotic
products such as yogurt, fermented meat, fermented vegetables…
These products are not only used as food but also used for treating

intestinal and stomach diseases because LAB can produce
antibiotics to prevent and skill pathogenic bacteria and germs.
Also, LAB are widely used in food fermentation because of their
ability to improve flavour, texture and safety of perishable raw
materials (Caplice and Fitzgerald, 1999).
Fruit juice is a good environment for the growth of bacteria
and probiotic products. Fruits and vegetables are good for health
because of antioxidants, vitamins, fibers and minerals in
components.
“Dragon fruit” are commonly known as “thanh long” as in
Vietnamese meaning “green dragon”. Dragon fruit contains
vitamins A, C, sugar, organic acids,… Dragon fruit juice is a
suitable medium for lactic acid bacteria to produce new probiotic
products. Therefore, the study “Fermentation of dragon fruit juice
by lactic acid bacteria” was carried out.

1


Objectives:
To isolate LAB strains from dragon fruit juice, and to study
fermented dragon fruit juice producing process by lactic acid
bacteria.
Contents made
- Isolation and identification of LAB isolates at genus level
- Study on the acidity tolerant ability of LAB isolates
- Study on application in producing of fermented dragon
fruit juice by LAB
- Study on dilution rates of dragon fruit juice and sucrose
rates

- Study on inoculum levels, incubation temperature and
fermentation time
- Study on storage temperature and storage time

2


2. MATERIALS AND METHODS
2.1 Materials
- Dragon fruit bought from Xuan Khanh market (Can Tho
City)
- Probiotics powders:
+ Antibio powder: Product of Han Wha Pharma Co., Ltd.
(Number 472, Namgog-Ri, Yangji - Myon, Yongin - Si,
Kyonggi - Do, Korea).
+ PROBIO powder: Made in the Corporation Imexpharm
(No. 4, 30/4, Cao Lanh Town, Dong Thap).
+ Lactomin powder plus: RexGene Biotech Co. products.,
Ltd. (Number 641-2, Ochang - Myun, Cheongwon - Kun,
Chungbuk, Korea)

- Medium:
+ MRS broth (Merck, Germany): Peptone from casein: 10
g/L, meat extract: 8 g/l, yeast extract: 4 g/L, D-glucose: 20 g/L,
dipotassium hydrogen phosphate: 2 g/L, tween 80: 1 mL,
diammonium hydrogen citrate: 2 g/L, sodium acetate: 5 g/L,
magnesium sulfate: 0.2 g/L, manganese sulfate: 0.04 g/L.
+ MRS agar: MRS broth + agar 14 g/L
- Chemicals: NaOH 0.1N, HCl 0.1N, H2O2 3%, NaHSO3, the
Gram stain (Crystal violet, Iodine, Acetone, Ethanol, Fuchsin)

-

Equipments

in

Food

Biotechnology

Laboratory,

Biotechnology Research and Development Institute, Can Tho
University.
2.2 Methods
2.2.1. Isolation and identification of LAB isolates at
genus level
3


- Sterilized dragon fruit juice was incubated at 37ºC for 24
– 48 hours. Fermented juice and probiotic powders were
incubated in MRS broth medium for 24 hours. LAB were isolated
by culturing the incubated media on MRS broth agar medium
until getting the pure bacteria.
- LAB isolates were identified at genus level through the
preliminary tests: Gram stain, catalase, oxidase test and inspection
capabilities resolution of CaCO3.
2.2.2. Study on the acidity tolerant ability of LAB
isolates

LAB isolates were inoculated in MRS broth medium
adjusted to different levels of pH (1.5, 2.5 and 3.5). Bacterial
density levels of LAB were determined at incubation time (T 0)
and after 2 hours of incubation by the counting living method.
Acidity tolerant LAB isolates were chosen.
2.2.3. Study on application in producing of fermented
dragon fruit juice by LAB
4 mL of LAB with 5 log cells/mL density were inoculated
to 36 mL of pasteurized dragon fruit juice. The inoculated juices
were incubated at 37ºC for 48 hours. After 48 hours of incubation,
the juices were analyzed the assessment criteria including:
- pH: measured by pH meter
- Soluble matter content (Brix): measured by Brix handheld
meter
- Content of generated lactic acid: based ion total acid
index
- Density of LAB: determined by plate counting method
- Sensory evaluation
4


2.2.4. Study on dilution rates of dragon fruit juice and
sucrose rates
0.4 mL of LAB was inoculated to 39.6 mL of pasteurized
dragon fruit juice adjusted to different dilution rates (0, 20, 30 and
40% w/v) and sucrose rates (6, 9, 12 and 15%). The inoculated
juices were incubated at 37ºC for 48 hours. After 48 hours of
incubation, the juices were analyzed the assessment criteria
including: pH, soluble matter content, content of generated lactic
acid, density of LAB and sensory evaluation.

2.2.5. Study on inoculum levels, incubation temperature
and fermentation time
0.4 mL of LAB was inoculated to 39.6 mL of pasteurized
dragon fruit juice adjusted to different levels of inoculum (4, 5,
and 6 log cells/mL). The inoculated juices were incubated at
different temperature levels (25, 30, and 37ºC) at 12, 24, and 36
hours. After incubation, the juices were analyzed the assessment
criteria including: pH, soluble matter content, content of
generated lactic acid, density of LAB and sensory evaluation.
2.2.6. Study on storage temperature and storage time
The fermented dragon juice products were stored at
different temperature levels including: 4 – 6ºC (refrigerator), 20 –
25ºC (cooler) and 28 – 32ºC (room temperature) in 1, 2, 3, and 4
weeks. After storage time, the juices were analyzed the
assessment criteria including: pH, soluble matter content, content
of generated lactic acid, density of LAB and sensory evaluation.

5


3. RESULTS AND DISCUSSION
3.1. Isolation and identification of LAB isolates at genus
level
Isolation results
The features of isolated bacteria colonies: round, glossy,
smooth, creamy white. The colonies of each bacteria strain are
identical. Colonies of bacteria isolated from Antibio and Probio
powder were larger than dragon fruit ones.
There were six isolated bacteria strains, two from dragon
fruit juice and four from probiotic powder. Cell morphology of

isolated bacteria was various as shown in Table 3’.
Table 3’. Cell morphology of isolated bacteria
No
1
2
3
4
5
6

Sources
Dragon fruit
Dragon fruit
Antibio probiotic powder
Probio probiotic powder
Lactomin plus probiotic powder
Biosubtyl probiotic powder

The isolates
TL1
TL2
A
Pro
Lac
Bio

Cell morphology
Rod
Pairing rod
Short rod

Short rod

streptococci
Long rod

Characteristics of isolated bacteria strains:
- Bacteria strains isolated from Antibio and Probio powder
were rod-shaped bacteria. They had the same characteristics of
Lactobacillus acidophilus, the bacteria in probiotic powder
components.
- Bacteria strains isolated from Lactomin were streptococci.
- Bacteria strains isolated from dragon fruit juice were rodshaped bacteria. However, the length of these bacteria strains was
shorter than the length of rod-shaped bactera strains isolated from
Antibio and Probio powder.
6


Genus identification
Some simple biochemistry experiments were carried out to
determine the isolated bacteria strains at genus level. The results
showed that:
- Gram staining: Bacteria strains remained purple color
after staining crystal violet, thus they were Gram-positive
bacteria.
- Catalase test: All six bacteria strains could not produce
bubbles when adding H2O2 3%, indicated negative results.
It can be concluded that these isolated bacteria strains
belonged to lactic acid bacteria because they:
- Are Gram-positive bacteria
- Has no enzyme catalase

- Are rod-shaped or streptococcus.
- Can grow on MRS medium.
3.2. Acidity tolerant ability of LAB isolates
According Guarner and Schaafsma (1998), one of the
criteria of probiotic products is survival ability of microorganisms
after consuming process. In human digestive system, the stomach
pH is very low, about 1 – 2, so majority of microorganisms are
difficult to survive in this pH. However, microorganisms existed
only 1 – 2 hours in the stomach and then move to the large
intestine with a neutral pH.
After 2 hours of incubation at low pH, the bacteria
increased concentration. Initial inoculum level of LAB was 4 log
cells/mL. But when inoculating to MRS medium with pH in the
range of 1.5 to 2.5, the bacterial density reduced significantly
(only 1.08 to 1.40 log CFU/mL), because of shock of bacteria
7


under low pH condition. But only after 2 hours incubation at
37ºC, they recovered and increased concentration (up to 6.31 to
6.62 log CFU/mL).
Almost bacteria could resist to pH 3.5. The decrease of
bacterial density was not significantly at T 0 (3.34 – 3.58 log
CFU/mL). After 2 hours of incubation, the concentration reached
to 6.45 – 6.66 log CFU/mL.
At T0, the concentration of 6 bacteria strains in pH 3.5
medium were higher than theirs in pH 2.5 medium. And the
lowest concentration of bacteria was in pH 1.5 medium. It was
showed that the lower pH affect to the survival ability of LAB.
Table 4. Density of bacteria in MRS broth with low pH

pH

1.5

2.5

3.5

Strains
A
Pro
TL1
TL2
Bio
Lac
A
Pro
TL1
TL2
Bio
Lac
A
Pro
TL1
TL2
Bio
Lac

T0
(Log CFU/mL)

1.26
1.08
1.16
1.19
1.12
1.21
1.40
1.37
1.36
1.35
1.39
1.37
3.44
3.58
3.34
3.37
3.36
3.43

T2
(Log CFU/mL)
6.62b
6.48g
6.56c
6.49g
6.48g
6.44i
6.48g
6.40j
6.55cd

6.31k
6.53def
6.31k
6.53def
6.66a
6.54de
6.46h
6.45hi
6.52f

Note: Value in the table was average value of triplication; the average values
with the same letter were not significantly different at the 95% confidence level.

8


All six LAB strains increased the density after 2 hours of
incubation at 3 different pH levels (ranging from 6.31 to 6.66 log
CFU/mL). The bacterial densities after fermentation were
significantly different at the 95% confidence level (Table 4’). The
Pro strain reached the highest value (6.66 log CFU/mL) at pH 3.5,
next to A strain at pH 1.5 (6.62 log CFU/mL) and TL1 strain at
pH 2.5 (6.55 log CFU/mL).
All isolated lactic acid bacteria strains were able to adapt
and develop in a low pH environment, so they could survive in
the environment of the stomach. Therefore, these strains were
able to apply in probiotic products.
3.3. Application in producing of fermented dragon fruit
juice by LAB
This experiment was carried out to determine whether the

lactic acid bacteria strains could be applied in the production of
fermented dragon fruit juice to create probiotic products, and to
compare individual application abilities of LAB strains together.
After 48 hours, all lactic acid bacteria strains increased
their concentration; solvent content (ºBrix) decreased; lactic acid
was produced to reduce the pH of medium (Table 5).
Table 5. Following indicators fermented dragon fruit juice
Bacteria
strains
A
Pro
TL1
TL2
Bio
Lac

Brix

pH

8.0
7.5
7.1
7.9
8.0
7.8

4.31
3.23
4.01

3.95
4.25
4.09

Acid
(% w/v)
1.38ª
1.36b
1.28c
1.24d
1.19e
1.23d

Log T0
(log CFU/mL)
3.86ab
3.84c
3.82d
3.85bc
3.87ª
3.84c

Log T48
(log CFU/mL)
8.44ª
8.36b
8.35bc
8.34c
8.37b
8.27d

Note: Value in the table was average value of triplication; the average values
with the same letter were not significantly different at the 95% confidence level.

9


Brix concentration of all treatments after fermentation
decreased insignificantly (from 8.9ºBrix to about 7.1 – 8.0ºBrix).
After 48 hours of fermentation, pH decreased from 4.52 to
about 3.23 – 4.31. At this pH, lactic acid bacteria could remain.
The pH decreased because due to the production of lactic acid
during development of bacteria. Concentration of lactic acid
produced from A strain (1.38%) and Pro strain (1.36%) were
higher than other strain’s and they were significantly different to
others at the 95% confidence level.
After incubation, densities of LAB increased significantly
(from 3.82 – 3.87 to 8.27 – 8.44 log CFU/mL). As statistical data
analysis results, at T 0, densities of Bio and A strains were highest
(3.87 and 3.86 log CFU/mL), they were significantly different to
others at the 95% confidence level.
After 48 hours, densities of LAB were higher than 6 log
CFU/mL. The highest concentration (8.44 CFU/mL) belonged to
A strain (Lactobacillus acidophillus) and it was significantly
different to others at the 95% confidence level.
Therefore, the Lactobacillus strain isolated from Antibio
powder (notated as A) was used in further experiments.
3.4. Dilution rates of dragon fruit juice and sucrose rates
The pH and Brix values in different dilution rates of dragon
fruit juice and sucrose rates before and after fermentation were

presented in Table 5.

10


Table 6. Effects of dilution rates of dragon fruit juice and
sucrose rates
pH
Brix concentration
Sugar
content
Before
After
Before
After
(%)
fermention fermention fermention fermention
6
3.73
12.4
12.0
9
3.72
19.1
18.5
Juice
4.35
12
3.80
17.4

16.8
15
3.71
17.5
17.0
6
3.72
11.6
11.0
Juice +
9
3.77
14.9
14.0
20% pure
4.42
12
3.78
17.8
17.0
water
15
3.75
19.4
19.0
6
3.69
11.5
11.0
Juice +

9
4.00
14.1
13.5
40% pure
4.35
12
3.73
15.6
15.0
water
15
3.87
19.7
19.1
6
3.81
10.8
10.1
Juice +
9
3.74
13.6
12.8
60% pure
4.36
12
3.74
16.4
16.0

water
15
3.94
17.6
17.0
Note: Values in the table were average values of triplications.
Types of
juice

After

48

hours

of

incubation

with

Lactobacillus

acidophilus strain isolated from Antibio powder, pH values were
reduced (from 4.35 – 4.42 to 3.69 – 4.0) due to bacteria growth
converting sugar to lactic acid. At the same time, solute
concentration (Brix) also decreased insignificantly.
The relationship between sugar concentration and acid
index was indicated in Figure 5.

11


density of acid (%w/v)

2.5
2

sugar 6%
1.5

sugar 9%
1

sugar 12%

0.5

sugar 15%

0

juice

juice+20%
water

juice+40%
water

juice+ 60 %
water

Figure 5: The change in concentration of acid in different
dilution rates of dragon fruit juice and sucrose rates
At the same dilution rate, the acid index dropped down
when sugar concentration grew up. Acid concentration reached
the highest value at 6 % of sugar concentration, and the lowest
value at 15% of sugar concentration due to the inhibition of high
concentration of sugar. Besides, at the same glucose concentration
level, acid indexes were different. In particular, the pure dragon
fruit juice had the highest acid index. It was suitable medium for
LAB growth.
The change of bacterial density was shown in Figure 6.

12


9.4
9.2

Log CFU/ml

9

sugar 6%

8.8

sugar 9%

8.6

sugar 12%
8.4

sugar 15%
8.2
8

juice

juice+20%
water

juice+40%
water

juice+ 60 %
water

Figure 6. The change in the density of bacteria
In general, the bacterial density decreased when sugar
concentration increased, with 12 – 15% of sugar concentration,
bacterial density was lower than 9%. The reason was that high
sugar concentration inhibited bacteria growth partly (Nguyen Thi
Hien, 2006). Overall, with 40% of dilution rate, bacterial density
was quite high, nearly equivalent to pure juice. However, in pure
dragon fruit juice with 9% of sugar conceatration, the bacterial
density achieved the highest value (9.3 log CFU/mL). Thus pure

dragon fruit juice was suitable environment for fermentation.
The sensory evaluation assessed according to smell, taste
and status criteria were presented in Table 6.
Table 6. Description of product evaluation criteria
Target
Smell

Description
Fragrance, specific characteristics of fruit, acid and
sugar concentrations are not strong

Taste

Sour harmony, not sourer or sweeter

Status

Homogeneous status, not washier or too condensed
13


The results of sensory evaluations of group of 5 people
were presented in Table 7.
Table 7. Sensory evaluation results with different dilution
rates and sugar content
Types of juice
Pure juice

Juice + 20% water

Juice + 40% water

Juice + 60% water

Sugar content (%)
6
9
12
15
6
9
12
15
6
9
12
15
6
9
12
15

Score
8.1
8.6
8.4
8.4
5.1
5.3
4.8

4.2
2.1
2.3
2.2
1.8
1.5
1.7
1.2
1.6

Note: Values in the Table were average values of 5 replications.

The sensory evaluation of pure dragon fruit juice was
higher than others. The lowest cores belonged to the samples with
40% and 60 %of dilution rates (1.2 to 2.3 per 10 points) due to
less of specific fragrance of fruit. The pure dragon fruit juice had
quite high evaluation (more than 8 per 10 points). In particular,
the 9%-concentration-rate sample got the highest score due to
harmonious flavor.
In summary, from the results of the acid index, the
concentration of bacteria and sensory evaluation, it could be
concluded that the pure dragon fruit juice with 9% of sugar
concentration was appropriate for fermentation.
14


3.5.

Inoculum

levels,

incubation

temperature

and

fermentation time
Results of fermentation in different inoculum levels,
incubation temperature and incubation time were shown in Table
9.
Table 9. Effects of inoculum levels and incubation temperature
Inoculum
concentratio
n
(log CFU
/mL)
4

5

6

Temperature
(oC)

pH

Brix

Acid
concentratio
n (% w/v)

25
30
37
25
30
37
25
30
37

3.54
3.53
3.52
3.53
3.52
3.48
3.55
3.36
3.20

23.73
23.69
23.66
23.72
23.69

23.67
23.64
23.61
23.54

0.54d
0.52e
0.55d
0.55d
0.56d
0.57c
0.54d
0.64b
0.74ª

Bacteria
concentratio
n
(log
CFU/mL)
4.21e
4.45e
4.51e
5.16d
5.72c
6.05bc
6.13b
6.36ab
6.71ª

Note: Values in the Table were average values of triplications.

The initial pH and Brix were 4.52 and 23.9, respectively.
After fermentation, pH and Brix tended to decline. Final pH of
fermentation ranged from 3.20 to 3.55. Brix reduction was not
significant and the values at the end of fermentation were in the
range from 23.54 to 23.73ºBrix.
Effects of incubation temperature and inoculum levels on
acid concentration were presented in Figure 7.

15


0.9

concentration of acid (%w/v)

0.8
0.7
0.6

25oC

0.5
0.4

30oC

0.3

37oC

0.2
0.1
0
4

5

6

strain density (log cell/mL)

Figure 7. Effects of incubation temperature and inoculum
levels on acid concentration
At the same inoculum levels, acid concentration at 25ºC
was lower than 30ºC’s and 37ºC’s. The acid concentration
reached the highest value at 37ºC since this was the optimal
temperature for the growth and development of acid lactic
bacteria. When inoculum level was 6 log cells/mL, the acid
concentration was higher than others at 37ºC. At this level of
inoculum, the acid concentration reached the highet value (0.74%
w/v) at 37ºC) and it was significantly different to others, next to
30ºC (0.64% w/v).
Effects of incubation temperature and inoculum levels on
bacterial concentration were shown in figure 8.

16

8

7

6

Log CFU/ml

5

25oC
4

30oC
37oC

3

2

1

0
4

5

6

density of strains (log cell/ml)

Figure 8. Effects of incubation temperature and inoculum
levels on bacteria concentration
As the results, at the same inoculum level, bacterial
concentration increased with the increase of

incubation

temperature. The lowest and highest bacterial density were at
25ºC and 37ºC, respectively. Suitable temperature for growth of
lactic acid bacteria was 37ºC. Similarly, bacterial concentration
accompanied with inoculum levels to increase. With the 6 log
CFU/mL of inoculum level, the bacterial concentrations reached
the highest values at 30 and 37ºC (6.71 and 6.36 log CFU/mL,
respectively) and the values were significantly different to others.
The

requirement

of

probiotic

products

was

the

concentration of bacteria must be more than 6 log cells/mL. In

this study, conditions satisfied the requirement of probiotic
products were 5 log cells/mL inoculum concentration + 37ºC of
incubation temperature or 6 log cells/mL + all 3 types of
incubation temperature (25, 30, and 37ºC).
17


The samples satisfied the requirement of probiotic products
were sensory evaluated. The results were presented in Table 10.
Table 10. Sensory evaluation results with different inoculum
levels and incubation temperature
Density of
strains
(log cell/mL)

Fermented temperature
(oC)

Marks

5

37

7.4

25

7.4

30

8.3

37

8.9

6

Note: Values in the Table were average values of 5 replications.

Results sensory evaluation showed that sample incubated at
37ºC with inoculum level of 5 log cells/mL and sample incubated
at 25ºC with inoculum level of 6 log cells/mL had the lowest
score. At 6 log cells/mL inoculum level, 30 and 37ºC of
incubation temperatures, the sensory quality of the products was
higher (8.3 to 8.9 / 10 points). Thus, conditions applied to dragon
fruit juice fermentation by LAB were 6 log cells/mL of inoculum
level and 37ºC of incubation temperature.
3.6. Storage temperature and storage time
Effects of storage temperature and time were presented in
Table 10.

18


Table 10. Effects of storage temperature and storage time
Acid concentration
(%w/v)

pH
Time
(weeks)

4
–
6ºC

0

20
–
25ºC

28
–
32ºC

4
–
6ºC

3.51

20
–
25ºC

28

–
32ºC

Bacteria
concentration
(log CFU/mL)
4
20
28
–
–
–
6ºC 25ºC 32ºC

0.634

6.87

1

3.53c

2.62f

2.67d

0.631e

1.54d

1.62b

6.86e

7.66b

7.92ª

2

3.58b

2.61g

2.63ef

0.622e

1.59c

1.62b

6.89d

5.87f

5.71g

3

3.66a

2.60g

2.64e

0.622e

1.59c

1.67ª

7.02c

5.42h

5.32i

(Note: Values in the Table were average values of triplications)

With the samples stored at cool temperature (20 – 25ºC)
and room temperature (28 – 32ºC), the bacterial concentrations
had increasing trend after 1 week (from 6.87 log cells/mL to 7.66
– 7.92 log cells/mL) an the acid concentrations increased (from
0.634% w/v to 1.54 – 1.67% w/v). It made products become
unpleasant and sour. But from the week 2 onwards, the
concentrations of bacteria began to reduce due to the inhibition of
acid against bacteria (less than 6.0 log cells/mL). Thus, these
temperatures were not suitable for storing products.
The samples stored at 4 – 6ºC condition had stable

bacterial density (6.86 – 7.02 cells/mL) and acid concentration
(0.622 – 0.631% w/v). Low temperatures inhibited microbial
activity and the products were kept the better features. The
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bacterial density had a trend to increase from week 1 to week 3 of
storage (from 6.87 to 7.02 log CFU/mL).
In summary, with products stored at 4 – 6ºC temperature,
the bacterial densities and acid concentrations did not change
largely comparing to the time of the end of fermentation. Thus,
the proper temperature for storage was 4 – 6ºC and the products
were maintained quality after 3 weeks of storage in this study.

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