Antibacterial Activity of Lactobacillus buchneri Bacteriocin against Vibrio parahaemolyticus

Abstract

Eleven yoghurt samples were collected from local markets in Baghdad to isolate Lactobacillus buchneri. Only 3 isolates of L. buchneri were found and the isolate No. 3 was the most producer of bacteriocin. Bacteriocin was adsorbed 100% onto silicic acid at pH 6.0-7.0. Below or above these pH values, adsorption was decreased, ranging between 35 and 90%. Therefore, pH 6.0 was used for the purification procedure. The purification procedure including silicic acid adsorption/desorption and Cation-exchange chromatography (CEC) resulted in a 11.11 fold increase in the final specific activity 1176.47 Au/mg of pure bacteriocin compared to the culture supernatant which was 32.64 Au/mg. The molecular weight was determined to be about 3.4 kDa. The bacteriocin lost its activity completely after treatment with proteolytic enzymes and it was resistant to non-proteolytic enzymes. The results indicated that bacteriocin in both concentrations (500 and 1000) µg/ml possesses significant antimicrobial activity against Vibrio parahaemolyticus in contrast with control P<0.01 and the antimicrobial activity of crude and purified bacteriocin at the concentration of 1000 µg/ml were higher than the other concentration 500 µg/ml. The antimicrobial activity of purified bacteriocin was significantly higher than that of crude bacteriocin (P<0.01).

Full text

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Antibacterial Activity of Lactobacillus buchneri Bacteriocin against Vibrio
parahaemolyticus
Likaa Mahdi*, Harith Jabbar Fahad Al Mathkhury1  -Kakei*2, Khetam Habeeb Rasool3, Luma Zwain4, Istabreq
Muhammed Ali Salman5, Nada Zaki Mahdi6, Sadeq Abdulridha Gatea Kaabi7, Nibras Nazar Mahmood8
AL-Mustansiriyah University, College of Science, Dept. of Biology, Baghdad, Iraq.*,2,3,5,6,7.8
University of Baghdad, College of Science, Dept. of Biology, Baghdad, Iraq.1
University of Baghdad, College of Education Ibn AL-Haitham, Biology Department, Baghdad, Iraq.4
Abstract
Eleven yoghurt samples were collected from local markets in Baghdad to isolate Lactobacillus buchneri.
Only 3 isolates of L. buchneri were found and the isolate No. 3 was the most producer of bacteriocin.
Bacteriocin was adsorbed 100% onto silicic acid at pH 6.0-7.0. Below or above these pH values,
adsorption was decreased, ranging between 35 and 90%. Therefore, pH 6.0 was used for the purification
procedure. The purification procedure including silicic acid adsorption/desorption and Cation-exchange
chromatography (CEC) resulted in a 11.11 fold increase in the final specific activity 1176.47 Au/mg of
pure bacteriocin compared to the culture supernatant which was 32.64 Au/mg. The molecular weight was
determined to be about 3.4 kDa. The bacteriocin lost its activity completely after treatment with
proteolytic enzymes and it was resistant to non-proteolytic enzymes. The results indicated that
bacteriocin in both concentrations (500 and 1000) µg/ml possesses significant antimicrobial activity
against Vibrio parahaemolyticus in contrast with control P<0.01 and the antimicrobial activity of crude
and purified bacteriocin at the concentration of 1000 µg/ml were higher than the other concentration 500
µg/ml. The antimicrobial activity of purified bacteriocin was significantly higher than that of crude
bacteriocin (P<0.01).
Keywords: Lactobacillus buchneri, Buchnericin LB, antimicrobial activity
_____________________________________________________________________________________
*Corresponding author: Assist. Prof. Dr. Likaa Hamied Mahdi
Tel.: ++9647901266314
E-mail: likaahamied@yahoo.com
Assist. Prof. Dr. Sana'a Noori Hussein
Tel.: ++9647714763199
E-mail: sanaa_kakei@yahoo.com
www.uomustensiriyah.edu.iq
rd@uomustansiriyah.edu.iq
1. Introduction
Lactic acid bacteria (LAB) have been studied extensively for bacteriocinogenicity, and numerous
bacteriocins are produced by LAB ([1], [2]). Lactobacilli that produce bacteriocins have been cultured
from naturally fermented dairy products, non-dairy fermentations (plant and meat), starter cultures, and
plant, animal, or human isolates. Among the numbers of LAB, the lactobacilli are composed of a diverse
group of homofermentative and heterofermentative species. They are most often cited for production of
bacteriocins [1]. Bacteriocins are antimicrobial proteinaceous compounds produced by bacteria [1].
Lactobacillus buchneri is a heterofermentative, facultative anaerobe that belongs to the lactic acid

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bacteria. Strains of L. buchneri have been described as having diverse activities, ranging from prevention
of silage spoilage by yeasts and molds ([3], [4], [5]) to histamine production in Swiss cheese [6]. Yildirim
and Yildirim [7] have described a bacteriocin, termed buchnericin LB produced by L. buchneri.
Buchnericin LB inhibited the growth of some species of Listeria, Bacillus, Micrococcus, Enterococcus,
Leuconostoc, Lactobacillus, Streptococcus and Pediococcus. In order to use buchnericin LB in the most
effective way, it is important to determine factors affecting its adsorption to indicator organisms. The
aims of this study were to determine factors affecting buchnericin LB adsorption to indicator bacteria
Vibrio parahaemolyticus.
2. Materials and Methods
2.1 Sample collection
Eleven yoghurt samples were collected from local markets in Baghdad to isolate L. buchneri. The
isolates were incubated in lactobacilli MRS broth as growth medium at 37 C for 24 h.
2.2 Identification of Lactobacillus buchneri
The properties of the isolates was investigated by testing Gram staining and microscopic observation after
cultivation on tryptic soy agar medium at 37 C for 24 h. Bergey's Manual of Systematic Bacteriology was
used to examine the morphological and physiological properties of the isolates [8]. The API 50 CHL-kit
(Bio-Merieux, France), an identification system for LAB was used to identify L. buchneri isolates.
2.3 Vibrio parahaemolyticus isolates
Six isolates of V. parahaemolyticus isolated from clinical sources were used in this study. All isolates
were confirmed with API 20E (Bio-Merieux, France). This system was used according to Bio-Merieux
company instruction.
2.4 Production of crude bacteriocin
MRS broth (Hi Media Laboratory Pvt. Ltd. India) pH 6.0 was seeded with 5% inoculum of L. buchneri
overnight culture and maintained anaerobically at 30 C for 48 h. After incubation, cells were removed
from the growth medium by centrifugation (10,000xg for 15 min. at 4 C). The cell-free supernatant was
adjusted to pH 6.0 using 1N NaOH and it was used as crude bacteriocin [9].
2.5 Purification of the bacteriocin
For adsorption of bacteriocin onto silicic acid (100 mesh), freeze-dried supernatants reconstituted with
150 ml of distilled water were fractionated into 10 ml. Bacteriocin preparations in 10-ml fractions were
adjusted to pH 2.0-9.0 with 5 M phosphoric acid or 5 M NaOH, and their volumes were brought up to 15
ml with distilled water. After silicic acid (2%) purification the bacteriocin produced by L. buchneri LB
was added into each sample. They were stirred overnight at 4 C and then centrifuged at 1,000 x g for 20
min. Bacteriocin adsorbed silicic acid was washed with sterile distilled water and re-suspended to the
original volume of 15 ml with 100 mM NaCl. In order to desorb the bound bacteriocin, the pH of the
silicic acid samples was adjusted to 2.0 with 5 M phosphoric acid. The samples were stirred for 2 h at 4 C
and heated at 80 C for 5 min. After centrifugation (1,000 x g for 20 min), the pH of the supernatants was
adjusted to 6.0 with 5 M NaOH, and bacteriocin activity was determined in all samples collected during
adsorption and desorption procedures ([10], [11]). After that, collected samples were prepared for cation
exchange chromatography for further purification.

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2.6 Cation-exchange chromatography (CEC)
The bacteriocin preparation (15 ml) was filter-sterilized (0.22 µm pore size). Then it was applied to a
Whatman carboxymethyl cellulose column CM-52 previously equilibrated with sodium phosphate buffer
(50 mM, pH 6.6). The column (30 cm x 2.5 cm) was washed with the same buffer, followed by a 500 ml
linear NaCl gradient (0-1 M) in sodium phosphate buffer. Fractions of 5 ml were collected at a flow rate
of 1.5 ml min-1, and monitored for absorption at 280 nm. For inhibitory activity against the indicator
isolate, V. parahaemolyticus, the agar well diffusion method was used. Fractions showing inhibitory
activity were pooled, dialyzed against distilled water and freeze dried ([10], [11]).
2.7 Detection of molecular weight
The molecular weight of bacteriocin was detected by gel filtration method on sephadexG-150 (0.75×100
cm) (Pharmacia Fine Chemical, Sweden) -amylase (Sigma, USA)
as reference proteins [12].
2.8 Effect of enzymes
Sensitivity of bacteriocin to different enzymes was determined using purified bacteriocin treated with the
following enzymes: pepsin, proteinase K, lipase -amylase and DNase (Sigma, USA) then boiled for 2
min. to inactivate the enzymes. After performing each treatment, bacteriocin was tested for antibacterial
activity against V. parahaemolyticus [13].
2.9 Effect of crude and purified bacteriocin on V. parahaemolyticus
Agar well diffusion method was used to detect antibacterial activity of the crude and purified bacteriocin
produced by L. buchneri against V. parahaemolyticus isolates at the concentrations of (1000 and 500)
µg/ml according to Batdorj et al. [14].
2.10 Statistical analysis
Results were expressed as the mean ± standard deviation (SD). The intergroup variation was assessed by
one way analysis of variance (ANOVA) P<0.01 using sigma state statistical software.
3. Results and Discussion
Out of 11 local yoghurt samples, only 3 isolates of L. buchneri were found and the isolate No. 3 was the
most producer of bacteriocin. Bacteriocin was adsorbed 100% onto silicic acid at pH 6.0-7.0 (Fig. 1).
Below or above these pH values, adsorption was decreased, ranging between 35 and 90%. Therefore, pH
6.0 was used for the purification procedure. Sixty-five percent of bacteriocin bound silicic acid was
desorbed at pH 2.0 with a combination of heat (80C for 5 min) and NaCl (100 mM). After the silicic acid
step, the specific activity increased to 105.92 (Au/mg) and purity of the bacteriocin increased 3.25 fold
(Table 1). Yildirim [15] reported that 85% of buchnericin LB bound silicic acid was desorbed at pH 2.0.
Janes et al. [16] reported that nisin, tetragenocin A and enterocin CS1 showed 90-94% desorption from
silicic acid, whereas pediocin RS2 showed only 50% desorption from silicic acid. After adsorption to
cation exchange chromatography (CEC), the bound bacteriocin was desorbed from the column by a linear
NaCl gradient, yielding a single peak of inhibitory activity.

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Figure (1): Effect of pH on adsorption of Lactobacillus buchneri bacteriocin to silicic acid
The molecular weight was determined to be about 3.4 kDa. The molecular size of buchnericin LB
produced by Yildirim and Yildirim [7] was about 3.5-4.5 kDa according to SDS-PAGE. The binding of
the bacteriocin to silicic acid and CEC is dependent on the pH. This indicated that L. buchneri bacteriocin
is a cationic molecule like other lactic acid bacteria bacteriocins ([10], [11], [17]). The purification
procedure including silicic acid adsorption/desorption and CEC resulted in a 11.11 fold increase in the
final specific activity 1176.47 Au/mg of pure bacteriocin compared to the culture supernatant which was
32.64 Au/mg (Table 1).
Table (1): Purification steps of Lactobacillus buchneri bacteriocin from isolate No. 3
Purification
stage
Volume(ml)
Total
activity(Au/ml)a
Protein
con.(mg/ml)b
Specific
activity(Au/mg)c
Purification
foldd
Recovery
(%)e
Culture
supernatant
100
7350
225.2
32.64
1
100
Silicic acid
25
2680
28.7
105.22
3.25
41.36
CEC
5
800
0.64
1176.47
11.11
26.32
a,b,c,d and e: These values calculated according to the international methods of protein activity measurement
The bacteriocin lost its activity completely after treatment with proteinase K and pepsin; however, it was
resistant to non-proteolytic enzymes such as lipase, - amylase and DNase (Table 2). These results
indicated that the inhibitory agent is a protein since protease sensitivity is a key criterion for the
characterization of a bacteriocin. After treatment with lipase and - amylase, bacteriocin did not lose its
inhibitory activity. These results showed that lipid and carbohydrate moieties were not responsible for its
antimicrobial activity.
0
20
40
60
80
100
120
2 3 4 5 6 7 8 9 10
Adsorption %
pH

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Table (2): Effect of enzymes on Lactobacillus buchneri bacteriocin activity
Enzymes
Activity %
Proteinase K
α-amylase
DNase
Pepsin
Lipase
0
100
100
0
100
The results indicated that bacterioin of both concentrations (500 and 1000) µg/ml possesses significant
antimicrobial activity against V. parahaemolyticus in contrast with control (P<0.01) and the antimicrobial
activity of crude and purified bacteriocin at the concentration of 1000 µg/ml was higher than that of the
other concentration (500 µg/ml). The antimicrobial activity of purified bacteriocin was significantly
higher than that of crude bacteriocin (P<0.01) as shown in (Table 3).
Table (3): Antibacterial activity of Lactobacillus buchneri bacteriocin against Vibrio parahaemolyticus
Treatment
Concentration
(µg/ml)
Inhibition zone (mm) Mean
±SD
Crude buchnericin LB
500
1000
17.44±1.95 a
21.98±0.63 a
Purified buchnericin LB
500
1000
24.35±1.66 a b
33.45±2.08 a b
Control D.W
0
0±0
a : Significant differences according to control ()
b : Significant differences according to control ()
4. Conclusions
Bacteriocin was adsorbed 100% onto silicic acid at pH 6.0-7.0. Below or above these pH values,
adsorption was decreased, ranging between 35 and 90%. Therefore, pH 6.0 was used for the purification
procedure. The molecular weight was determined to be about 3.4 kDa. The bacteriocin lost its activity
completely after treatment with proteolytic enzymes and it was resistant to non-proteolytic enzymes.
These results indicated that the bacteriocin is a protein. The antimicrobial activity of purified bacteriocin
was significantly higher than that of crude bacteriocin.
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