Analysis of Exopolysaccharides in Lactobacillus casei group
Probiotics from Human Breast Milk
Nur Kusmiyati1, Yuni Puspitasari2, Ulfah Utami3, Anggeria Oktavisa Denta4
1

Agricultural Product Technology
Department, Faculty of Agricultural
Technology, Universitas Brawijaya, Malang,
East Java, Indonesia
2

Biology Study Program, Faculty of Science
and Technology, Universitas Islam Negeri
Maulana Malik Ibrahim, Malang, East Java,
Indonesia
3

Biology Study Program, Faculty of Science
and Technology, Universitas Islam Negeri
Maulana Malik Ibrahim, Malang, East Java,
Indonesia
4

Nursing Study Program, Politeknik Negeri
Madura, Sampang, East Java, Indonesia
Correspondence:
Nur Kusmiati,
Jl. Veteran, Lowokwaru, Malang, East Java,
Indonesia
Zip Code: 65145
Email: kusmiy4tinur@gmail.com
Received: April 16, 2022
Revised: January 6, 2023
Accepted: March 27, 2023
Published: April 29, 2023
DOI: 10.33086/ijmlst.v5i1.2872

Abstract
Exopolysaccharides get a lot of attention because they can
improve the host immune system. Exopolysaccharide is a
polysaccharide that is produced and secreted from microbes
outside the cell, usually found on the outside of the bacterial
structure. The Lactobacillus casei group from human breast
milk is thought to have the ability to produce
exopolysaccharides. The purpose of the study was to
examine the exopolysaccharide of the L. casei group that
was isolated from breast milk. The methods used include the
gravimetric, the phenol-sulfuric acid and the Fourier
Transform Infra-Red (FTIR). The results showed that the L.
casei group could produce exopolysaccharides, and had
high exopolysaccharide total sugar content. Lactobacillus
paracasei had the highest exopolysaccharide and total sugar
content of 3660 mg/L and 80.6%, respectively. The FTIR
results of the L. casei group exopolysaccharides showed the
presence of hydroxyl functional groups O-H (3425.763295.98 cm-1), methyl C-H (2930.86-2856.70 cm-1),
carbonyl C=O (1660.11-1647.27 cm-1), C-H (1456.161373.44 cm-1) and C-O-C ether (1071.08-1056.82 cm-1)
which are specific characters of exopolysaccharides. Since
the FTIR profile demonstrates that the L. casei group can
produce exopolysaccharides, it has greater potential as a a
probiotic.
Keywords
Exopolysaccharide, Functional
casei.

Ggroup,

Lactobacillus

Citation: Kusmiyati N, Puspitasari Y, Utami U, Denta AO. Analysis of Exopolysaccharides in Lactobacillus casei group Probiotics from
Human Breast Milk. Indones J Med Lab Sci Technol. 2023;5(1):29–41. DOI: 10.33086/ijmlst.v5i1.2872
This is an open access article distributed under the Creative Commons Attribution-ShareAlike 4.0 International License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
©2023 by author.

INTRODUCTION

decrease in the body's immune system (1).

Changes in lifestyle and poor diet in the

One of the prevention efforts against this is

community can disrupt the balance of the

by restoring the balance of the immune

digestive tract microbiota, resulting in a

system, like improving the health of the
29

Nur Kusmiyati, et al.

digestive tract. According to Ahamad et al.

probiotic. L. casei group consisting of

(2), the digestive tract is a reflection of the

Lactobacillus casei, Lactobacillus paracasei,

immune system because almost 80% of the

Lactobacillus rhamnosus can be used as

components of the immune system are found

candidates to produce EPS.

in the digestive tract. Improving the health of

EPS analysis can be done through EPS

the digestive tract can be done by consuming

production and total sugar analysis (5). In

probiotics.

addition to these two analyzes, to ensure the

Lactobacillus is one of the largest genera

compound is an EPS is also needed. One

of lactic acid bacteria (LAB) and is often used

method

as

to

characterization of the functional group of a

Lactobacillus probiotics is limited to the

compound using Fourier Transform Infra-

requirements that must be met as a probiotic,

Red (FTIR) spectrophotometry (6). EPS

namely acid and bile salt resistance, can

functional group analysis using FTIR can

adhere to intestinal cells and survive in the

provide complete information about the

intestinal tract, able to produce antimicrobial

functional groups contained in the structure

compounds, antagonists against pathogens,

of an EPS compound.

a

probiotic.

Research

related

safe in food and clinically, proven to affect

that

The

can

be

exploration

of

the

probiotic

microorganisms

advantage of the Lactobacillus genus is that it

increasing because the ability of these

has been declared a safe microorganism when

microorganisms

added to food because it does not produce

considered very important for health. This

toxins, otherwise known as Generally

study

Recognized

(GRAS)

exopolysaccharide profile of the L. casei

microorganisms (3). According to Teame et

group derived from human breast milk based

al. (4), several microorganisms are probiotic

on parameters of crude extract content, total

which can produce exopolysaccharides.

sugar,

As

Safe

Exopolysaccharides (EPS) are sugar
polymers or polysaccharides secreted by

and

to

produce

is

health, and comes from humans (1). The

aimed

that

used

EPS

synthesize

to

EPS

analyze

identification

is

is

the

of

exopolysaccharide functional groups using
FTIR.

microorganisms out of cells. EPS has a health

30

effect because it can improve the immune

MATERIALS AND METHODS

system, so it is essential to analyze probiotics

Materials used in this study are L. casei

in producing EPS. According to Ahamad et

group (L. casei, L. paracasei, L. rhamnosus)

al. (2), the Lactobacillus casei group derived

isolated from human breast milk (7), deMan

from human breast milk is eligible as a

Rogosa

and

Sharpe

Broth

(MRSB)

Ina. J. Med. Lab. Sci. Tech. 2023; 5(1): 29–41

Nur Kusmiyati, et al.

(Himedia), deMan Rogosa and Sharpe Agar
(MRSA)

(Merck),

sucrose,

mg
EPS dry weight (mg)
)=
L
media volume (L)

EPS content (

ethanol,

trichloroacetic acid 10% (TCA), 70% alcohol
(OneMed), spirits, distilled water, phenol

Analysis of total sugar content
The total sugar content of EPS was

5%, concentrated H2SO4.

determined by the phenol sulfuric acid

Bacterial Cultivation
This study used the L. casei group

method using glucose as the standard curve.

isolated from human breast milk in the study

The standard curve was made by means of 1

of Kusmiyati et al. (7). The cultivation of the

mL of a standard glucose solution containing

L. casei group was carried out through

10, 20, 30, 40, 50 and 60 ppm. Glucose was

culture. One ose of pure culture was taken

put into a test tube and 1 mL of 5% phenol

and grown on 5 mL of MRSA-tilted media at

solution and 5 mL of concentrated sulfuric

37 ºC for 24 hours. The rejuvenated species

acid were added. The solution was heated in

were used for inoculum stock preparation and

a water bath at 40 ºC for 15 minutes and the

exopolysaccharide

absorbance was measured at 490 nm (9).

(EPS)

production

analysis (8).
Exopolysaccharide production analysis

Exopolysaccharide: as much as 2 mg of
crude EPS was dissolved in 50 mL of distilled

One ose of the isolate was inoculated into

water. A total of 1 mL of EPS solution was

20 mL of MRSB, which 5% sucrose had been

quickly added to 1 mL of 5% phenol and 5

added and incubated at 37 ºC for 24 hours.

mL of concentrated sulfuric acid and left for

Inoculum was added to 20 mL of 10% TCA

10 minutes. Next, the solution was heated in

and homogenized with a 90 rpm shaker

a water bath at 40 ºC for 15 minutes. The

incubator for 30 minutes. Then it was

absorbance was measured at 490 nm (9).

centrifuged for 30 minutes at 4 ºC at 4500

Identification

rpm. The supernatant was taken and added to

compound profile with Fourier Transform

96% cold ethanol twice. The sample was

Infra-Red (FTIR)

allowed to stand at 4 ºC for 24 hours, then

The

Fourier

of

exopolysaccharide

Transform

Infra-Red

centrifuged at 4 ºC at 4500 rpm for 30

provides complete information about the

minutes. The pellets were dried in an oven at

functional groups contained in the structure

60 ºC and the EPS dry weight was weighed.

of a compound. Thus, in this analysis, it can

EPS levels were calculated based on the

be ascertained that the isolate group can

method of Nurhasanah et al. (5) and are

produce exopolysaccharides (10). 2 mg of

presented in Equation 1.

dry exopolysaccharide was put into an agate

bromide (KBr). After that, the material is
Ina. J. Med. Lab. Sci. Tech. 2023; 5(1): 29–41

31

mortar and mixed with 200 mg of potassium

Nur Kusmiyati, et al.

ground until homogeneous. Next, the mixture

data is normal and homogeneous, then it is

is pressed and formed into pellets. The pellet

analyzed using the Analysis of Variance

is placed in the cell holder of the FTIR

(ANOVA). If the variance in the data gives a

instrument. Dry exopolysaccharides and KBr

significant difference, then the Duncan

were suppressed with a hydraulic suppressor

Multiple Range Test (DMRT) is carried out

with an FTIR spectrum at a wave number of

with a level of = 5%.

500-4500 cm-1 (11).
Data analysis
Data

RESULTS

analysis

descriptively,

was

carried

Exopolysaccharide production analysis

and

Exopolysaccharide production analysis

qualitatively. Quantitative data analysis used

showed that the L. casei group could produce

the

exopolysaccharides

product

quantitatively

out

and

total

sugar

of

(Figure

1).

The

exopolysaccharides, while qualitative data

calculation results show that L. paracasei can

analysis used the results of the FTIR profile

produce the highest

of exopolysaccharide compounds. Product

which is 3660 mg/L. Based on statistical

data and total sugar exopolysaccharide were

analysis, the results were not different with L.

tested for normality using Shapiro-Wilk and

casei and different

homogeneity tested using Levene Test. If the

rhamnosus.

exopolysaccharide,

(P<0.05) with L.

32

Figure 1. Exopolysaccharide content of the L. casei group

Ina. J. Med. Lab. Sci. Tech. 2023; 5(1): 29–41

Nur Kusmiyati, et al.

Exopolysaccharide total sugar analysis
The

total

sugar

content

of

Identification

of

the

functional

exopolysaccharide group of Lactobacillus

Transform

casei showed different results (Figure 2). The

spectrophotometer

calculation results showed that the total sugar

The

exopolysaccharide

groups

using

a

Fourier

Infra-Red

analysis

of

(FTIR)

exopolysaccharide

content of the highest exopolysaccharide in

spectra produced by the L. casei group

L. paracasei was 80.6%. This value was not

showed different wave numbers (Figure 3).

significantly different in L. casei by 72.6%,

All species have functional groups indicating

and

L.

the presence of an exopolysaccharide in

rhamnosus. The research of Xu et al. (12)

general, namely O-H (hydroxyl), C=O

showed that the total sugar content of L. casei

(carbonyl), C-H (methyl), and C-O-C (ether).

significantly

different

from

exopolysaccharide isolated from sauerkraut,
northeastern China, by the phenol-sulfuric
acid method was 88%.

Figure 2. Total sugar content of the L. casei group of exopolysaccharides

The results of the exopolysaccharide

stretching

molecular

vibration.

The

spectra produced in the L. casei group of

absorption band in the 1660.11 cm-1 region

bacteria, namely the spectra at a wave

represents the stretching C=O vibration of

number of 3295.98 cm-1, indicated the

the carboxyl group. Spectra at wave numbers

presence of a hydroxyl group (O-H) with the

1453.31 and 1373.44 cm-1 indicate the

type of stretching molecular vibration. The

presence of a group (C-H) with a bending

absorption band in the wave number region

molecular

of 2926.59 and 2856.70 cm-1 represents the

absorption band at the wave number of

methyl group (C-H) with the type of

1071.08

cm-1

type.

indicates

the

The

sharp

stretching

33

Ina. J. Med. Lab. Sci. Tech. 2023; 5(1): 29–41

vibration

Nur Kusmiyati, et al.

vibration of C-O-C of the ether functional

casei group regarding the similarity of the

group. The results of the FTIR spectra

wave numbers obtained for each functional

showed the presence of exopolysaccharides

group is presented in Table 1. The L. casei

in the L. casei group.

group has eleven to twelve functional groups.

An analysis of the results of the FTIR
spectra of exopolysaccharides from the L.

L. casei had more functional groups than L.
paracasei and L. rhamnosus.

Figure 3. FTIR spectra of L. casei group exopolysaccharide;
(a) L. casei, (b) L. paracasei, (c) L. rhamnosus.
Table 1. FTIR spectra of exopolysaccharides from the L. casei group

34

Functional
groups
(O-H)
Hydroxyl
(C-H)
Methyl
(C=O)
Carbonyl
(C-H)
(C-O-C)
Ether

Vibration
Stretching
Stretching
Stretching
Bending
Stretching

L. casei
3295.9

Wave number (cm-1)
L. paracasei
L. rhamnosus
3425.7
3412.9

Wave number
(cm-1) (13)
3650-3200

2926.5 and
2856.7
1660.1

2929.4

2930.8

3000-2850

1650.1

1647.2

1850-1630

1453.3 and
1373.4
1071,08

1454.7 and
1414.8
1058.2

1456.1 and
1414.8
1056.8

1465-1370
1090-1000

Ina. J. Med. Lab. Sci. Tech. 2023; 5(1): 29–41

Nur Kusmiyati, et al.

Exopolysaccharide FTIR results in L.

Anindita (16) reproted that four strains of

casei have similarities to previous studies.

Weisella confusa derived from breast milk

Differences in previous studies often occur in

produced

shifting wavenumbers but still show the

namely W. confusa AS3 (1883 mg/L), W.

similarity of functional groups. The L. casei

confusa AS14 (1570 mg/L), W. confusa

group has all the functional groups required

AS18 (1369 mg/L) and W. confusa AS21

for a polysaccharide compound. According

(1480 mg/L). These results indicate that the

to Zhang et al. (14) functional groups that

ability to produce exopolysaccharides is

indicate

an

influenced by differences in strains. The

exopolysaccharide are O-H (hydroxyl), C=O

same species have varying capabilities in the

(carbonyl), C-H (methyl), and C-O-C (ether)

production of exopolysaccharides. Fatih (17)

groups. This is following the research of

added that the differences in the production

Chambi et al. (15), who stated that FTIR

of exopolysaccharides were caused by the

analysis of exopolysaccharides had several

different enzyme activities of each species.

the

presence

carbohydrate-related

peaks,

of

different

exopolysaccharides,

including

The exopolysaccharide content of L.

hydroxyl, methyl, carbonyl, and ether groups.

casei isolated from palm sap was lower
(106.33 mg/L) (10) compared to the

DISCUSSION

fermented colostrum kefir (1340 mg/L). In

Exopolysaccharide production analysis

another

study,

L.

paracasei

Genetics and individual phenotypes

exopolysaccharide levels were lower after 48

influence differences in the production of

hours of fermentation (376.4 mg/L) (18)

exopolysaccharides in LAB (16). Genetic

compared to fermented yoghurt (932.0 mg/L)

traits are inherited traits of each bacterial

(19). This study showed that L. paracasei

species

gene

produced an exopolysaccharide of 3660

composition, while phenotypic traits tend to

mg/L, which is a fairly high value compared

be influenced by environmental factors. In

to existing studies. The production of

addition, the origin and species differences of

exopolysaccharides in the L. casei group

LAB strains will be able to contribute to the

resulted in a high value because the MRSB

richness and diversity of glucosyltransferase

medium was added with 5% sucrose.

(gtf) genes, which will affect the diversity of

According to Malick et al. (20), bacterial

enzymes in exopolysaccharide synthesis so

culture

that

composition,

that

are

variations

influenced

in

by

exopolysaccharide

Ina. J. Med. Lab. Sci. Tech. 2023; 5(1): 29–41

pH,

such

as

medium

temperature,

and

incubation period have been shown to

35

production occur.

conditions

Nur Kusmiyati, et al.

significantly

affect

exopolysaccharide

production.

the late logarithmic or stationary growth

The exopolysaccharide content of L.

phase, while others are synthesized during

rhamnosus with the addition of hydrogen

the microbial growth process. The quantity of

peroxide (H2O2) supplementation after 24

exopolysaccharide produced varies between

hours of incubation was 567 mg/L (21). The

strains, media composition, and culture

same

conditions such as pH, temperature and

study

showed

an

increase

in

exopolysaccharides when given the addition

carbon ratio.

of calcium chloride (CaCl2) and H2O2 after

According to Al-Manhel (26), the

12 hours, namely 2498 mg/L. Research by

concentration of carbon sources is one of the

Rajoka et al. (22) showed that L. rhamnosus

most important factors in achieving high

could

mg/L

levels of exopolysaccharide production. In

exopolysaccharides, while the study of

this study, the 5% sucrose addition increased

Bertsch et al. (23) L. rhamnosus could

the production of exopolysaccharides more

produce 1095 mg/L exopolysaccharides

than previous studies, which were without the

without

addition of sucrose. According to Bibi et al.

produce

the

1808

addition

of

media

supplementation.

(27) Weisella cibaria C43-11 grown in liquid

Another factor that affects the production

media and enriched with 10% sucrose

of exopolysaccharides is incubation time. In

showed high exopolysaccharide production.

this study, the incubation time used was 24

The addition of excessive sugar in the culture

hours. Incubation time must pay attention to

medium also increased the production of

the adequacy of nutrients in the media to

LAB exopolysaccharides. The increase in

ensure that bacteria can still grow by

sucrose concentration in MRS media was

producing exopolysaccharides and do not

suitable for exopolysaccharides production in

reach a static or death phase when harvested.

Lactobacillus confusus TISTR 1498. The

According to Angelin & Kavitha (24),

exopolysaccharide produced by the L. casei

exopolysaccharides are synthesized in the

group in this study showed that the L. casei

logarithmic or final logarithmic, and the

group had the potential to be developed other

stationary

than as a probiotic.

phase.

However,

maximum

production occurs only in the late logarithmic
phase rather than the stationary phase.

36

exopolysaccharides are only synthesized in

Exopolysaccharide total sugar analysis
The

total

sugar

content

of

According to Fan et al. (25), the production

exopolysaccharides is influenced by the

of exopolysaccharides is often associated

amount of sugar contained in the production

with the growth phase of bacteria. Some

medium and the activity of the invertase
Ina. J. Med. Lab. Sci. Tech. 2023; 5(1): 29–41

Nur Kusmiyati, et al.

enzyme, which plays a role in converting the

different. Fatih (17) added that the number of

substrate into its constituent sugar monomers

exopolysaccharides produced by different

(28). According to Imran et al. (29), the total

LAB was caused by inherited traits or genetic

sugar content in the exopolysaccharide L.

traits. The analysis of the total sugar

plantarum NTMI05 was 95.45% and L.

exopolysaccharide of the L. casei group in

plantarum NTMI20 was 92.35%. This

this study showed a relationship between the

indicates the presence of total sugar in the

total sugar content of the exopolysaccharide

exopolysaccharide

and

produced

by

each

the

crude

production

of

different strain. In the same study, the total

exopolysaccharides. This analysis supports

sugar content of L. helveticus MB2-1 was

the presence of polysaccharides produced by

obtained at 95.45%. In another study, the

the L. casei group and improves the quality

total sugar content of L. pentosus 14FE was

of the probiotics of the L. casei group.

81.38%, L. plantarum 47FE was 83,28%, L.

Identification

pentosus

85.19%,

functional

indicating that all exopolysaccharides consist

Transform

of carbohydrates (30).

spectrophotometer

68F

was

obtained

The total sugar content of Bacillus

The

of

groups

exopolysaccharide
using

a

Fourier

Infra-Red

FTIR

results

(FTIR)

of

this

subtilis exopolysaccharide in the research of

exopolysaccharide can provide information

Razack et al. (31) with media without

that the L. casei group is capable of

supplementation showed a yield of 53%,

producing

while media supplemented with 2% sucrose

Exopolysaccharides can be used as a form of

showed a yield of 76%. This indicates that the

self-defence by the L. casei group. In

medium has an affect on the total sugar

addition, exopolysaccharides in lactic acid

content of the exopolysaccharide.

bacteria can also be used to improve the host

exopolysaccharides.

and

immune system. According to Jurášková et

exopolysaccharide sugar content obtained

al. (32), exopolysaccharides produced by

from the three bacteria in the L. casei group

lactic acid bacteria can increase macrophage

showed varying results due to different

activity

species or strains. According to Nurhasanah

stimulate

et al. (5) differences in yield and total sugar

immunoglobulin-A (IgA).

Exopolysaccharide

content

of

production

exopolysaccharides

due

and

cytokine
the

production
formation

and
of

According to Mundiri et al. (33),

differences in strains between species.

exopolysaccharides are known to have the

Differences in bacterial strains cause the

potential to act as immunomodulators that

number of metabolites produced to also be

play a role in the innate immune system in

Ina. J. Med. Lab. Sci. Tech. 2023; 5(1): 29–41

37

to

Nur Kusmiyati, et al.

digestion. Exopolysaccharides of lactic acid

immunomodulatory in functional foods has

bacteria can enhance the innate immune

potential in the future.

system through the role of gut-associated
lymphoid tissue (GALT). The use of

CONCLUSIONS

exopolysaccharides as immunomodulators

The L. casei group was able to produce

has been widely carried out and has been

exopolysaccharides (EPS). L. paracasei was

proven in-vitro and in-vivo to increase

able to produce the highest EPS (3660 mg/L)

macrophage activity, cytokine production

and total sugar (80.6%). Confirmation by

and ability to stimulate IgA formation. In the

FTIR showed that the L. casei group could

research

(34),

produce EPS characterized by hydroxyl,

exopolysaccharides from lactic acid bacteria,

methyl, carbonyl and ether functional groups.

Bifidobacterium longum can increase the

This study shows that the L. casei group has

production of IFN- γ , IL-1 β , and IL-6.

more value than just being a probiotic.

of

Inturri

et

al.

According to Domingos-Lopes et al. (35),
exopolysaccharides from the lactic acid
bacteria Leuconostoc citreum L3C1E7 can
suppress the synthesis of allergen-specific
IgE and exopolysaccharides from lactic acid
bacteria

have

been

shown

to

have

immunomodulatory activity with non-toxic
effects. According to Rajoka et al. (36), the
exopolysaccharides of Lactobacillus contain
various functional groups (such as hydroxyl
groups, phosphate groups, and carbonyl
groups),

which

help

immunomodulatory,
antioxidant,

and

to

exert

their

antimicrobial,
anticancer

activities.

Exopolysaccharides from Lactobacillus can
be used as nutritional and therapeutic agents
to regulate the immune system, which can
help fight various diseases such as cancer,

38

diabetes, and hypertension. Therefore, the
addition

of

exopolysaccharide

Lactobacillus

as

AUTHOR CONTRIBUTIONS
Nur Kusmiyati: substantial contributions
to conception and design, acquisition of data,
or analysis and interpretation of data, drafting
the article or revising it critically for
important intellectual content, final approval
of the version to be published, agreement to
be accountable for all aspects of the work in
ensuring that questions related to the
accuracy or integrity of any part of the work
are appropriately investigated and resolved.
Yuni Puspitasari: rafting the article or
revising it critically for important intellectual
content. Ulfah Utami and Anggeria Oktavisa
Denta: agreement to be accountable for all
aspects of the work, ensuring that questions
related to the accuracy or integrity of any part
of the work are appropriately investigated
and resolved.

agents
Ina. J. Med. Lab. Sci. Tech. 2023; 5(1): 29–41

Nur Kusmiyati, et al.

ACKNOWLEDGEMENTS
This research was funded by Lembaga
Penelitian

dan

Pengabdian

through

Bantuan

Tinggi

Negeri

(BOPTN)

Litapdimas Kementerian Agama Indonesia.

Masyarakat

(LP2M) UIN Maulana Malik Ibrahim
Malang

Perguruan

CONFLICT OF INTEREST

Operasional

The authors declare no conflict of
interest.

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