Safety Assessment of Plantaricin from Lactobacillus plantarum S34:

Isolated From Indonesia Traditional Food, Bekasam

ARIDO YUGOVELMAN AHADDIN1♥, SRI BUDIARTI2, A. ZAENAL MUSTOPA3,♥♥, HUDA S DARUSMAN4, LITA
TRIRATNA3
1
School of Biotechnology, Bogor Agricultural University. Jl. Raya Dramaga Bogor 16680, West Java Java, Indonesia. Tel./Fax. +62-251-8622642,
♥
email: a.yugovelman@gmail.com.
2
Research Center for Bioresource and Biotechnology, Bogor Agricultural University, Indonesia, Bogor 16680, West Java, Indonesia.
3
Research Center for Biotechnology, Indonesian Institute of Sciences, Cibinong, Bogor 14430, West Java, Indonesia, ♥♥email: azmustopa@yahoo.com.
4
Physiology, Farmacology and Toxicology Departement, Veterinery Faculty of Medicine, Bogor Agricultural University,Indonesia, Bogor 16680, West
Java, Indonesia.

Manuscript received: DD MM 2020 (Date of abstract/manuscript submission). Revision accepted: .................... 2020.

Abstract. Plantaricin S34 is a bacteriocin produced by Lactobacillus plantarum S34 and have antimicrobial or antifungal activity which
is deserve to be an antimicrobial candidate. The aims of this study was to determine antimicrobial activity of plantaricin S34 and its
safety using animal model ddY mice. Identification of plantaricin on crude extract was conducted using Tricine SDS-PAGE.
Antimicrobial activity of crude extract plantaricin S34 was observed using disk diffusion against EPEC K1.1, S. aureus, S. typhosa, S
typhimurium, and Proteus as the bacterial challenge. The safety of plantaricin S34 crude extract was evaluated in ddY mice with doses
of 50, 100, 1000, and 5000 mg/kgBW. The identification of plantaricin showed an active molecule with molecular weight of 7.34 kDa.
Plantaricin S34 has activity to inhibit pathogens used in these experiments. The safety test showed that crude extract plantaricin S34
caused no abnormalities to experimental mices even after 5000 mg/kgBW treatment. Blood analysis results show both hematology and
blood biochemical are in the normal range. The histopathological analysis show no tissue damage to intestine, liver and kidney. From
these experiments we conclude that crude extract plantaricin S34 has potential as antimicrobial agents with no toxicity effect to the
experimental animals.

Key words: Antibiotics, Bacteriocin, Lactobacillus plantarum, Plantaricin, Toxicity.

Abbreviations (if any): Aspartate transaminase (AST), Alanine transaminase (ALT), Multiple drug resistant (MDR), Packed cell
volume (PCV).

Running title: Safety of Lactobacillus Antimicrobial peptide

INTRODUCTION

Multiple drugs resistant is a big problem that continues to grow with the times. This problem causes various
antibiotics can no longer be used to overcome the infection. Research from WHO shows there are 12 types of bacteria that
have MDR (WHO 2017). Various studies have been conducted to find antibiotic replacement agents, one of which is an
antimicrobial peptide. Antimicrobial peptides (AMPs) are peptides that are produced by organisms and have antimicrobial
activity. The nature of antimicrobial activity possessed by AMPs is almost the same as antibiotics, which has a broad
spectrum. This broad spectrum can be used to fight both positive and negative gram bacteria, viruses, fungi and parasites
(Dosler and Mataraci 2013). This ability makes bacteriocin one of the potential alternative to substitute antibiotics. One
group of bacteria that can produce bacteriocin is lactic acid bacteria.
Lactic acid bacteria (LAB) is a group of gram-positive bacteria that naturally found in processed milk or fermented
food (Nur 2005). Some BAL genera that are known to have antimicrobial, antifungal, or antioxidant activity are
Lactobacillus, Lactococcus, Streptococcus, Leuconostoc, Pediococcus, Bifidobacterium and Propionibacterium (Utami
2011). From those research, Lb. plantarum is the most developed bacteria which reported to has antimicrobial activity that
known as plantaricin.
In this research we divide it into three steps. The first step identified the presence of plantaricin in media overgrown
with LAB using Tricine SDS-PAGE. Identification of the presence of plantaricin becomes important because it used to
prove the activity occurred in the study is caused by the presence of these compounds. The extract that have been
identified the presence of plantaricin were tested for their activity against test bacteria, such as EPEC K1.1, S. aureus, S.
typhosa, S typhimurium, and Proteus. The use of test bacteria is based on the presence of MDR in the environmental. The
last step was evaluated the safety of plantaricin S34 crude extract on ddY mice. As a new candidate for antibiotic, safety of
plantaricin is needed to make sure no abnormalities will occur during treatment.
 Plantaricin is an exoprotein belong to class II bacteriocin. Class II bacteriocins are bacteriocins that have low
molecular weight (<10 kDa) and heat resistance 100-121 °C (Karaoglu et al. 2003; Savadogo et al. 2006). The
antimicrobial activity of plantaricin was reported able to inhibit Candida albicans (Sharma and Srivastava 2014), Listeria
innocua NRLL B33314, Micrococcus luteus MTCC 106, Enterococcus casseliflavus NRRL B3502, Lactococcus lactis
NRRL 1821 Lactobacillus curvatus NRRL B4562, and Lactobacillus plantarum NRRL B4496 (Pal et al. 2014). In
Indonesia, research on plantaricin S34 was started by isolating Lb. plantarum S34 from bekasam, a traditional Indonesia
fermented food (Mustopa 2013). On the genetical level, identification of plantaricin gene showed that plantaricin S34 pln
gene encode plantaricin E and F (Mustopa et al. 2016), and W (Umami et al. 2017). These gene that encode plantaricin
were transform into Lactococcus lactis and the presence of each peptide was evaluated. The recombinant protein of
plantaricin has ability to inhibit Entherophatogen Escherichia coli both in vitro and in vivo test (Hanny et al. 2019). As the
previous study success in inhibit pathogens, the antinicrobial produce by Lb. plantarum S34 was assess as a new candidate
for anibiotic replacement.
As a new antimicrobial candidate, activity and safety of plantaricin S34 needs to be evaluated. These study aimed
to analyze the antimicrobial activity of plantaricin S34 both in vitro and its safety in vivo by using the animal model of
ddY mice.

MATERIALS AND METHODS

Materials
Material used in this study are Ammonium sulfat (Merck, Denmark), Agarose (Himedia, India), de Man Rogosa
Sharpe Broth (Himedia, India), Luria Bertani (Himedia, India), Hematoxylin-Eosin, formalin 4%, ethanol absolute
(Merck, Denmark), xilol, parafin, gliserin 99.5%, NaCl (Merck, Denmark), and TCA, TBA, BHT, Tris-HCl (Vivantis,
USA).

Mikroorganism
Lactobacillus plantarum S34 was obtained from the Biotechnology Laboratory, Indonesian Institute of Sciences (LIPI)
Cibinong. The inoculum was stored in de Man Rogosa Sharpe broth (MRS) media at 4 oC. S. aureus, S. typhosa, S.
typhimurium, and Proteus bacteria were obtained from the LIPI collection and stored in Luria Bertani (LB) media. EPEC
K1.1 bacteria were obtained from the Animal Biotechnology Laboratory, Bogor Agricultural University and stored in
nutrient broth media (NB).

Procedures
Production of Plantaricin S34
The production of plantaricin S34 was using Sukmarini et al. (2011) and the isolation was using Xie et al. (2011).
Lactobacillus plantarum S34 was cultured in MRS medium and incubated overnight at 37°C. After the incubation, Lb.
plantarum was centrifuged at 10000 × g in 4°C for 30 min. The supernatant was collected and adjusted with 0.1 N NaOH
until pH 6.5 reached. The supernatant was precipitated with 60% (NH 4)2SO4. The precipitation was done at 4°C, overnight.
The precipitate was collected by centrifugation at 10000×g in 4°C for 30 minutes. The pellet was collected and
resuspended in Tris HCl buffer pH 7.4.

Identification of Plantaricin S34 Using Tricine SDS-PAGE and Zymograms

Plantaricin identification using tricine sodium dodecyl sulfate polyacrylamide gel (Tricine SDS-PAGE) with a
concentration of 4% (staking gel) and 16% (separating gel) (Schagger 2006). Polyacrylamide gels are made by mixing
acrylamide: bis-acrylamide (29:1), aquadest, glycerol, TEMED, and ammonium persulfate. The gel staining was using
silver stain reagent (Thermo Fisher). The zymograms was done by placing the gel above agar media which contain 10 6
EPEC K1.1 bacteria.The gel was fixated using 25% ethanol and 5% formaldehyde for 30 minutes th aen washed using
sterilized water for 3 h. After that, it was washed three times using Tween-80 for 40 min before laid out in pathogens
media. The gel was incubated at 37 ºC for 24 h.

Antibacterial Activity Test

Antibacterial activity was carried out by using agar diffusion method (Arief et al. 2015). The test bacteria was cultured
on LB for 16 h and dilluted with physiological NaCl until it has reached a concentration of 10 6 CFU/mL. The bacteria was
incubated in Luria Bertani agar (LA). Incubation was carried at 37 °C for 16 h and the clear zone was meassured.

Experimental Animals Preparation

Male ddY mices were obtained from the Experimental Animal Laboratory Biofarma, Bandung. These mice were used
for this experiment. Mice weighed between 15 and 30 g were kept in plastic boxes with feed and water ad libitum. These
animals were used in experiments after a period of 7 days of adaptation in captivity, with 12 h light–dark periods at 20ºC.
The procedures was approved by Bogor Agricultural University Ethics Commission with ethic number 80-2017IPB.

In vivo Toxicity of Plantaricin S34 Crude Protein

The toxicity assay was following Almeida vaucher et al (2011). Male ddY mice aged 4-8 weeks were kept in group
and given standard food and drink. The treatment group was divided into 6 treatment groups. The experimental was using
simple random sampling method. Plantaricin S34 was given orally. The clinical symptomps (skin and fur, eyes, lethargy,
convulsions (seizures), tremors (trembling), diarrhea, and death) was observed at 2, 12, 24, and 48 h post-treatment. The
weight measurement was done every 24 h. After 48 h post-treatment, mice were euthanized (exanguination) and the blood,
liver, and kidney were collected for further analyses.

Hematology Analysis
The hematology analysis was following Aboderin and Oyetayo (2016) methods. Collected mice blood was analize for
hematology parameters (PCV, hemoglobin, eritrocyte, leucocyt, and trombocyt) using hematology analyzer Hemavet
HV950FS (Drew Scientific Inc, German).

Biochemical Analysis
Collected mice blood was centrifuged at 6000 × g and the serum was separated from the blood cell. The serum was
tested for biochemoical parameters (AST, ALT, ureum, and creatinine) using hematology analyzer Hemavet HV950FS.

Histopathological analysis
The histopathological sample was prepared using Prophet et al. (1992) methods. Tissue sample that collected was fixed
in 10% of buffer formaline and processsed for parafin embedding. The histological sections were stained with
hematoxylin–eosin (HE). The slides were coded and analyzed at the Primate Study Center (Bogor Agricultural University,
Bogor).

Data analysis
The results were expressed as mean ± standard deviation of the groups and subjected to analysis of variance (ANOVA)
and Tukey’s test using software Minitab 17.0. The differences were considered statistically significant when p < 0.05.

RESULTS AND DISCUSSION

Identification and antimicrobial activity of Plantaricin S34

Plantaricin S34 was gained using 60% precipitation of ammonium sulfate. The concentration of crude plantaricin was 66
mg/mL. The molecular identification using Tricine SDS-PAGE showed the presence of two bands lower than 10 kDa at
positions approximately 7.34 kDa (red arrow) and 5.82 kDa (green arrow) (Fig 1). The zymogram showed that the 7.34
kDa band has activity against EPEC K1.1 while the 5.82 kDa band has no activity to it.
kDa

~40

~25

~15

~10

4.6
1.7

(a) (b)
Figure. 1 Separation of plantaricin S34 using Tricine SDS-PAGE(a) and zymograms (b)

Antimicrobial activity of plantaricin S34 crude protein was measured against several pathogens. The result was shown
in table 1. The plantaricin S34 crude protein has better activity against EPEC K1.1 but have different activity to the other
pathogens.

Table 1. Antimicrobial activity of plantaricin S34

Pathogen Clear zone (mm)
 Ampicillin Plantaricin
EPEC K1.1 1.5 ± 0.29 6 ± 0.82
S. aureus 12 ± 0.41 7 ± 0.32
S. typhimurium 18 ± 0.00 4 ± 0.96
Proteus 13.5 ± 1.25 4 ± 0.50

Plantaricin S34 Safety in ddY mice

The toxicity test of plantaricin crude extract was showing no changes in clinical observation (data not showed). The
mice body mass measurement show a slight rising but for the treatment is decreasing (Figure 2A). All the body mass have
no significant difference compared to control groups. The organ weight was also has no significant difference to the
control (Figure 2B).
27.5
26.5
Mass (g)

25.5
24.5
23.5
0 48
Time (h)

Control 5000 1000

100 50

Figure 2. Effect of plantaricin S34 crude protein administration to body and mass organs

The plantaricin treatments have significant effect to lowering leucocyte and PCV level and rising trombocyte and
eritrocyte. While the concentration increased, the leucocyte level was gradually decrease. The leucocyte was lowered for
0.8 thous/µL to 1.6 thous/µL. It was contrary to PCV level which could rise the PCV level in low concentration and have a
little effect while the concentration was increase. The highest level of PCV was observed in 50 mg/kgBW and the lowest
was at 100 mg/kgBW plantaricin S34 crude protein administration. The highest level of trombocyte and eritrocyte could
rise were 524.67±43.25 thous/µL and 8.93±0.03 mill/µL and the lowest were 378.00±26.06 thous/µL and 35.03±1.97
mill/µL. There are signifficant difference compared to control (Table 2). The normal levels of leukocytes is between 1.5–
4.8 thous/µL platelet levels between 325–888 thous/µL erythrocytes between 6.1-10.7 mill/µL, and PCV between 33.5–
47.8% (Santos et al. 2016).

Table 2. Hematology analysis of ddY after 48 h treated with plantaricin S34 crude protein
Hemoglobin Leucocyte Trombocyte Eritrocyte PCV
Groups
(g/dL) (thous/µL) (thous/µL) (mill/µL) (%)
Kontrol 12,73±1.31a 3,10±0.10a 473,33±15.31a 7,02±0.64a 34,93±3.23a
NaCl 11.05±0.31a 1.50±0.20b 451.00±39.74a 7.10±0.47a 34.13±1.35a
 S34 5000 12.60±0.10a 1.95±0.53bc 378.00±26.06b 8.30±0.22b 40.73±0.55b
S34 1000 13.43±0.59a 2.20±0.10c 466.00±8.54a 8.58±0.28b 42.00±2.19b
S34 100 12.00±1.35a 2.10±0.85bc 524.67±43.25a 6.49±0.13a 35.03±1.97a
a c a b
S34 50 13.13±0.78 2.30±0.10 436.67±65.96 8.93±0.03 44.63±2.69b
*The number shared the same character have no significant result with P < 0.05
Blood serum analysis was also performed to see the effect of plantaricin on liver and kidney performance. Table 3
shows an increase in ureum, creatinine, AST, and ALT levels. The rising in ureum and AST not always followed with the
rising of creatinin and ALT. The 1000 mg/kgBW of plantaricin S34 crude protein administered could rising most of the
blood serum components. There was a significant difference to all observed components.
Table 3. Blood serum analysis of ddY after 48 h treated with plantaricin S34 crude protein
Ureum Creatinin AST ALT
Groups
(g/dL) (g/dL) (U/L) (U/L)
Control 46.67±8.14a 0.39±0.03a 207.25±74.43a 52.33±1.15a
NaCl 85.33±5.51b 0.43±0.02a 250.00±37.47a 80.33±7.77b
S34 5000 80.67±3.06b 0.65±0.06b 230.67±36.67a 83.87±7.76b
S34 1000 95.33±10.41b 0.53±0.08b 395.00±92.07b 128.67±26.10c
S34 100 61.67±1.15d 0.45±0.04a 304.00±31.19a 64.33±4.93d
S34 50 90.67±1.53bc 0.41±0.03a 288.00±42.58a 131.33±20.21c
*The number shared the same character have no significant result with P < 0.05
Observation of liver cells shows a picture of normal hepatocyte and central venous cells (Figure 2F). Kidney organs
were also not found to have damage to kidney cells, tubules or glomerulus (Figure 2E). Observation of mice intestinal
organs showed no damage to the epithelium or villous small intestine (Figure 2D).
A A B B C C

D D E E F F

Figure 3. Histopathology of intestine, kidney and liver of ddY mice after 48 hours of plantaricin S34 crude protein administration. A-C
is a control organ and D-F is an organ with plantaricin 5000 mg/kgBW

Discussion
The identified protein in Tricine SDS-PAGE predicted as two type of class II bacteriocin based on the molecular
weight gained from it. The low molecular weight identified from Tricine SDS-PAGE was suspected as plantaricin S34.
From zymograms it was confirmed that plantaricin S34 used in this research only has one active band which is 7.34 kDa
band.
Plantarcin is able to inhibit bacterial growth through the mechanism of electrolyte efflux, disrupt membrane potential
(Zhang et al. 2015), and inhibit 14-α demethylase enzyme (Omar and Yadav 2018). Plantaricin S34 crude protein was
better than ampicillin in inhibit EPEC K1.1. Arivo et al. (2011) reported that EPEC K1.1 has resistencies against
ampicillin while for the others still lower than ampicillin. The presence of plantaricin S34 crude protein activity against
pathogens makes this compound has potencies to substitute antibiotic.
All mice groups that had plantaricin S34 crude protein treatments showed a significant difference in leukocytes,
platelet, eritrocytes, and PCV. However, the changes still in normal level for each components. Thus showed that
plantaricin S34 crude protein did not cause organs damage which made changes from normal range. As protein plantaricin
S34 is easily to degradate by proteolitic enzymes. The residues from degradable plantaricin did not cause elevation of
hematology profile exceed the normal range.
Blood serum analysis was also performed to see the effect of plantaricin on liver and kidney. The increase in urea
indicates that plantaricin can be digested by enzymes and converted to amino acids and ammonia. The presence of
protease enzymes in the body can degrade plantaricin so that the level of ureum in the blood increases (Hata et al. 2010).
Changes in urea levels are influenced by protein consumption and digestion of proteins in the digestive tract. Increased
urea levels can be also caused by the breakdown of muscle protein as indicated by increased creatinine levels. Increased
creatinine levels are influenced by muscle mass, body metabolism, or infection or inflammation (Martono and Satino
2014).
Changes in AST and ALT are related to the secretion of protease enzymes carried out by the liver. Changes in AST and
ALT can be caused by protein consumption, activity, damage to body cells, or infections. Tissue damage or liver disorders
can be indicated by an increase in activity up to 5-15 times normal activity. The use of drugs that can cause liver disorders
incresing ALT/AST activity up to ≥20 times normal activity (Aleya and Berawi 2015).
Observation of liver cells shows a picture of normal hepatocyte and central venous cells (Figure 2F). Kidney organs
were also not found damaged for kidney cells, tubules or glomerulus (Figure 2E). Observation of mice intestinal organs
showed no damage to the epithelium or villous small intestine (Figure 2D). However, intestinal organs show a slight
infiltration of mononuclear inflammatory cells. The low damage caused by the administration of plantaricin S34 crude
protein to a dose of 5000 mg/kgBW indicates the similarity of safety with commercial bacteriocin which has a toxicity of
up to 6950 mg/kgBW (Jozala et al. 2007).
From these research we know that plantaricin S34 was observed as exoprotein with molecular weight of 7.34 kDa. The
antimicrobial activity of plantaricin can inhibit EPEC K1.1, S. typhi, S. typhosa, S. Aureus, and Proteus grow. Analysis of
hematology and blood biochemical did not show changes that exceeded the normal limits of blood of mice while the
histopathology analysis show no abnormalities at kidney, liver, and intestine. It means crude extract plantaricin has no
toxicity effect to the experimental animals.

ACKNOWLEDGEMENTS

Acknowledgments are expressed in a brief; all sources of institutional, private and corporate financial support for the
work must be fully acknowledged, and any potential conflicts of interest are noted.

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