fermentation of pineapple 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 PINEAPPLE JUICE
BY LACTIC ACID BACTERIA

SUPERVISOR

STUDENT

Dr. NGO THI PHUONG DUNG

MACH TU UYEN
Student code: 3082566
Session: 34 (2008-2013)

Can Tho, May 2013



APPROVAL

SUPERVISOR

STUDENT

Dr. NGO THI PHUONG DUNG

MACH TU UYEN

Can Tho, May 2013
PRESIDENT OF EXAMINATION COMMITTEE

i



Abstract
Lactic acid bacteria are used popularly for fruit juice
fermentation because they are good source of probiotics for
human. In this study, lactic acid bacteria were isolated from
pineapples and probiotic products. The isolated bacterial strains
and some strains from the previous research were screened for
application on pineapple fermentation process. There were 4
strains of lactic acid bacteria isolated from pineapple juice. All of
them could grow in pH 1.5 medium and reached high density
(over 6 log CFU/mL). Moreover, Lactobacillus acidophilus strain
produced highest concentration of lactic acid, so it was the most
favorable strain for pineapple fermentation. The different diluted
ratios of pineapple juice (0%, 10%, 20% and 30% w/v) and
different sucrose supplements (0%, 3%, 6%, and 9% w/v) were
used for testing fermenting capacity of Lactobacillus acidophilus
strain. The pure pineapple juice with 9% of sucrose supplement
was found to be suitable for fermentation. Based on the results of
sensory evaluation and bacterial density determination, the
favorable conditions for pineapple fermentation were determined

as follow: temperature at 37°C, fermentation time for 36 hours
and initial bacterial level at 5 log cells/mL. The results of storage
testing showed that the suitable temperature for product storage
was 4 – 6°C, bacterial density (8.057 log CFU/mL) of final
product maintained after 3 weeks.
Key words: fermentation, lactic acid bacteria, Lactobacillus
acidophilus, pineapple, probiotic.

iii


Contents
Abstract ........................................................................................ iii
Contents........................................................................................ iv
1. Introduction .............................................................................. 1
Objectives ................................................................................. 3
2. Materials and methods .............................................................. 4
2.1 Material............................................................................... 4
2.2 Methods .............................................................................. 4
2.2.1 Isolation and identification of LAB isolates at genus
level...................................................................................... 4
2.2.2 Study on acidity tolerant ability of LAB isolates........ 5
2.2.3 Study on application in producing of fermented
pineapple juice by lactic acid bacteria ................................. 5
2.2.4 Study on dilution ratio of pineapple juice and sucrose
supplement ratio................................................................... 6
2.2.5 Study on inoculum density, temperature and
fermentation time ................................................................. 7
2.2.6 Study on storage temperature and storage time .......... 7
3. Results and discussion.............................................................. 9

3.1 Results of isolation and identification of LAB isolates at
genus level ................................................................................ 9
3.2 Acidity tolerant ability of lactic acid bacterium isolates .. 12
3.3 Application in producing of fermented pineapple juice by
lactic acid bacteria .................................................................. 14
3.4 Dilution ratio of pineapple juice and sucrose supplements
ratio ......................................................................................... 15
3.5 Study on inoculum density, temperature and fermentation
time ......................................................................................... 18
3.6 Storage temperature and storage time .............................. 21
4. Conclusions and suggestions ................................................... 24
4.1 Conclusions ...................................................................... 24
4.2 Suggestions ....................................................................... 24
References ................................................................................... 25

iv


1. Introduction
Nowadays, scientists as well as nutritional experts always
research to find bio-products which are good for health and
nutritional. These products are called probiotic products.
Probiotics are bacteria that help maintain the natural balance of
organisms. A number of probiotic strains have been introduced in
the market in dietary and pharmaceutical forms. Lactic acid
bacteria (e.g. Lactobacillus) and Bifidobacterium constitute the
main

group

of

probiotics

commercialized

for

human

consumption. The treatment of gastrointestinal infections
continues to be complicated due to the expansion of antibiotic
resistances. Of the benefits of probiotics, those related to their
preventive and therapeutic uses against gastrointestinal infections
have an outstanding position, as reflected in a large number of
patents. The mechanisms of action of probiotics against
gastrointestinal pathogens addressed in diverse patent applications
include:

modification

of

the

environmental

conditions,

competition for nutrients and adhesion sites, production of

antimicrobial metabolites and modulation of the immune and nonimmune defense mechanisms of the host (Sanz et al., 2007).
Currently, the probiotic product is commonly used, which
is type of fermented milk and yogurt. However, many consumers
are intolerant of lactose as well as cholesterol in the milk product.
Thus, the fruit juice is recommended as a good medium to
produce fermented probiotic products (Mattila-Sandholm et al.,
2002).
Lactic acid bacteria (LAB) are part of the macrobiotic on
mucous membranes, such as the intestines, mouth, skin, urinary and

1


genital organs of both humans and animals, and may have a
beneficial influence on these ecosystems. They are used widely in

probiotic products such as yogurt, fermented meat, fermented
vegetables… LAB strains are supported the immunomodulatory
role attributed. These products are not only used as food but also
used for treating intestinal and stomach diseases because lactic
acid bacteria can produce antibiotics to prevent and skill
pathogenic bacteria.
Fruit juices are good media for bacteria’s growth and
probiotic products. Fruits and vegetables are good for health as
they contain antioxidants, vitamins, fibers and minerals. There is
no allergen in their components.
Pineapple is one of the most popular fruits in tropical
countries especially in Vietnam. Pineapple, Ananas comosus,
belongs to the Bromeliaceae family, from which one of its most
important health-promoting compounds, the enzyme bromelain,

was named. The Spanish name for pineapple, pina, and the root of
its English name, reflects the fruit's visual similarity to the
pinecone.There are many vitamins (A, B1, B2, C et al.), enzyme
bromelain and minerals (Fe, Mg, K, Zn, Ca et al.) in pineapple.
So many health benefits of using pineapple such as potential antiinflammatory, digestive benefits, antioxidant protection, immune
support and protection against macular degeneration (Ensminger
et al., 1986; Cho et al., 2004; Maurer, 2001). Besides, pineapple
juice is a suitable medium for lactic acid bacteria to produce new
probiotic products. Therefore, the research “Fermentation of
pineapple juice by lactic acid bacteria” was carried out.

2


Objectives
To isolate lactic acid bacterium isolates from pineapple, and
to study on conditions for fermented pineapple juice by lactic acid
bacteria.
The content of this study include:
- Isolation and identification of LAB isolates at genus level
- Study on acidity tolerant ability of LAB isolates
- Study on application in producing of fermented pineapple
juice by lactic acid bacteria
- Study on dilution ratio of pineapple juice and sucrose
supplement ratio
- Study on inoculum density, temperature and fermentation
time
- Study on storage temperature and storage time

3

2. Materials and methods
2.1 Material
-

Pineapple bought from Hung Loi market, Ninh Kieu

District, Can Tho City.
-

Six lactic acid bacterium isolate was isolated from five

probiotics powder and fermented papaya juice ( Luong Phuoc
Truong, 2012)
-

Sucrose

-

Medium
 MRS broth (De Man, Rogosa and Sharpe-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, di-ammonium 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.

- Chemical: NaOH 0.1N, HCl 0.1N, H2O2 3%, NaHSO3,
the Gram stain (Crystal violet, Iod, Acetone, Ethanol, Fuchsin).

- Equipment in Food Biotechnology laboratory.
2.2 Methods
2.2.1 Isolation and identification of LAB isolates at genus level
-

Pineapple juice solution was contained in a glass, which

was sterilized. Then, incubated for naturally fermented at 37ºC for
24 – 48 hours. Transferred 1 mL of fermentation solution above
into a tube containing MRS broth medium. After 24 hours,
transferred bacteria from MRS broth medium to MRS agar
medium.

4


-

Observation and photograph the bacteria under the light

microscope with objectives 100X.
-

Genus identification by some tests: Gram stain and

catalase, oxidase, dissolution of CaCO3.

2.2.2 Study on acidity tolerant ability of LAB isolates
Inoculated 1 mL of LAB isolates (6 log cells/mL) into 9
mL MRS broth medium with different pH levels (1.5; 2.5 and
3.5). There were two factors (bacterium strains and pH) and
triplication in this experiment. Then, bacterial density of each
treatment was determined at initial inoculating time (at T0) and 2
hours after inoculating (at T2) by plate counting method on MRS
agar.
-

Evaluation criterion: cell density of the bacterial strains.

-

Results: Chose bacterial strains tolerant ability of lowest

-

Using Statgraphics Centurion XV software to analysis

pH.
data.
2.2.3 Study on application in producing of fermented
pineapple juice by lactic acid bacteria
-

Pineapple was pressed and put into the tubes with 50 mL

volume.
-

Pasteurized by 140 mg/L of NaHSO3 in 20 minutes, then

inoculated the strains of LAB into tubes, each tube contained 36
mL of pineapple juice and 4 mL of LAB. Inoculum density of all
samples were 5 log cells/mL.
-

There were one factor and triplication in this experiment.

Incubated at 37ºC for 48 hours and then analyzed the assessment
criterion.

5


 The evaluation criterion
-

pH: Measure by pH meter.

-

Soluble matter content (ºBrix): Measured by Brix

handheld meter.
-

Lactic acid content: Based into total acid index.

-

Density of LAB: Plate counting method.

-

Sensory evaluation.

2.2.4 Study on dilution ratio of pineapple juice and sucrose
supplement ratio
Experiments were conducted randomly with two factors, at
4 levels and triplication. Total number of treatments: 4x4x3 = 48
X factor (%): Percentage of dilution ratio
X1 = 0%

X2 = 10%

X3 = 20%

X4 = 30%

Y factor (%): Percentage of sucrose ratio
Y1 = 0%

Y2 = 3%

Y3 = 6%

Y4 = 9%

 Processing:
Pineapple juice was diluted at four levels (0%, 10% 20%,
30% w/v). Pasteurized by NaHSO3 (140 mg/L) for 20 minutes.
Then, distributed into 48 tubes, each tube contained 40 mL. Each
dilution rate was mixed with four sucrose levels. Then they were
measured pH and ºBrix. Inoculated 1% bacteria with 7 log
cells/mL into each tube (0.4 mL/tube). Then, all of tubes were
incubated at 37ºC for 48 hours.
 The evaluation criterion
-

pH

-

Soluble matter content (ºBrix)

-

Lactic acid content

-

Density of LAB

6


2.2.5

Sensory evaluation
Study

on

inoculum

density,

temperature

and

fermentation time
The experiment was conducted in three factors and
duplication.
Density of LAB (log cells/mL):
X1 = 3

X2 = 5

X3 = 7

Incubating temperature factor (ºC):
Y1 = 30

Y2 = 37

Y3 = 28 – 32 (room temperature)

Fermentation time factor (hour):
Z1 = 24


Z2 = 36

Z3 = 48

Processing:
Selected the most appropriate treatments in experiments in

section 2.2.4, fixed 2 treatments and continued to do this
experiment. Performed the same experiment above but density of
LAB are diluted with saline for 5, 7, 9 log cells/mL. Then
inoculated into the tube 1% of bacteria (initial concentration the
bacteria corresponding 3, 5, 7 log cells/mL). Divided each group
and incubated conducted at different time and different
temperatures.


The evaluation criterion:

-

pH

-

Soluble matter content (ºBrix)

-

Lactic acid content

-

Density of LAB

-

Sensory evaluation

2.2.6 Study on storage temperature and storage time
The experiment included two factors and triplication.

7


Factor 1: Storage temperature: the refrigerator (4 – 6ºC),
cool room (20 – 25ºC) and room temperature (28 – 32ºC).
Factor 2: Storage time: 1, 2 and 3 weeks.
The total number of experimental units: 3 x 3 x 3 = 27.


The evaluation criterion:

- pH
- Soluble matter content (ºBrix)
- Lactic acid content
- Density of LAB

- Sensory evaluation

8


3. Results and discussion
3.1 Results of isolation and identification of LAB isolates at
genus level
From naturally fermented pineapple juice four bacterial
strains were isolated. Besides, this project used six lactic acid
bacteria, taken from thesis of Luong Phuoc Truong (2012). The
aim of isolation was to compare the usability of pineapple juice
by four isolated strains and known strains.
Determination of whether these isolated strains in
experiment 1 depended on lactic acid bacteria was tested by some
basically physical and biochemistry properties.
Gram staining
Results of gram staining gave information that bacterial
species remained purple color after staining crystal violet, thus
they were gram positive bacteria.

Figure 10. Gram-positive bacteria (X100)
Dissolution of CaCO3
Lactic acid from bacteria was dissolved CaCO3 and formed
clear zones around colonies on the medium (MRS agar with 1.5%
CaCO3).
9


Figure 11. Reduction of CaCO3 by lactic acid

Catalase test
The results showed that
the formation of bubbles did
not occur in them, indicated
negative results.

Figure 12. Catalase-negative

Oxidase test
Because color changes
of filter paper strips were
absent in surface of agar plate
(Figure 13). Therefore, these
bacteria

did

cytodrome

not

contain

oxydase

in

Figure 13. Oxidase-negative

metabolism.

Morphology characteristics of bacteria were briefly
described by Table 2.

10


Table 2. Morphology characteristics of bacteria
LAB
Numbers

Sources

Colony

Cell shape

isolates
Antibio
1*

2*

probiotic

White color, circular
A

shape, convex elevation,

power

smooth texture.

Probio

White color, circular

probiotic

P

shape convex elevation,

power

Short rod

Short rod

smooth texture
White color, circular

Ybio probiotic
3*

Y

shape, convex elevation,

Short rod

power
smooth texture
Lactomin plus
4*

5*

probiotic

White color, circular
L

shape, convex elevation,

power

smooth texture

Biosubtyl

White color, irregular

probiotic

Bio

shape, convex elevation,

power

Cocci

Long rod

smooth texture
White color, circular

6*

Papaya juice

DD

shape, convex elevation,

Rod in pair

smooth texture
White color, circular
Pineapple
7

K1

shape, convex elevation,

Short rod

juice

smooth texture
White color, circular
Pineapple
8

K2

shape, convex elevation,

juice
smooth texture

11

Long rod


LAB
Numbers

Sources

Colony

Cell shape

isolates
White color, circular
Pineapple
9

K3

shape, convex elevation,

Short rod

juice
smooth texture
White color, circular
Pineapple
10

K4

shape, convex elevation,

Short rod

juice
smooth texture

Due to the above mentioned characteristics, four isolated
bacteria were lactic acid bacteria with following characteristics
such as: gram-positive bacteria, catalase-negative, reducing lactic
acid to dissolution of CaCO3, cocci or rod shape and growth in
MRS agar.
3.2 Acidity tolerant ability of lactic acid bacterium isolates
The results were shown in Table 3.
Table 3. The evaluation criterion of LAB density

pH

1.5

LAB density at T0

LAB density at T2

(log CFU/mL)

(log CFU/mL)

Strains
ij

6.85efg

A

1.545

P

1.53jk

6.87cde

Y

1.555i

6.82ghi

L

1.52k

6.81 ij

Bio

1.53jk

6.93a

DD

1.515k

6.86def

K1

1.55ij

6.825hi

K2

1.56i

6.81 ij

K3

1.53jk

6.86abcd

K4

1.51k

6.8j

12


pH

2.5

3,5

CV%

LAB density at T0

LAB density at T2

(log CFU/mL)

(log CFU/mL)

A

1.925gh

6.89bc

P

1.96e

6.88bcd

Y

1.91h

6.89bc

L

1.93fgh

6.85efg

Bio

1.91h

6.86def

DD

1.955e

6.8j

K1

1.915h

6.83ghi

K2

1.95ef

6.87cde

K3

1.92gh

6.82hij

K4

1.94efg

6.825hi

A

4.28a

6.9b

P

4.235d

6.95a

Y

4.24cd

6.93a

L

4.25bcd

6.935a

Bio

4.26abc

6.84fgh

DD

4.28a

6.81 ij

K1

4.23d

6.88bcd

K2

4.26abc

6.87cde

K3

4.26abc

6.83ghi

K4

4.27ab

6.81ij

47.038%

0.6403%

Strains

*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.

After incubation at 37ºC for 2 hours with different pH
levels, all strains increased densities.

13


At pH 1.5 and 2.5, the bacterial densities reduced
significant at initial time point (T0) with value (1.51 – 1.96 log
CFU/mL), but they recovered, increased and achieved value (6.8
– 6.93 log CFU/ml) after incubation at 37ºC for 2 hours. Each
bacterial species were specialized to live in basic pH. If they
moved stress pH conditions, metabolic imbalances of bacteria and
environment occurred, that led to the death of bacteria.
At pH 3.5, most of bacteria resistanted and reduced slightly
at initial time point (T0) with value (4.23 – 4.28 log CFU/mL), as
well as, increased and achieved value (6.8 – 6.95 log CFU/mL)

after incubation at 37ºC for 2 hours.
3.3 Application in producing of fermented pineapple juice by
lactic acid bacteria
The result was shown in Table 4.
Table 4. The evaluation criterion after fermentation
of pineapple juice
LAB
strains

ºBrix after
fermentation

pH after
fermentation

Lactic
acid
(% w/v)

A

8a

3.417bc

0.786cd

4.247b

7.207de

P

8a

3.513a

0.768cd

4.223bc

7.28bc

Y

8a

3.39bc

1.203a

4.353a

7.38a

L

8a

3.407bc

0.882b

4.263b

7.163e

Bio

8a

3.383bc

0.882b

4.247b

7.233cd

DD

8a

3.417bc

0.732d

4.247b

7.31b

K1

7,333c

3.35c

0.84bc

4.247b

7.257cd

K2

7,833ab

3.523a

0.81bcd

4.237bc

7.21de

K3

7,667b

3.26d

0.773cd

4.067bc

7.283bc

K4

7,833ab

3.437b

0.81bcd

4.18c

7.247cd

14

LAB density at LAB density at
T0
T48
(log CFU/mL) (log CFU/mL)


CV%

3.310%

2.370%

16.003%

1.274%

0.885%

*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.

Over view, ºBrix and pH after fermentation were reduced
after inoculating for 48 hours because of converting sucrose to
lactic acid by LAB. L. acidophillus from Ybio probiotic powder
had a preponderance of lactic acid content. LAB density at initial
inoculum was close to inoculum density. The Y strain included
highest LAB density (4.353 log CFU/mL) and the difference was
significant with other strain.
After 48 hours incubated, LAB density was increased
above 6 log CFU/mL. That satisfied the requirement of probiotic
product. There for, all of these LAB strains could use to apply in
producing of fermented pineapple juice. Besides, acid lactic
content of L. acidophillus from Ybio probiotic powder was
highest (1.203% w/v) than others. So this bacterium was chose to
apply in producing of fermented pineapple juice.
3.4 Dilution ratio of pineapple juice and sucrose supplements
ratio
The result indicated that L. acidophilus isolated from Ybio

probiotic powder could be applied in fermentation of pineapple
juice.
At the same dilution of juice, the lowest bacterial density
was measured in the treatment of 0% of sugar. The reason is that
nutrient-depleted environment after converting all sugar into
lactic acid inhibited the bacterial growth and development.
Besides, lactic acid itself could inhibit the development of
bacteria (Nguyen Thi Hien, 2006). In contrast, the treatment of
15


9% of sugar content exhibited the highest bacterial density, which
means this was optimal for fermentation of pineapple juice.
Moreover, bacterial density at all dilution ratios was above 6 log
CFU/mL, this will be probably considered for further research of
probiotic production.

Figure 15. Changes of LAB density
by different dilution ratio and sugar content
Choosing suitable dilution ratios and sugar content as well
as appropriate sensory evaluation determine fermented juice
quality. The evaluation was carried out with 5 parameters by 5
undergraduates of Biotechnology (Table 6).
The results showed that fermented juice samples at initial
dilutions were statistically significant different (p<0.05). The
treatment of 0% of sugar was given the lowest score because of
bland taste. The treatments of 0% of initial dilution were more
favorable than others. Particularly, the treatment of 0% of initial

16

dilution and 9% of sugar content had the highest score because of
gentle smell and comfortable sweet-sour taste.
Table 6. The result of sensory evaluation of product
Dilution

Sucrose

ratio (%

ratio (%

w/v)

0

10

20

30

Color

Smell

State of

Average

solution

of score

Taste

CV%

w/v)
0

5

5.0

2.0

4

4.00g

5.0%

3

5

3.8

3.6

4

4.10e

9.756%

6

5

4.8

3.8

4

4.40b

2.272%

9

5

4.6

5.0

4

4.65a

1.075%

0

5

3.2

2.6

4

3.70l

5.405%

3

5

4.2

2.6

4

3.95h

1.265%

6

5

4.0

3.0

4

4.00g

7.5%

9

5

4.6

3.8

4

4.35c

5.747%

0

5

3.8

2.0

4

3.70l

1.351%

3

5

3.0

2.0

4

3.50o

1.428%

6

5

3.8

3.8

4

4.15d

3.614%

9

5

3.8

3.8

4

4.15d

1.204%

0

5

4.8

2.4

4

4.05f

1.234%

3

5

3.8

2.4

4

3.80j

2.631%

6

5

2.6

3.4

4

3.75k

4.0%

9

5

2.8

3.6

4

3.85i

6.493%

*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.

In short, fermented juice samples in treatment of 0% of
initial dilution and 9% of sugar were better than those in other

treatments. Hence, they were chosen for next experiments.
17


3.5 Study on inoculum density, temperature and fermentation
time
Figure 16 indicated that with the same inoculum density and
temperature with the same inoculum density and temperature.
This proved that 36 hours was optimal for the development of
bacteria in pineapple juice.
Figure 16. Changes of lactic acid content

by different inoculum density and fermentation time
In general, after 48-hour incubation, lactic acid content in
sample-7 log cells/mL-37ºC was higher than that in sample-5 log
cells/mL-37ºC after 36-hour incubation, 1.89% w/v and 1.65%
w/v, respectively. In addition, with the same inoculum and
incubation time, lactic acid content at 37ºC was higher than that at
30ºC and 28 – 32ºC, showing that 37ºC was optimal temperature

18


for the growth and development of bacteria. This could lead to
high lactic acid was converted.
At the same temperature and fermentation time, inoculum
density of 7 log cells/mL gave higher lactic acid content than
inoculum of 3 and 5 log cells/mL. This might result from the fact
that the higher the inoculum density, the higher lactic acid
content.

Similarly, bacterial density was highest at sample 36-hour
incubation-7 log cells/mL-37ºC (8.43 log CFU/mL), and sample
48-hour incubation-7 log cells/mL-37ºC (8.38 log CFU/mL).
Besides, bacterial density incubated at 37ºC was higher than that
at other temperatures. Therefore, 37ºC was optimal for the growth
and development of bacteria.

Figure 17. Changes of LAB density by different inoculum
densities, fermentation time and temperatures
Figure 17 showed that bacterial density increased in
proportion with the rise of incubation temperature, except for
19