Enhancing Anti-pathogenic Bacteria Activity of Lactobacillus

Plantarum AKK-30 Cultured on the Medium Containing

Fructose-Oligosaccharides

Nisa Grendpina

, Dyah Fitri

, Hardi Julendra

, Ahmad Sofyan

and Ema Damayanti

Faculty of Biology, Jenderal Soedirman University, Purwokerto, Central Java, Indonesia

Research Division of Natural Product Technology (BPTBA) - Indonesian Institute of Sciences (LIPI), Indonesia

Keywords: Antimicrobial Metabolites, Fructose-Oligosaccharide, L. plantarum, Probiotic.

Abstract: The purpose of this study was to evaluate the concentration of Fructose-Oligosaccharides (FOS) in correlation

with incubation time for growing - Lactobacillus plantarum AKK-30, and to assess metabolites of L.

plantarum AKK-30 as antimicrobial substances against to pathogenic bacteria. L. plantarum AKK-30 was

isolated from the small intestine of native chicken. L. plantarum AKK-30 was grown on MRSB medium

containing FOS (0%, 0.5%, 1%, and 1.5%), and incubated at different times (6, 12, 18, and 24 hours) at 37°C.

Antimicrobial activity of L. plantarum AKK-30 metabolites was tested on four species of pathogenic bacteria

consisted of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Salmonella pullorum.

The results showed that the concentration of 1% FOS and 24-hours incubation were most effective in

increasing L. plantarum AKK-30 growth (2.11 x 10

CFU/ml). Antimicrobial activity extract of L. plantarum

AKK-30 metabolites was able to inhibit the growth of E. coli, P. aeruginosa, S. aureus, and S. pullorum. The

highest inhibition of bacteria was observed on S. aureus which was 10.8 mm, followed by E. coli at 9.9 mm,

S. pullorum at 9.083 mm, and P. aeruginosa 8.783 mm.

1 INTRODUCTION

Lactobacillus plantarum AKK-30 is a lactic acid

bacteria (LAB) isolated from Indonesian native

chicken (Damayanti et al., 2014) and has been

identified microbiologically, biochemically, and

molecularly (Istiqomah et al., 2017). This species

reported that has an activity of enzyme cholesterol

reductase (Julendra et al., 2017; Palaniyandi et al.,

2019). L. plantarum AKK-30 has an inhibitory agent

for pathogenic bacteria and produces antimicrobials

(Julendra et al., 2018; Sophian et al., 2018) and could

be used as probiotics for poultry (Wei et al., 2018).

Previous studies have reported that potency

antibacterial activity of L. plantarum (Kabir et al.,

2009), (Yang et al., 2017), (Lin and Tzu-M, 2017).

The growth of probiotic bacteria can be increased by

the addition of oligosaccharides in their medium

(Pranckute et al., 2016). The use of inulin and mono-

oligosaccharides as prebiotics has been investigated

for increasing viability of L. plantarum AKK-30

(Julendra et al., 2018). However, Julendra et al.

(2018) reported that a combination of L. plantarum

AKK-30 and oligosaccharides were not significant

influences of antibacterial activity. Addition of

mannan oligosaccharides (MOS) at 0.5 - 2% could

increase L. plantarum AKK-30 growth with 0.5%

MOS. However, the possibility of improving L.

plantarum growth by combining FOS has not been

reported. FOS is an oligosaccharide composed of 2-

10-unit fructose monomers with bonds -(2-1)

glycoside and one glucose monomer with bonds -(2-

1) glycoside at the ends (Yuliana et al., 2014).

Addition of FOS in probiotics was for microbial

nutrition (Setiarto et al., 2017), it could improve

metabolism of probiotic bacteria and increase the

number of bacterial cell biomass, bacteriocin

increases (Ogunbanwo et al., 2003), inhibited the

growth of pathogenic bacteria (Pranckute et al.,

2016).

Addition of FOS prebiotic in media is expected to

stimulate the growth of L. plantarum AKK-30

because a FOS was more soluble than inulin (about

80% in water at room temperature) (Gibson et al.,

2017). Therefore, a current study was conducted to

evaluate the effect of FOS addition in growth medium

Grendpina, N., Fitri, D., Julendra, H., Sofyan, A. and Damayanti, E.

Enhancing Anti-pathogenic Bacteria Activity of Lactobacillus Plantarum AKK-30 Cultured on the Medium Containing Fructose-Oligosaccharides.

DOI: 10.5220/0009991800002964

In Proceedings of the 16th ASEAN Food Conference (16th AFC 2019) - Outlook and Opportunities of Food Technology and Culinary for Tourism Industry, pages 205-209

ISBN: 978-989-758-467-1

205

on enhancing the anti-pathogenic bacteria activity of

L. plantarum AKK-30.

2 MATERIALS AND METHODS

2.1 Materials and Research Design

The research was conducted at the Research Division

of Natural Product Technology (BPTBA)-

Indonesian Institute of Sciences (LIPI) at the

Microbiology Laboratory, from November to

December 2018 using L. plantarum AKK-30 isolates

belonging to the BPTBA-LIPI Microbiology

Laboratory and the commercially obtained of Fructo-

oligosaccharides (FOS). FOS was dissolved in

distilled water until homogeneous and then sterilized

using 0.22 µm Millipore then implanted in MRSA. 2

mL of MRSB and 1% of L. plantarum AKK-30 were

added into microtube and then were vortexed and

incubated 24 hours at 37ºC. A series of falcon tubes

filled with 10 mL of sterile MRSB were added with

FOS (0%, 0.5%, 1%, 1.5%) and 1% of L. plantarum

AKK-30 culture (Setiarto et al., 2017). Pathogenic

bacteria used were Escherichia coli FNCC 0194,

Staphylococcus aureus FNCC 6049, Pseudomonas

aeruginosa FNCC 0063, and Salmonella pullorum

ATCC 13036, the bacteria were grown in nutrient

agar (NA) [Merck].

The study used an experimental method with two

stages using a Factorial-Completely Randomized

Design (FCRD). The experiment was arranged using

two stages; the first stage was optimization of L.

plantarum AKK-30 growth added with FOS 0%,

0.5%, 1%, and 1.5% and the second stage using a

Completely Randomized Design (CRD) tested the

antimicrobial activity of metabolites from the best

growth results of L. plantarum AKK-30 in the first

stage of which each treatment consisting of 4

replications. The parameters measured were bacterial

growth and antibacterial activity.

2.1.1 Total Plate Count (TPC)

Colonies of L. plantarum AKK-30 were enumerated

by the TPC method as previously reported by Setiarto

et al. (2017). Briefly, 1 ml of L. plantarum AKK-30

culture (6, 12, 18 and 24 hours) was diluted with 9 ml

of sterile distilled water until 10

-7

. 1 ml of culture was

inoculated on MRSA by pour plate method, then

incubated at 37°C for 48-hours.

2.1.2 Antimicrobial Activity

Antimicrobial activity was assessed according to

Damayanti et al. (2014). Briefly, L. plantarum AKK-

30 culture was centrifuged at 12.500 rpm and 4°C for

15 minutes. The supernatant was neutralized using

0.5N NaOH and sterilized using 0.22 μL millipore.

Agar diffusion method (Bonev et al., 2008) was used

to test the inhibitory activity of pathogenic bacteria

by inoculating pathogenic bacteria on NA media

(Merck) and 50 μL supernatant dripped on sterile disk

paper. Then incubated for 24-hours at 37°C. Positive

results of antimicrobial activity were revealed by the

formation of clear zones around the disk paper.

2.2 Data Analysis

Quantitative data from bacterial growth and

antibacterial activity were analyzed by using analysis

of variance (ANOVA) and followed by Duncan’s

multiple range test to distinguish the effect of

different treatment mean using CoSTAT statistical

software (Cohort, 2008).

3 RESULT AND DISCUSSION

3.1 The Growth of Lactobacillus

plantarum AKK-30

The results of the growth L. plantarum AKK-30 with the

addition of different FOS in MRSA were presented in

Figure 1.

Figure 1: Growth of Lactobacillus plantarum AKK-30 at

different FOS and incubation times.

The results showed that the growth of L.

plantarum AKK-30 with FOS 1% better than other

treatments, it was seen starting from 6-hours of

incubation and the highest number of bacterial

colonies of L. plantarum AKK-30 (2.11 x 10

CFU/ml) occurred when 24-hours incubation. It was

16th AFC 2019 - ASEAN Food Conference

206

explained that the growth of L. plantarum AKK-30

was influenced by the addition of 1% FOS and

significantly different (P <0.05) with no addition of

FOS. The interaction between FOS and incubation

time effect on L. plantarum AKK-30 growth was

explained in Table 1.

Table 1: Interactions Between FOS Concentration and

Incubation Time (Log CFU/mL).

FOS

(%)

Time of Incubation (hours)

6 12 18 24

7.411 ±

0.052

7.639 ±

0.059

7.722 ±

0.129

7.738 ±

0.117

0.5

7.417 ±

0.140

7.718 ±

0.03

7.886 ±

0.129

7.929 ±

0.040

1.0

7.758 ±

0.03

7.834 ±

0.009

7.926 ±

0.129

8.261 ±

0.274

1.5

7.225 ±

0.249

7.815 ±

0.068

8.039 ±

0.129

7.736 ±

0.223

a,b,c,d

: Means in the same column and row differ significantly

(P<0.05).

The results showed that at the 24-hour incubation

of L. plantarum AKK-30 with the addition of FOS

were as follows FOS 0% (7.738 ± 0.117), FOS 0.5%

(7.929 ± 0.040), FOS 1% (8.226 ± 0.274) and FOS

1.5% (7.736 ± 0,223). At 24-hour incubation, growth

of L. plantarum AKK-30 adding 1% FOS was

significantly higher (P<0.05) (8.226 ± 0.274) than

other treatments.

3.2 Antibacterial Activity

The antibacterial in Lactobacillus is obtained from its

metabolite compounds (Setiarto et al., 2017) called

bacteriocin (Rawal et al., 2013).

Table 2: The Diameter of Inhibition Zones of L. plantarum

AKK-30 with FOS 1%.

Metabolic

Extract

The Diameter of Inhibition (mm)

aureus

aeru

ino

ullorum

E. coli

plantarum

-30

10.8

8.783

9.083

9.9

a,b

; Means in the same column differ significantly (P<0.05).

In Table 2, L. plantarum AKK-30 with FOS 1%

demonstrated antibacterial ability as evidenced by the

inhibition zone in the growth of pathogenic bacteria,

Staphylococcus aureus FNCC 6049, Pseudomonas

aeruginosa FNCC 0063, Salmonella pullorum ATCC

13036 and Escherichia coli FNCC 0194. The

antibacterial activity of L. plantarum AKK-30 against

Staphylococcus aureus was significantly higher

(P<0.05) of 10.8 ± 3.59 compared to other pathogenic

bacteria. In Table 2, it can be said that the bacteriocin

in L. plantarum AKK-30 has inhibited Gram-positive

or Gram-negative bacteria. Bacteriocin has a broad

spectrum and could inhibit the growth of pathogenic

bacteria (Sifour et al., 2012; Arief et al., 2013;

Khikmah, 2015; Sulistiani, 2017).

The bacteriocin was an extracellular protein that

has antimicrobial activity (Sari et al., 2018).

Mechanism of inhibition of microbial growth by

bacteriocin is the cell wall damage and have causing

lysis (Pranckute

et al., 2016), the cell's metabolic

system was disrupted by inhibiting the activity of

intracellular enzymes (Pelczar and Chan, 1998) and

disruption of cytoplasmic membrane permeability

(Hasan and Wikandari, 2018).

Figure 2: Inhibitory Activity Metabolic Extracts of L.

plantarum AKK-30 (mm) (a) Staphylococcus aureus (b)

Pseudomonas aeruginosa (c) Escherichia coli and (d)

Salmonella pullorum.

In Figure 2. it was shown that all pathogenic

bacterial growth was inhibited by L. plantarum AKK-

30 metabolite, the widest inhibitory zone was

Staphylococcus aureus and the lowest was

Pseudomonas aeruginosa. Inhibition zones were

influenced by bacteriocin concentrations (Julendra et

al., 2018), bacteriocin activity (Forte et al., 2016),

types of lactic acid bacteria (Kasi et al., 2017) and

different bacterial lipid layers (Jawetz et al., 2005),

(Radji, 2011). The active substance in bacteriocin

from L. plantarum was plantaricin (Gonzalez et al.,

14

8.1

9.2

10.5

11.2

9.35

9.95

9.25

7.7

7.9

10.1

8.25

Enhancing Anti-pathogenic Bacteria Activity of Lactobacillus Plantarum AKK-30 Cultured on the Medium Containing

Fructose-Oligosaccharides

207

1996), antibacterial (Lim et al., 2007) that could lysis

cell membranes of pathogenic bacteria (Lu et al.,

2017).

The difference in width of the inhibition zone can

be caused by the bacterial lipid layer (Radji, 2011).

Gram-negative bacteria have thin peptidoglycan but

there are three polymers outside of peptidoglycan

namely lipoprotein, outer membrane, and

lipopolysaccharide. Permeable outer membranes are

resistant to low molecular weight substances and

hydrophilic solutes but are relatively quickly

penetrated by high molecular weight substances such

as bacteriocin (Jawetz et al., 2005). The mechanism

of bacteriocin is to damage the cell wall causing lysis

(Hasan and Wikandari, 2018), and inhibit cell wall

growth, change the permeability of cytoplasmic

membranes, denaturation of cell proteins, and

damage the metabolic system (Pelczar and Chan,

1998).

The action of plantaricin, in inhibition of

Staphylococcus aureus is by blocking the

permeability of the cytoplasmic membrane and

inducing the release of Adenosine Triphosphate

(ATP), chloroplast factor (CF), and glutamate

(Gonzalez et al., 1996). Plantaricin can disrupt cell

membranes of Gram-negative bacteria such as

Escherichia coli and Pseudomonas aeruginosa (Lu et

al., 2017), and causes the release of intracellular

components of enzymes and ions (Lim and Im.,

2007).

4 CONCLUSIONS

The highest total plate count of L. plantarum AKK-

30 was found at the medium containing 1% FOS with

24-hour incubation. The highest inhibition of L.

plantarum AKK-30 was observed against

Staphylococcus aureus (10.8 mm), followed by

Escherichia coli (9.9 mm), Salmonella pullorum

(9.083 mm), and Pseudomonas aeruginosa (8.783

mm).

ACKNOWLEDGMENTS

This research was financially supported by the Insinas

research program from the Indonesian Ministry of

Research, Technology and Higher Education. The

authors addressed to thank to drh. Ade Ema Suryani,

M.Sc., Mrs. Lusty Istiqomah, M. Biotech., Mrs.

Rumini., Mr. Nurhadi., Fitri Nurhayati., Wahyu Dwi

Saputra, S.Si., Awwaluz Zahroh Mahya A, S.Si., and

Isna Fitriana, S.Si for technical assistance and

supporting during the experiment.

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Enhancing Anti-pathogenic Bacteria Activity of Lactobacillus Plantarum AKK-30 Cultured on the Medium Containing

Fructose-Oligosaccharides

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