Immobilizing lactobacillus plantarum bacteria on sodium alginate and its application to tomato juice fermentation for making probiotic products

Paper Number THE UNIVERSITY OF DANANG, JOURNAL OF SCIENCE AND TECHNOLOGY, NO 6(79) 2014, VOL 1 39 IMMOBILIZING LACTOBACILLUS PLANTARUM BACTERIA ON SODIUM ALGINATE AND ITS APPLICATION TO TOMATO JUICE F[.]

THE UNIVERSITY OF DANANG, JOURNAL OF SCIENCE AND TECHNOLOGY, NO 6(79).2014, VOL 1 39

IMMOBILIZING LACTOBACILLUS PLANTARUM BACTERIA ON SODIUM

ALGINATE AND ITS APPLICATION TO TOMATO JUICE FERMENTATION

FOR MAKING PROBIOTIC PRODUCTS

NGHIÊN CỨU CỐ ĐỊNH VI KHUẨN LACTOBACILLUS PLANTARUM TRÊN NATRI

ALGINATE VÀ ỨNG DỤNG LÊN MEN TẠO NƯỚC CÀ CHUA BỔ SUNG PROBIOTIC

Ngo Thi Minh Phuong, Nguyen Thanh Hoi

The University of Danang, College of Technology; Email: nthoi@ud.edu.vn

Abstract - Immobilizing bacteria on sodium alginate brings about

some incomparable advantages such as enhancing cell viability,

strengthening spore stability, protesting against a number of

physicochemical conditions, assuring simple implementation

procedures and low-cost preservation Lactobacillus plantarum,

considered to be a probiotic, brings out many benefits This study

aims at examining the survivability of Lactobacillus plantarum on

sodium alginate and its possible applications in fermenting tomato

juice to create probiotic products The research results showed that

the capsules prepared with 3% (w/v) sodium alginate and 1,5%

(w/v) CaCl 2 were most appropriate for cell immobilization, and the

density of Lactobacillus plantarum was 1,1.107 cells/ml after it had

been stored at room temperature for three months The bacterial

motility revealed no significant change before and after the

immobilization process Also, the sodium alginate capsules which

had been reused three times still maintained high lactic acid

production ability, creating new products that benefit our health

Tóm tắt - Cố định vi khuẩn trên chất mang natri alginate có nhiều

ưu điểm vượt trội như: khả năng sống sót của tế bào cao, tăng cường sự ổn định của tế bào và chống lại một số điều kiện hóa lí, phương pháp đơn giản, dễ thực hiện, giá thành bảo quản thấp Vi khuẩn Lactobacillus plantarum có nhiều lợi ích và được xem như một probiotic Nghiên cứu này nhằm mục đích khảo sát khả năng sống sót của vi khuẩn Lactobacillus plantarum khi cố định trên natri alginate và ứng dụng lên men nước cà chua tạo sản phẩm probiotic Kết quả nghiên cứu cho thấy: Hạt gel được thử nghiệm với natri alginate 3% và CaCl2 1,5% tốt nhất cho việc cố định tế bào, mật độ của Lactobacillus plantarum sau ba tháng bảo quản ở nhiệt độ thường là 1,1.107 tế bào/ml, hoạt lực của vi khuẩn so với trước khi cố định thay đổi không đáng kể, các hạt natri alginate qua

3 lần tái sử dụng vẫn sinh axit lactic tốt, đồng thời đã tạo ra một sản phẩm mới tốt cho sức khỏe người tiêu dùng

Key words - lactobaccillus plantarum; immobilized; sodium

alginate; tomato juice; probiotic

Từ khóa - lactobaccillus plantarum; cố định; natri alginate; nước

cà chua; probiotic

1 Introduction

Preservation of microorganism strains is particularly

important It serves as the basis for research and

applications related to many fields such as: biology,

medicine, agriculture, food technology, etc There are

many methods of preservation microorganisms such as

freezing, freeze-drying and so on

Microorganisms preserved by freeze-drying can be

retained for 10-20 years However, this method requires

high cost and when strains are taken out to use, the rest is

highly likely to be infected Therefore, we have always

found new, innovative methods for optimum preservation

Among the various techniques of immobilization,

immobilizing on alginate gel has outstanding advantageous

features It is simple, and generally nontoxic, and allows

easy control of particle characteristics such as permeability

or thickness of the gel membrane Besides the fact that it is

a light and inexpensive method, the capsules can be re-used

many times in the production process

We use strains of L plantarum for our research because

they have proven their ability to survive gastric transit and

colonize the intestinal tract of humans and other animals

[6] Various special therapeutic or prophylactic properties

have been associated with L plantarum, such as reducing

diarrhea infection, stomach pain and constipation

associated with irritable bowel syndrome, flatulence and

positively affecting the immune system of children

infected with HIV, they are considered a probiotic

Because of the above reasons we chose the theme:

"Immobilizing Lactobacillus plantarum bacteria on

sodium alginate and its application in tomato juice fermentation making probiotic products”

2 Materials and methods

2.1 Materials

➢ Lactobacillus plantarum is activated on the strains

taken from the laboratory of Food Technology – Da Nang College of Technology

➢ Tomato

2.2 Methods

2.2.1 Microbiological method [3]

The number of cells was identified by counting the number of colonies growing on agar The number of bacterial cells in 1 ml of the sample is calculated as follows:

C

N CFU gorCFU ml

=

+ +

− N: Number of cells (colony forming units) of bacteria

in 1g or 1ml of sample

− C: Total number of colonies counted on one selected petri dish

− ni: Number of petri dish implanted at ith dilution rate

− di: Corresponding dilution factor

− V (ml): Volume of sample solution implanted in each petri dish

2.2.2 Biochemical method [3]

The produced lactic acid was quantified by titration

40 Ngo Thi Minh Phuong, Nguyen Thanh Hoi with solution of NaOH of 0.1 N

The equation to calculate the total acid content is:

𝑣 𝑘 𝑛 1000 𝑉 v: Volume of NaOH consumed for sample preparation

k: Coefficient of acid (for lactic, k = 0.009)

1000: Calculation coefficient (by g/l)

n: Dilution factor

V: Volume of sample

2.2.3 Immobilizing Lactobacillus plantarum bacteria

- Observations were made with Sodium alginate is of

different concentrations, i.e 1%; 1.5%; 2%; 2.5%; 3%;

3.5%; 4% and CaCl2 0.5%; 1 %; 1.5%; 2%; 2.5%; 3%;

3.5%

- The capsules were then created and Lactobacillus

plantarum was immobilized on the capsules gel according

to the diagram in Figure 1

Figure 1 Diagram of the package granulating

2.2.4 Examining the viability of Lactobacillus plantarum

on sodium alginate gel capsules during storage time

The steps were carried out in the following order:

- Survey the density of Lactobacillus plantarum cells

during storage time

- Soak five sodium alginate capsules which had just

been immobilized with bacteria in tubes containing 9 ml of

distilled water for 40 minutes

- Dilute the samples decimally and spread them on petri

dishes to determine the initial cell by counting the number

of the formed colonies

This procedure was periodically repeated after 7 days,

14 days, 30 days, and 1 month

2.2.5 Physical method

The number of apparent cells of Lactobacillus

plantarum was determined by measuring the optical

density OD600 on a UV - Vis spectrophotometer

2.2.6 The experiment procedure for the production of

tomato juice fermentation making probiotic products

Figure 2 Process of tomato juice fermentation

3 Results and discussions

3.1 The activation of Lactobacillus plantarum strains

After the activation process, we obtained separated, circularly-shaped, pure, milky, shiny, and smooth colonies that were stuck on the surface of the activation environment (Figure 3)

Observing the cells by microscope, we found that they are rod-shaped and sequencing They were concluded to be

L.plantarum bacteria

After being activated, the L.plantarum bacteria were

preserved as in Figure 4

preservation in tubes

3.2 Lactic acid produced by Lactobacillus plantarum

A solution with bacteria density of 109 CFU.ml-1 was put into 100ml flask of milk to be fermented The lactic acid productivity was measured as described in Section 2.2.2 From the experimental results we can draw the following conclusions:

Figure 5 Acidity in every 12 hours

4.7

7.5

0 2 4 6 8 10

g/l

time, h

Sodium alginate

CaCl2

Cacilum alginate capsules with immobilized bacteria

Classification Washing Tomato juice Sterilization (1210C, 20 min)

Mixing Fermentation L.p bacteria

Filtration Fermented tomato juice

Sugar Water v

Tomato

THE UNIVERSITY OF DANANG, JOURNAL OF SCIENCE AND TECHNOLOGY, NO 6(79).2014, VOL 1 41

On the 1st, 2nd, 3th days, acid production increased

rapidly; especially, after 48 hours, the content of acid was

7.5 g l-1 However, after the 4th day, it decelerated This

happened as the amount of bacteria had been increasing

while the amount of substrate remained unchanged When

the substrate content gradually decreased, the amount of

acid was also reduces It is suggested that the content of

lactic acid produced during fermentation is relatively larger

than it is mentioned in the literature [5]

capsules production

From the results in Table 1, the optimal concentration

of sodium alginate is 3% This concentration was selected based on some literature references, especially [1], in which it is advised the harder capsules are, the lower the ability of diffusion of substrate into the capsules via microbial cell wall is The formed capsules, however, are strong, and also, the ability to protect immobilized cells from external effects is good, and vice versa The capsules formed by Sodium alginate 3% are spherical and slightly solid, so they have better capacity for cell protection and substrate diffusion during the fermentation process in comparison with hard gel capsules

Table 1 Examined concentrations of Sodium alginate and CaCl 2

The ability to

create particles

Create particles less

Create a soft particle and

shrinkage

Soft, firm Create

good particles

Create good particles

broken

Spherical particles, shrinkage

Spherical Spherical Spherical Undefined

shape

Undefined shape

Structure of

capsule

Particles are broken

Soft, firm Soft, firm Soft, firm Soft, rather

solid

Concentration of CaCl 2 (%)

The ability to

create particles

Particles are broken

Create rather good particles

Creating good particles

Creating good particles

Create good particles

Create good particles

Create good particles

Structure of

capsule

Particles are broken

Create soft gel

Soft, firm Soft, firm Soft, firm Solid Solid

3.4 Lactobacillus plantarum encapsulation procedure

The solutions of Sodium alginate 3%, and of CaCl2

1,5% were prepared At the same time, we prepared a

solution of Lactobacillus plantarum cells at the density of

109 cells ml-1 (the density is determined by measuring the

optical density and calculating the apparent number of cells

afterwards) The apparent number of cells ml-1 can be

calculated through the optical density (OD) using the

following equation [7]:

− X: Apparent number of cells ml-1

− OD: Optical density values measured at a wavelength

of 600nm

Next, the bacteria solution was mixed with CaCl2

Then, capsules were created by the drip method The

formed capsules were removed to drain then put in an oven

for drying at a temperature of 380C until constant mass is

reached, and finally stored According to [8], the drying

temperature of 380C was chosen because the drying time is

ensured without inactivating bacteria From the obtained

results in Figure 6, it shows that L.plantarum bacteria are

very convenient for later storage and use after being immobilized on sodium alginate capsules

Figure 6 Capsules with immobilized bacteria

3.5 The viability of Lactobacillus plantarum on sodium alginate capsules during storage time

The viability of lactic bacteria on Sodium alginate capsules was examined by sowing the samples in a proper nutrition environment and observing the growth and development of colonies on a petri dish

42 Ngo Thi Minh Phuong, Nguyen Thanh Hoi

Figure 7 Survivability of immobilized bacteria

during storage

The results in Figure 7 show that the number of viable

Lactobacillus plantarum bacteria is large After 3 months

being stored at room temperature (300C), their survival

rates remained high (1,1.107 cells ml-1)

It can be said this method of microorganism

preservation allows high productivity, easy

implementation and and applications In this method, the

survival rate is high because the combination between

sodium alginate and Ca2+ helps forms an area of alginate

and create gels with “egg-box” model

The formation of gel particles requires linking

mechanism between two or more alginate chains The

chain of alginate molecules has folds and clefts so that Ca2+

can get into, locate and make connections Ca2+ links the

alginate molecules into the alginate chains, making the

chains remain stable Lactobacillus plantarum blended

with CaCl2 solution also gets into the clefts and stays

immobilized there Accordingly, the gel particles can hold

numerous bacterial cells The outer protection layer

protects the cells from external conditions, so the bacteria

can be preserved in a long period without being infected or

inactivated

3.6 Lactic fermentation ability of the gel capsules during

the preservation process

As described in section 2.2.2, five immobilized gel

capsules preserved for one week were put into 100 ml flask

of fresh milk to study their lactic fermentation ability The

productivity of the bacteria was checked in every 12 hours,

which is presented in Figure 8

Figure 8 Productivity of bacteria

From the graph, we can infer that:

- Compared to the sample before storage (Figure 5), the

productivity of Lactobacillus plantarum decreased

slightly

- After 24 hours of fermentation, the immobilized cells, which were preserved for 3 months produced less acid (2.7

g l-1) than free cells (4.32 g l-1) However, after 96 hours the difference is not significant (8.3 g l-1 vs 8.8 g l

-1,respectively) It means that the preservation process did affect the motility of bacteria, or in other words, the bacteria were inhibited by storage conditions In the first

24 hours, the bacteria needed some time to re-activate The duration was not enough for the bacteria to adapt to the fermentation solution

- After 96 hours, the bacteria stored for 3 months became active and adapted to the medium, so the fermentation abilities of all the samples were the same This suggests that under room-temperature storage condition, the preservation

process does not considerably affect Lactobacillus plantarum As a result, we are able to conclude that with this

preservation method allows the productivity of immobilized bacteria to remain relatively stable after 3 months of storage

It can be explained as follows: after the bacterial cells had been immobilized in the gel capsules, the gel matrix surrounded the cells Such structure created pores, enabling substrates and fermentation products to be easily diffused out of the gel capsules Thus, we can well control the reaction and separate the products from the reaction vessel Moreover, because of the gel layer surrounding bacterial cell, the productivity of the immobilized bacteria is stable during storage

3.7 Producing fermented tomato juice making probiotic products

3.7.1 The content of produced lactic acid during reuse L.plantarum strains preserved for over 3 months were

used to ferment tomato juice Afterthat, lactic acid formed

in the product was quantified and qualified (Figure 9)

Figure 9 Content of acids (g/l)

As can be seen in Figure 9, the content of lactic acid tends to increase over time This is because right at the end

of the first fermentation, we implemented the second and then the third fermentation In fact, this strain had been activated in the previous fermentation, so it did not require activation for the next one Therefore, the amount of acid produced later was greater than that of the previous fermentation

0

1

2

3

4

5

6

7

8

9

Time, h

Sau 1 tuần Sau 1 tháng Sau 3 tháng

After 1 week After 1 month After 3 months

CFU/ml

1 week 1 month 2 month 3 month

Tim

e

THE UNIVERSITY OF DANANG, JOURNAL OF SCIENCE AND TECHNOLOGY, NO 6(79).2014, VOL 1 43 Also, reusing strains 3 times did not reduce the acid

content generated

3.7.2 Tomato juice fermentation making probiotic

products

The Lactobacillus plantarum strain stored for 3 months

on sodium alginate capsules was used to ferment tomato

juice, the result of which is shown in Figure 10

Figure 10 Fermented tomato juice

The tomato juice was then examined as is revealed in

Tables 2 and 3

Table 2 Target indexes of products after 72 hours

of fermentation

Density of Lactobacillus plantarum

cells

109 cells/ml

Table 3 Sensory evaluation of formed products

Colour Brick red

Flavor Typical odor of fermented lactic

(Smell of tomatoes no longer exists)

Savour Sweet and sour

4 Conclusion

From what have been discussed, we have successfully

immobilized Lactobacillus plantarum on substances

containing sodium alginate (Sodium alginate 3% and CaCl2 1.5%).The density of Lactobacillus plantarum was

1,1.107 cells/ml when stored at room temperature after three months, proving this method is much more convenient than others

The bacterial productivity has no significant change in comparison with that of non-immobilized bacteria after three months of immobilization

After the immobilization process, Lactobacillus plantarum can be consecutively reused three times but still maintains high acid fermentation ability Also, the tomato juice fermented by Lactobacillus plantarum strains ensures good- quality probiotic products

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(The Board of Editors received the paper on 28/02/2014, its review was completed on 13/05/2014)