J. Life Sci. Biomed. 6(1): 15-21, Jan 30, 2016  
JLSB  
© 2016, Scienceline Publication  
Journal of  
ISSN 2251-9939  
Life Science and Biomedicine  
Characterization of Bacteriocin Lactobacillus casei on Histamine-  
Forming Bacteria  
  
Amidya Nugrahani1 , Hardoko2, Anik Martinah Hariati3  
1Master Program, Faculty of Fisheries and Marine Sciences, University of Brawijaya, Malang-65145, Indonesia.  
2Department of Fisheries Product Technology, Faculty of Fisheries and Marine Sciences, University of Brawijaya, Malang-65145, Indonesia.  
3Head of the Laboratory of Fish Nutrition, University of Brawijaya, Malang-65145, Indonesia.  
ABSTRACT: Tuna which has undergone a process of decay will be poisoned if being consumed. It caused by  
the contamination of pathogenic bacteria such as Escherichia coli, Salmonella, Vibrio cholerae,  
Enterobacteriacea etc. Some types of fish contain histidine family scmbroidae high free, such as yellow tail  
tuna 740 mg/100g of meat, bigeye tuna 491 mg/100 g, mahi-mahi 344 mg/100 g, mackarel 600 mg/100 g,  
skipjack 1192 mg/100 g and albakor highest to 2 g/100 g. The formation process of histamine in fish is  
influenced by the activity of the enzyme L-Histidine Decarboxylase (HDC). Bacteriocin Lactobacillus casei  
extract is able to inhibit the activity of Pseudomonas sp, Proteus morgani and Micrococcus sp. The extract of  
bacteriocin Lactobacillus casei has a high temperature stability which has inhibitory activity against bacteria  
test at a temperature of 90oC. Bacteriocin Lactobacillus casei from bacteria Pseudomonas sp has the optimum  
activity at pH 5 with inhibition diameter of 8.25 mm, while the bacteria Micrococcus sp has the optimum  
activity at pH 4 with a inhibition diameter of 9.25 mm. Bacteriocin Lactobacillus casei has a molecular weight  
of 14.34 kDa which included in the group of class III bacteriocins, generally has a large size (> 10 kDa), and can  
not survive against the heat. Bacteriocins Lactobacillus casei extract can inhibit the activity of histamine-  
forming bacteria growth and have stable properties to high temperature and pH. Future research is  
recommended to do the production of bacteriocins Lactobacillus casei optimization and its application in  
fishery product.  
Keywords: Characterization, Lactobacillus casei, Bacteriocin, Histamine Forming Bacteria  
INTRODUCTION  
Swordfish which belonging to the family of scombroidae will being easily decayed at the room temperature,  
and also the high water content in fish would be a suitable medium for the metabolism of spoilage bacteria that  
will causing the process of decay and the fish will no longer fresh anymore. The swordfish that has been decayed  
will be poisoned if being consumed. It caused by the contamination of pathogenic bacteria such as Escherichiacoli,  
Salmonella, Vibrio cholerae, Enterobacteriacea etc. Poisoning that often occurs by tuna were the histamine  
(scombroid fish poisoning) [1]. The process of formation of histamine in fish is influenced by the activity of the  
enzyme L-Histidine Decarboxylase (HDC). Various types of bacteria are capable to producing the enzyme HDC,  
including the Enterobacteriaceae, for example: Enterobacter agglomerans, Enterobacter cloacae, Enterobacter  
intermedium, Hafnia alvei, Klebsiella pneumoniae, and Morganella morganii [2].  
To inhibit the bacterial growth, it is necessary to do the preventive measures in order to slow down the  
change of histidine so it will not cause the allergie such as by using bacteriocins as an antibacterial agent.  
Bacteriocins is one of the antimicrobial compounds that produced by lactic acid bacteria. Bacteriocins is defined  
as the active peptides or peptide complexes that were synthesized at the ribosomes, and also have the activity of  
bakteriostatic and bactericidal [3]. Bacteriocin that produced by LAB is a secondary metabolite produced by  
ribosomes, sensitive to proteolytic enzymes and may be inactivated by the digestive tract protease enzyme,  
°
°
generally has a heat resistance (60 C or 100 C for 30 minutes or more), stable at acid pH and neutral,  
inactivated at pH above 8.0 [4].  
It is known that Lactobacillus casei bacteria can produce the bacteriocin that has antibacterial activity  
against several common pathogens and spoilage microorganisms in food production. It was explained by Chotiah  
[5] with the result that the crude bacteriocin Lactobacillus casei has antagonistic properties against pathogens  
(S.typhimurium; E. coli; B. cereus and S. enteritidis. Inhibitory activity against E.coli K99 enterotoxigenic and S.BCC  
aureus B2062 / ATCC 25923 is not visible. Based on the result above, this research will discuss about the chemical  
and physical characteristics of bacteriocins of Lactobacillus casei and its application on histamine-forming  
bacteria.  
To cite this paper: Nugrahani A, Hardoko, Martinah Hariati A. 2016. Characterization of Bacteriocin Lactobacillus casei on Histamine-Forming Bacteria. J. Life Sci.  
Biomed. 6(1): 15-21.  
Journal homepage: www.jlsb.science-line.com  
15  
MATHERIAL AND METHODS  
The Growth character of Lactobacillus casei  
The growth curve of bacteria for 24 hours of incubation and sampling every 1 hour to determine the  
existing phases. A total of 5% (v / v) of the cultivation Lactobacillus MRS agar were grown in MRS broth and  
incubated at 37ºC. Bacterial growth is followed every hour to observe the optical density value or optical density  
(OD) of the starter on MRS media with turbidimetric method with a wavelength of 620 nm [6].  
Making the Cultivation  
The isolates of lactic acid bacteria that used in the form of agar slant culture MRS broth were rejuvenated  
with yeast addition as much as 0.5% (w / v). Rejuvenation or activation of Lactobacillus casei activation were  
done by growing a loop isolates grown in an agar medium slant into 10 ml MRS broth, then it were incubated at  
37 ° C for 24-48 hours. After that, 1 ml of MRS Broth cultivation were taken to be grown in 10 ml MRS broth, then  
°
it were incubated at 37 C for 24-48 hours to obtain a cultivation [7]. Rejuvenation is also were done on bacteria  
test using Tryptone Soya Agar (TSA) media.  
Extraction of Bacteriocin  
The active cultures of Lactobacillus casei as much as 10% (v / v) were propagated in the 1000 ml of MRS  
broth (pH 7.0; glucose 0.25% w / v; peptone 0.5% w / v) for 20 hours at a temperature of 30°C. Culture  
°
centrifuged at a speed of 10,000 rpm for 20 min at 4 C and neutralized using 1M NaOH to pH 7.0. The solution  
that were obtained then being filtered using a 0.2 μm membrane filter to obtain the cell-free supernatant [8]. Cell-  
free supernatant obtained from the extraction process that were precipitated with ammonium sulfate saturated  
°
solution of 60% (w / v) and were homogenized with a magnetic stirrer for 24 hours at a temperature of 4 C.  
o
Then, the precipitation solution was centrifuged for 30 minutes at 12000 rpm at 4 C. The pellets were dissolved  
in 25 ml of 0.05 M potassium phosphate buffer (pH 7.0) [9]. The pellets obtained from the bacteriocins with  
ammonium sulfate precipitation that were dialyzed using a dialysis membrane (1.0 kDa). The buffer that being  
used were potassium phosphate buffer (pH 7.0) for 18 hours, then buffer have to be replaced at every 6 hours at a  
temperature of 4°C to obtained the bacteriocins extract [8]. Bacteriocins solution that obtained by dialysis was  
centrifuged at ultracentrifugation at a speed of 50,000 rpm for 30 min at 4°C and the supernatant that obtained  
were the bacteriocins extract [10].  
Biological Characterization (Inhibitory Activity Bacteriocin)  
Media MHA which have been sterilized then were put into petri dishes 20 ml each and allowed to solidify at  
room temperature. The medium were inoculated with 0.1 ml of bacterial suspension test and trimmed with a  
hockey stick, then allowed to stand to dry for 15 minutes. Bacteriocins extract as much as 50 mL were dripped on  
paper discs. Then placed on a paper disk media that has been inoculated by bacteria MHA test, then it were  
°
incubated at the temperature of 37 C for 24 hours. The clear zone that being formed indicates the existence of  
barriers of the growth of test bacteria by supernatant. We then measured the diameter of clear zone (mm) using  
calipers. The diameter of each inhibition zone was measured three times in different areas and then the results  
are averaged [11, 12].  
Stability against temperature and time  
°
This test were done by heating the bacteriocins as 400μl at a temperatures of 45 C, 70 ° C and 95 ° C for 15,  
30 and 45 minutes. Then, the bacteriocins extract were tested about its inhibitory activity by disc diffusion  
method.  
Stability against pH  
This test were performed by addition of 0.1 M NaOH or 0.1 M HCl to make a difference in the pH level of 2  
to 9. The volume of bacteriocins used is 400μl. Then, a solution of NaOH or HCl bacteriocins were homogenized  
and allowed to stand for a few minutes before being tested to its inhibitory activity against histamine-forming  
bacteria.  
Chemical characterization (Molecular weight Bacteriocin)  
Determination of molecular weight bacteriocins were using Deodecyl Sodium Sulfate Polyacrilamide gel  
electrophoresis (SDS-PAGE) [13].  
RESULTS AND DISCUSSION  
The growth phase of Lactobacillus casei  
During the growth of lactic acid bacteria, the maximum production occurs at the end of the exponential  
phase or early stationary phase. The incubation period were used at the 19th hour which this phase occurs  
production of bacteriocins. The best production of bacteriocins was when it reaches the end of the exponential  
phase or early stationary phase. The growth curve of Lactobacillus casei [14] can be seen in Figure 1.  
To cite this paper: Nugrahani A, Hardoko, Martinah Hariati A. 2016. Characterization of Bacteriocin Lactobacillus casei on Histamine-Forming Bacteria. J. Life Sci.  
Biomed. 6(1): 15-21.  
Journal homepage: www.jlsb.science-line.com  
16  
2.500  
2.000  
1.500  
1.000  
0.500  
0.000  
0
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24  
TIME (hours)  
Figure 1. The growth curve of Lactobacillus casei  
Biological Characterization  
The diameter of inhibition zone was looked like diameter of the clear zone around the well which exhibits  
bactericidal (killing bacteria) or pseudo-diameter zone that showed bacteriostatic properties (inhibit microbial  
growth). The Clear zone formed by the secondary metabolites or another antimicrobial active compound that  
were produced. The test results inhibitory activity can be seen in Figure 2.  
12  
10.20 ± 0.15c  
10  
8.28 ± 0.21b  
8.10 ± 0.31b  
8
6
4
2
0
0.00  
Histamin-forming bacteria  
Figure 2. Graph bacteriocins extract inhibitory activity of Lactobacillus casei  
On inhibition test bacteria Staphylococcus sp (Figure 3) there is no clear zone so that it can be stated that  
the bacteriocins of Lactobacillus casei can not inhibit the test bacteria Staphylococcus sp due to Staphylococcus sp  
has resistance to acids and has a strong cell wall (covalently bonded) so that this bacteria is more resistant to  
acids and other substances that were produced by antagonic Lactobacillus casei [15]. Bacteriocins extract of the  
bacterium Lactobacillus casei test has inhibitory activity which has inhibition diameter between 6-11 mm.  
Description:  
(A) Pseudomonas sp  
(B) Proteus morgani  
(C) Micrococcus sp  
(D) Staphylococcus sp  
Figure 3. Results of bacteriocins inhibition diameter Lactobacillus casei against histamine-forming bacteria  
To cite this paper: Nugrahani A, Hardoko, Martinah Hariati A. 2016. Characterization of Bacteriocin Lactobacillus casei on Histamine-Forming Bacteria. J. Life Sci.  
Biomed. 6(1): 15-21.  
Journal homepage: www.jlsb.science-line.com  
17  
Based on the results of bacteriocin inhibitory activity against histamine-forming bacteria, bacteriocins can  
be concluded that the extract can inhibit the growth of Lactobacillus casei histamine-forming bacteria. Inhibition  
of the enzyme L-histidine Decarboxylase will affect in delayed or no product formation so that leads to the  
reduction or even the histamine can not be produced [16]. Bacteriocins will affect the membranes, DNA synthesis  
and protein synthesis. In general, bacteriocins showed bactericidal or bacteriostatic activity against other  
bacteria that are closely related to the producing strain. The main mechanism of bacteriocin was varied, they are  
the formation of pores in the cytoplasmic membrane or cell wall biosynthesis and inhibition of enzyme activity  
(RNAse or DNAse) in target cells [5].  
Stability against the temperature  
From the test results of the temperature characteristics of these bacteriocins, bacteriocins obtained  
inhibitory activity in bacteria Pseudomonas sp results can be seen in Figure 4 and the bacteria Micrococcus sp that  
the results can be seen in Figure 5.  
9.00  
8.00  
7.00  
6.00  
temperature 45 C  
5.00  
temperature 70 C  
4.00  
temperature 95 C  
3.00  
2.00  
1.00  
0.00  
15  
30  
45  
Time (minute)  
Figure 4. Graph bacteriocin inhibitory activity against the treatment temperature and heating time in bacteria  
Pseudomonas sp  
10.00  
9.00  
8.00  
7.00  
6.00  
5.00  
4.00  
3.00  
2.00  
1.00  
0.00  
temperature 45 C  
temperature 70 C  
temperature 95 C  
15  
30  
45  
Time (minute)  
Figure 5. Graph bacteriocin inhibitory activity against the treatment temperature and heating time in bacteria  
Micrococcus sp  
To cite this paper: Nugrahani A, Hardoko, Martinah Hariati A. 2016. Characterization of Bacteriocin Lactobacillus casei on Histamine-Forming Bacteria. J. Life Sci.  
Biomed. 6(1): 15-21.  
Journal homepage: www.jlsb.science-line.com  
18  
°
°
Bacteriocin Lactobacillus casei did not lose the inhibitory activity in the temperature range of 45 C, 70 C  
and 90 ° C, however, these bacteriocins decreased inhibitory activity against both the histamine-forming bacteria,  
the Pseudomonas sp and Micrococcus sp. Bacteriocin inhibitory activity decline continued with the increasing of  
heating temperature and the longer it has been used. It also can be seen in Figures 4 and 5 that the higher  
temperature was used, the smaller of diameter of the inhibition of bacteriocins against histamine-forming  
bacteria, as well as time. The longer it is used, the smaller the diameter of the inhibition of bacteriocins against  
°
histamine-forming bacteria. Bacteriocins of lactic acid bacteria resistant to the temperature of 100 C for 30  
minutes even until the temperature of the autoclave. This is because in these bacteriocins may exist compounds -  
small globular compounds and their strong hydrophobic regions. It can be distinguished that from bakteriophage  
that is not heat resistant to autoclaving [17].  
Stability against pH  
The pH factor is often a consideration for preservatives to be used in food, especially food for livestock with  
low pH conditions such as beef, ham, meatballs, milk, butter, cheese etc. [18]. From the results of testing the  
characteristics of bacteriocins against this pH value, obtained bacteriocin inhibitory activity on histamine-forming  
bacteria (Pseudomonas sp and Micrococcus sp), the result can be seen in Figure 6.  
Pseudomonas sp  
Micrococcus sp  
10.00  
9.00  
8.00  
7.00  
6.00  
5.00  
4.00  
3.00  
2.00  
1.00  
0.00  
2
3
4
5
6
7
8
9
pH  
Figure 6. Graph bacteriocins extract inhibitory activity against pH  
Bacteriocin Lactobacillus casei bacteria Pseudomonas sp test has optimum activity at pH 5 with inhibition  
diameter of 8.25 mm, while the test bacteria Micrococcus sp has optimum activity at pH 4 with a diameter of 9.25  
mm inhibition. Bacteriocins produced by Lactobacillus acidophilus has optimum activity in the range of pH values  
of 4-5. Bacteriocin inhibitory activity decreased with increasing pH values (pH approaching the base) and active  
at acidic pH. The higher of the pH so the bacteriocin activity will be reduced, as seen in bacteriocins lost piscicolin  
activity at high pH near the pH 8 [19].  
Chemical characterization (Molecular Weight)  
Bacteriocins of Lactobacillus casei has a molecular weight of 14.34 kDa which can be seen in Figure 3. The  
molecular weight of bacteriocins were different by Lactobacillus species such as have a broad molecular weight  
range. Based on the size, morphology and physical, bacteriocins [20] produced by the bacterium Lactobacillus  
casei are grouped in Class III bacteriocins, which were generally large (> 10 kDa), and are not heat resistant  
consisting of two types. Type IIIa is bakteriolisin which is an enzyme of bakteriolitic. Examples are studied in this  
type is lisostaphin. Type IIIb is non-lytic type bacteriocins, one of which is helvetisin J (37 kDa) that were  
produced by Lactobacillus helveticus.  
To cite this paper: Nugrahani A, Hardoko, Martinah Hariati A. 2016. Characterization of Bacteriocin Lactobacillus casei on Histamine-Forming Bacteria. J. Life Sci.  
Biomed. 6(1): 15-21.  
Journal homepage: www.jlsb.science-line.com  
19  
B
M
kDa  
148  
98  
64  
50  
36  
22  
14,34 kDa  
(bacteriocin like)  
16  
6
Figure 7. Result of elektroforesis SDS-PAGE  
CONCLUSION  
Bacteriocin Lactobacillus casei extract is able to inhibit the bacteria Pseudomonas sp, Proteus morgani and  
Micrococcus sp. Extract of bacteriocin Lactobacillus casei has high temperature stability which at a temperature of  
95 ° C still has inhibitory activity against bacteria test. Bacteriocin Lactobacillus casei bacteria Pseudomonas sp test  
has optimum activity at pH 5 with obstacles measuring 8.25 mm, whereas the test bacteria Micrococcus sp has  
optimum activity at pH 4 with obstacles measuring 9.25 mm. Bacteriocin Lactobacillus casei active at acidic pH.  
Bacteriocin Lactobacillus casei has a molecular weight of 14.34 kDa were included in the group of class III  
bacteriocins, generally large (> 10 kDa), and are not heat resistant.  
Competing interests  
The authors declare that they have no competing interests  
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Biomed. 6(1): 15-21.  
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To cite this paper: Nugrahani A, Hardoko, Martinah Hariati A. 2016. Characterization of Bacteriocin Lactobacillus casei on Histamine-Forming Bacteria. J. Life Sci.  
Biomed. 6(1): 15-21.  
Journal homepage: www.jlsb.science-line.com  
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