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Characterization and purification of a bacteriocin-like substanceproduced by Lactobacillus crispatus LBS 17-11 isolated from anoral cavity of human subject
Gaku Akimaru, DDS (1), Susumu Imai, PhD (2), Kaoru Sogabe, DMD, PhD (2), Ayako
Okada, DDS, PhD (2), Tomohiko Terai, PhD (3), Takekazu Okumura, PhD (3),Nobuhiro Hanada, DDS, PhD (2), and Hiroshi Kawahara, DDS, PhD (1)(1) Department of Dental Anesthesiology, (2) Department of Translational Research, Tsurumi
University School of Dental Medicine, Yokohama, and (3) Yakult Central Institute, MicrobiologicalResearch Department, Kunitachi, Japan
Purpose: Lactobacillus crispatus LBS 17-11 was isolated from an oral cavity of human subject, and possess
growth inhibition activity against Streptococcus pneumoniae. The objective of this study was to purify abacteriocin-like substance, and to determine its characteristic properties.
Materials and Methods: Antibacterial activities against S. pneumoniae ATCC 33400 during purification steps
and against several oral bacteria were determined by a radial diffusion assay. Purification procedure forbacteriocin-like substance was achieved with ammonium sulfate precipitation of culture supernatant, andsubsequent extraction with chloroform. Minimum inhibitory concentration (MIC) was determined by liquid
microdilution method.
Results: The culture supernatant of L. crispatus LBS 17-11 showed a bacteriostatic activity against S.
pneumoniae. This substance was partially purified up to 2045-fold specific activity (unit/mg protein). The
inhibitory activity was shown against only S. pneumoniae, but Gram negative species. The antimicrobial activity
was heat stable and partially lost by the treatment with proteases (trypsin and proteinase K). The molecular massof bacteriocin-like substance was estimated to be approximately 7 kDa based on tricine SDS-PAGE. Binary
combination of 1/2-MIC level of sodium lactate (56 mM) and the bacteriocin-like substance showed lower MIC
level compared to that of the addition of the bacteriocin-like substance alone.
Conclusion: Bacteriocin-like substance produced by L. crispatus LBS 17-11 showed bacteriostatic activity
against S. pneumoniae. The partially purified bacteriocin-like substance was heat stable and degraded byproteases. Molecular mass was estimated to be approximately 7 kDa.
(Asian Pac J Dent 2017; 17: 9-14.)
Key Words: bacteriocin, Lactobacillus crispatus, probiotics, Streptococcus pneumoniae

Introduction
Bacteriocin is natural antibacterial peptides ribosomally biosynthesized by bacteria. The proteinaceous nature ofbacteriocin is shown to be degraded easily, and not to remain in human body. Therefore, they are recognized as asafer conventional food preservation [1,2]. Various bacteriocins have been identified in Gram positive bacteria,
including lactic acid bacteria. They are classified into two groups, class I and class II, and subclasses based on its
structure [1,2]. Nisin is classified as a Type I lantibiotic and containing several unusual amino acids. Class IIbacteriocins, nonlantibiotic bacteriocins, are further divided into four subclasses. Class IIa shows anti-Listeria
activity. Class IIb bacteriocins consist of two peptides and Class IIc bacteriocins are circular peptide andremaining other bacteriocins are classified as Class IId. All bacteriocins shows heat stable nature.
Oral lactobacilli were previously believed to be the cariogenic microorganism because of their high numbers
in carious lesions and their acidogenic properties. Subsequent research has shown that they are associated withthe caries progression in dentine rather than with the caries initiation in the enamel [3,4]. Lactobacilli are also
believed to be important organisms for modification of the flora in the human body based on their ability tosuppress growth of pathogens [5]. One of the main mechanisms for Lactobacillus strain to inhibit growth ofpathogens is a production of antimicrobial substances, such as organic acid, hydrogen peroxide and bacteriocins[4, 6-10].

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In our laboratory, we have been investigating the bacteriocins isolated from oral microorganisms for theapplication to oral health, such as prevention of dental caries, periodontal diseases and infection of respiratoryorgans [11]. Several oral bacteria are thought to cause systemic diseases, such as bacteremia, endocarditis,
pneumonia and sepsis. Streptococcus pneumoniae is one of pathogens involved in upper respiratory tract
infections in humans [8,12,13]. Therefore, it is important to eliminate oral pathogens for health. In this study, weinvestigated the purification of bacteriocin-like substance produced by L. crispatus LBS 17-11, which wasisolated from an oral cavity of human subject, and determined its characteristic property of the bacteriocin-like
substance against S. pneumoniae.

Materials and Methods
Chemicals
Trypsin, Proteinase K and sodium lactate (50%) were purchased from Wako Pure Chemical Industry, Ltd.
(Osaka, Japan). Nisin was purchased from MP Biomedicals, LLC (Solon, OH, USA) and used as a comparative
control.
Bacterial strains and culture conditions
S. pneumoniae ATCC 33400, Streptococcus mutans ATCC 25175T, Streptococcus sobrinus ATCC 33478T,
Fusobacterium nucleatum JCM 8532T, Prevotella intermedia ATCC 25261T, and Porphyromonas gingivalis
ATCC 33277T were the stock culture collection of Tsurumi University (Yokohama, Japan). Each strain was
inoculated into Triptic-Soy broth (TSB; Becton, Dickinson & Co. Detroit, MI, USA) and incubated in anaerobox(Concept Mini, Ruskinn, Central Scientific Commerce, Inc., Tokyo, Japan) under an atmosphere comprising
N2/CO2/H2 (80:10:10) at 37˚C for 24-48 hours. P. gingivalis, P. intermedia, and F. nucleatum were incubated in
TSB supplemented with 1 µg/mL of menadione and 5 µg/mL of hemin and incubated at 37˚C for 48 hours underanaerobic condition.
Determination of bacteriocin-like activity
L. crispatus LBS 17-11 was cultured in Lactobacilli; MRS Broth (MRS; Becton, Dikinson Co.) for 24 hours.
The culture broth was then centrifuged at 1,710 × g for 30 minutes. Antibacterial activities against S.
pneumoniae ATCC 33400 during purification steps and against several oral bacteria were determined by a radial
diffusion assay. Aliquots (100 µL) of overnight culture (16 hours) of S. pneumoniae, as the indicator strains,
were mixed into 10 mL TS agar (1.0% agar, w/v) and poured into sterile Petri dishes (85 mm diameter). Aliquotsof fresh overnight cultures or concentrated fractions (5-10 µL) from different steps of purification were poured
into wells. Plates inoculated with S. pneumoniae were incubated for 16 hours at 37˚C, and then inhibition zones
were scaled.
Minimum inhibitory concentration (MIC) was determined by liquid microdilution method in accordance with
the Standard Method of the Japan Society of Chemotherapy (1990) with minor modification. A 20 µL of
two-fold serial dilution of samples was put into microtiter wells. Bacterial suspensions adjusted to absorbanceturbidity. Then, 200 µL of these bacterial suspensions was added to 20 mL of the assay media, 180 µL of
aliquots were put into microtiter plate wells. The culture was incubated anaerobically for 16-24 hours at 37˚C.
MIC was defined as the lowest concentration for inhibiting visible growth.
Purification of bacteriocin-like substance
One liter of the supernatant of L. crispatus LBS 17-11 was precipitated with 35-80% ammonium sulfate with10

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stirring. The precipitated proteins were collected by centrifugation of 1,710 × g at 4˚C for 40 minutes and thenresuspended in 10 mL of phosphate buffered saline (PBS; pH 7.0). The precipitated proteins were dialyzed twice
against one L of PBS for 24 hours using a dialysis bag. The dialyzed proteins were mixed violently with an equalvolume of chloroform and then centrifuged with 1,710 × g at 4˚C for 30 minutes. Remaining precipitatedproteins was also extracted an equal volume of chloroform. The total organic phase was evaporated using arotary evaporator.
Determination of protein concentration and tricine-sodium dodecyl sulfate polyacrylamide gelelectrophoresis (Tricine-SDS-PAGE)
Protein content was estimated using Takara BCA Protein Assay Kit (Takara Bio Inc., Kusatsu, Japan), according
to manufacturer’s instruction. Tricine-SDS PAGE were performed with 15% acrylamide gel (e-PAGEL, ATTO
Corp. Tokyo, Japan). The gel was cut into two parts; one was stained with EzStain (AE-1340 EzStain AQua,
ATTO Corp.), the other was rinsed in 0.1 % Triton X100 in sterile water for 30 minutes, followed by three times
rinsing in sterile water for 30 minutes, finally rinsed in PBS for 30 minutes. The gel was overlaid on test agar
plate include S. pneumoniae indicator, and incubated in anaerobic condition at 37˚C for 16 hours.
Characterization of bacteriocin-like substance
The heat stability and proteinase sensibility were investigated. The partially purified protein (120 µg/mL)
obtained by final step and proteinase K (1 mg/mL) or trypsin (1 mg/mL) in PBS were incubated at 37˚C for 3
hours. Remained activity was measured by the diameters of inhibition zone of a radial diffusion assay, andpercentage of remained activity was calculated by a standard curve on semi-logarithmic plot of diameter-dose
responses.

Results
Purification of the bacteriocin-like substance of L. crispatus LBS 17-11
Indicator strain was S. pneumoniae ATCC 33400 for purification of this substance. Bacteriocin-like activity was
found in the supernatant of overnight culture of L. crispatus LBS 17-11. The growth inhibition activity showed apeak in the supernatant at 16 hours bacterial culture and its activity was lasting for more 2 days (data not shown).
The strong antimicrobial activity was recovered in the precipitation of 35 to 80% of ammonium sulfate. The
recovery of protein from culture supernatant was 5.2%, and purification of antimicrobial activity was 15-foldincreased. The ammonium sulfate precipitated protein was dialyzed and further extracted with chloroform. The
purification of antimicrobial activity was up to 2045-fold increased, and its recovery of activity was 19% of
initial culture supernatant (Table 1).
Table 1 Purification stage and antimicrobial activity of L. crispatus LBS 17-11
Purification step
Culture supernatantAmmonium sulfate
precipitation (35-80%)
Chloroform extraction(unit/mL)3

Total
protein(mg)
7,751

Proteinrecovery
(%)
100

40

60

404

6

95

0.72

VolumeActivity

(mL)
1,000

1

Recoveryof activity
(%)
100

5.2

15

80

0.01

2,045

19

Purification(fold)

Characterization of the bacteriocin-like substance of L. crispatus LBS 17-11
Anti-microbial activity against S. pneumoniae was observed, but this activity was bacteriostatic. Slight visible

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bacterial cells were observed in a clear zone of a radial diffusion assay, and in the well of higher concentration
than MIC in a liquid microdilution assay. S. mutans, S. sobrinus, and Gram negative bacteria, such as P.
gingivalis, P. intermedia, and F. nucleatum were not sensitive to this substance using a radial diffusion assay.
The only one band was found to be molecular mass of approximately 7 kDa based on Tricine-SDS-PAGE (15%)
with a low range molecular weight standard. The Ez-stained band coincident with the bacteriostatic spot of agar
plates inoculated with S. pneumoniae seemed to be high purified bacteriocin-like substance (Fig. 1). The
antimicrobial activity was heat stable at 100˚C for 10 minutes. The characteristic of this substance showed
proteinaceous nature, because the activity was partially lost (60 or 55% of control) by the treatment of trypsin orproteinase K, respectively (Table 2).
Effect of combined use of the bacteriocin-like substance and sodium lactate
Because anti-microbial activity against S. pneumoniae was bacteriostatic, we determined the combination of thissubstance and sodium lactate, which is recognized as safe compound for food preservation. Nisin, which is abacteriocin produced by a group of Gram-positive bacteria that belongs to Lactococcus and Streptococcus
species, was used as comparative control. Addition of 1/2-MIC level of sodium lactate (56 mM) and thebacteriocin-like substance showed lower MIC level compared to that of the addition of the bacteriocin-like
substance alone. The combination effect of nisin and sodium lactate was not observed (Table 3).
Table 2 Effect of protease and heat treatment on partiallypurified bacteriocin-like activity
Treatment
Antimicrobial activity (%)
Control100
Trypsin 1 mg/mL, 3 hours
60
Proteinase K 1 mg/mL, 3 hours
55
100˚C, 10 minutes
100
Table 3 Effect of combination of sodium lactate with bacteriocinlike substance and nisin against S. pneumoniae
MIC
Sodium lactate
112 mM
Bacteriocin-like substance (BLS)
1.5 µg protein /mL
Nisin
0.63 µg protein /mL
BLS + Sodium lactate (56 mM)
0.375 µg protein /mL
Nisin + Sodium lactate (56 mM)
0.63 µg protein /mL
MIC was determined by liquid microdillution method against
S. pneumoniae ATCC 33400.
Fig. 1
Tricin-SDS PAGE analysis of partial purified bacteriocin-like substance from Lactobacillus crispatus LBS 17-11. Lanes: M,
molecular mass standard (ATTO AE-1440 EzStandard) with sizes given on the left. Lane 1: chloroform extracted bacteriocin-like
substance stained with EzStain. Lane 2: gel overlaid with 1% agar plate containing S. pneumoniae ATCC 33400.

Discussion
In this study, we reported on purification of the bacteriocin-like substance against S. pneumoniae produced by L.
crispatus LBS 17-11, which was isolated from an oral cavity of human subject. The definition of the bacteriocin
is an antimicrobial peptide and proteins ribosomally synthesized by bacteria. All bacteriocins are heat stable [1].
In the present study, the bacteriocin-like substance was heat stable, and showed proteinaceous nature, because
activity was partially lost by the treatment of trypsin or proteinase K. However, the stained band in the
Tricin-SDS-PAGE was disappeared gradually. Strangely, this substance did not show bactericidal activity, but

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show bacteriostatic activity against S. pneumoniae. The growth of the other oral bacteria, Gram positive bacteriasuch as S. mutans and S. sobrinus, and Gram negative bacteria, such as P. gingivalis, P. intermedia and F.
nucleatum were not inhibited. This property was similar to bacteriocin, but it was slightly different frompreviously described definition of bacteriocin. Therefore, we thought this substance was “bacteriocin-like” and“partial purification substance”.
In the purification procedure, the chloroform extraction of the ammonium precipitated protein increased theantimicrobial activity, suggesting a high degree of purification of this substance. It may be explained by thatsome bacteriocins appear in their native state as aggregates with other molecules such as lipid materials. These
aggregates may mask the antimicrobial activity partially. Such complexities can be eliminated by extraction withchloroform. Contreras et al. demonstrated that a simple one-step methanol-chloroform extraction could remove
most fatty acid contamination from the ammonium sulfate precipitated bacteriocin, which resulted in a purebacteriocin [14]. Wannun et al. also reported the purification of bacteriocin produced by oral Lactobacillus
paracasei SD1 by using the chloroform extraction method. In the present study, the bacteriocin-like substancewas extracted with chloroform, which indicated this substance has a hydrophobic character [9].
Generally, the bacterium is pH sensitive, and its growth rate decreased at acidic pH. Usually, the screening
method of probiotic candidate was performed after adjustment of culture supernatant with NaOH to eliminate abactericidal effect by organic acid. However, sodium lactate was potent growth inhibitor, as well as sodium
citrate against S. pneumoniae and several oral bacteria [15]. Moreover, the synergistic action of nisin and sodium
lactate on the growth of Listeria innocua and Shewanella putrefaciens were reported, previously [16].
S. pneumoniae is prevalent in the oral cavity and/or the upper respiratory tract and it is a leading cause ofpneumonia, meningitis and septicemia in humans [12]. The nature of bacteriostatic substance may not besuitable for the application for the prevention of pneumococcal diseases, directly. Ooshima et al, reported thatthe mutacin, which was the bacteriocin produced by S. mutans, completely killed the cells grown in sucrose-free
medium, whereas the bacteriocin did not kill the S. mutans cell grown in a sucrose-containing medium [17]. The
inhibitory mechanism of the bacterioci-like substance in this study may be different from that of other known
bacteriocins. Some papers described that bacteriocins may have a function as signaling peptides, either signalingother bacteria through quorum sensing and bacterial cross talk within microbial communities or signaling cellsof the host immune system [18,19].
The partial purification of bacteriocin-like substance enable the further purification and bacteriostatic
characterization may contribute to an analysis of oral bacterial ecosystems, and may be an alternative approachfor promoting oral health or prevention of oral diseases.
Acknowledgments
We thank staffs of the Department of Dental Anesthesiology and Department of Translational Research, School of Dental Medicine,
Tsurumi University for valuable advice and great assistance to complete this study. This study was supported in part by JSPS KAKENHI
Grant Number 15H06700.
Conflict of Interest
N. Hanada and S. Imai received research fund from Yakult Central Institute (Tokyo, Japan). All remaining authors declare no potential
conflicts of interest with respect to authorship and publication of this article.

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Correspondence to:
Dr. Susumu Imai
Department of Translational Research, Tsurumi University School of Dental Medicine,
2-1-3, Tsurumi, Tsurumi-ku, Yokohama 230-8501, Japan
Fax: +81-45-573-2473 E-mail: imai-s@tsurumi-u.ac.jp
Accepted December 9, 2016.
Copyright ©2017 by the Asian Pacific Journal of Dentistry.
Online ISSN 2185-3487, Print ISSN 2185-3479

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