Determination of the Antibacterial Constituents Produced by Lactobacilli against a Periodontal Pathogen: Sodium Lactate and a Low Molecular Weight Substance

Background and objectives: Probiotics are living bacteria which can improve the balance of microbiota. There are many recent studies on the effects of probiotics, including oral health promotion and the prevention of oral diseases. However, the mechanisms that underlie the activity of probiotic bacteria against periodontal pathogens have not been clearly elucidated. The purpose of this study was to examine the effects of lactic acid bacteria as probiotics in the prevention and treatment of periodontal disease. Material and Methods: The growth inhibitory effects of the culture supernatants of 50 strains of lactobacilli on the periodontal pathogen Porphyromonas gingivalis ATCC33277 were examined. To obtain a substance with antibacterial properties under neutral pH conditions, each culture supernatant was neutralized and purified by gel filtration column chromatography and reverse-phase HPLC. The molecular weight of purified substances was analyzed with LC-MS. Results: The results showed that two strains of Lactobacillus plantarum 122 (derived from the oral cavity) and L. fermentum ALAL020 (derived from fermented soy milk food products) had strong growth inhibition effects. The major antibacterial substance produced by L. plantarum 122 was thought to be sodium lactate. On the other hand, the molecular weight of the major antibacterial substance produced by L. fermentum ALAL020 we purified was 226.131 Da. An LC-MS analysis revealed that it had the following composition: C11H18O3N2. Conclusion: The antibacterial substance of L. plantarum 122 against P. gingivalis was sodium lactate, and that of L. fermentum ALAL020 we purified was a novel low molecular substance. This antibacterial substance has a possibility for using periodontal disease prevention.


Introduction
The oral cavity is a very complex ecosystem which harbors more than 700 bacterial species [1,2]. These bacteria form specific microbial flora in several different habitats, including the tooth surface, the gingival sulcus and the dorsum of the tongue. The onset and development of periodontitis are associated with various factors. The bacterial factor is specific Gram-negative anaerobic bacteria such as Porphyromonas gingivalis [3].
Periodontitis is a recurrent disease with acute and chronic phases. In general, non-surgical periodontal treatments such as scaling and root planning are used for the chronic phase of periodontitis. In the acute phase, systematic and topical antibiotic therapies are applied to reduce the causative bacteria and prevent recurrence. However, antibiotic therapy has some harmful side effects, including possible allergic reactions, and antibiotic-resistant bacteria may appear after the prolonged use of antibiotics [4][5][6]. To develop an alternative therapy, there has been a focus on the usefulness of Lactobacillus and Bifidobacteria as probiotics [7]. Probiotics are defined by Fuller as, "a live microbial feed supplement which beneficially affects the host by improving its intestinal microbial balance" [8]. The effects of probiotics in the prevention of gastrointestinal infections and the improvement of allergy symptoms have been reported [9][10][11]. Recently, in the dental field, there have been several attempts to examine the possibility that probiotics may act directly in the oral cavity to prevent oral diseases such as dental caries and periodontal diseases. A number of clinical studies have already reported promising findings [12][13][14]. However, the antibacterial mechanism of probiotic bacteria against periodontal pathogens has not been clearly elucidated. The purpose of this study was to identify new probiotic strains that exhibit antibacterial activity against the representative periodontal pathogen, P. gingivalis, and to identify the antibacterial substances produced by the strains that may be applied to the prevention or treatment of periodontal disease.

Material and Methods
The purification procedure of the antibacterial constituents against a periodontal pathogen produced by lactobacilli was shown in a supplemental file.

Screening of probiotic strains by antibacterial activity
Each lactic acid bacterial culture broth was centrifuged to obtain a culture supernatant. After adjusting the pH to 7.0 with 10N NaOH, the supernatant as test samples were serially diluted (two-fold) to 1/256 and P. gingivalis culture (10 7 CFU/ml) was inoculated. The growth inhibition was determined as the absorbance value below 0.1 at 620 nm. Antibacterial activity against P. gingivalis was detected by the dilution folds as Units.

No.
Strain/species Site of isolation Highest inhibitory dilution (Units)  Table 1: Antibacterial activity of lactobacilli expressed as highest inhibitory dilution.
-; Antibacterial activity was not detected.  After shaking an equal amount of ethyl acetate or acetone with the culture supernatants of each strains, the water soluble phase was applied to a Sephadex G-25 gel filtration column (100 mmφ × 350 mm) and eluted with deionized water (flow rate 5 ml/min). The total carbohydrate level (420 nm) of the peak fraction was detected using the phenol-sulfuric acid assay method [16]. The acetone supernatant of L. fermentum ALAL020 Sephadex G-25 fraction was analyzed by HPLC (JASCO Corporation, Tokyo, Japan) in a reverse-phase preparative ODS (C18) column (Wakoshil 5C18 AR Prep 20.0 mmφ × 250 mm), using gradients of (B) 50% acetonitrile in (A) 10% acetonitrile, both containing 0.05% trifluoroacetic acid: 10-30% of B over 20 min, in a 50% step-wise manner over 10 min, at a flow rate of 5 ml/min.

Determination of the antibacterial activities in the chromatography fractions of L. fermentum ALAL020.
After adjusting the pH to 7.0, 20 µl of each fractionated sample were aliquoted into a 96-well plate. Subsequently, 160 µl of GAM broth (Nissui, Tokyo, Japan) supplemented with 5 µg/ml hemin, 1 µg/ml menadione and 20 µl of the P. gingivalis culture (1 × 10 6 -10 7 CFU/ml) were added into the wells. The mixtures in the 96-well plates were anaerobically cultured at 37°C for 48 h. The cultures were measured at an absorbance of 620 nm with a microplate reader. The antibacterial activity was measured as the Units used in screening or by the sodium lactate equivalence (SLU). The SLU was determined by dividing (L) mg/ml by (C) mg/ml, where (L) was the minimum inhibitory concentration (MIC) of sodium lactate against P. gingivalis and (C) was the concentration of the fraction that inhibited P. gingivalis to below the detection limit. The detection limit was determined as the absorbance value below 0.1 at 620 nm. To determine the antibacterial activity, the reciprocal value of (C) was divided by the reciprocal value of (L), using the following formula:

Quantification of sodium lactate and an analysis of the amino acids
Sodium lactate was quantified using an HPLC NANOSPACE SI-1 system (Shiseido, Tokyo, Japan) on a CAPCELL PAC C18 TYPE MG column (1.5×250 mm; Shiseido, Tokyo, Japan) equilibrated with 50 mM NH 4 H 2 PO 4 -H 3 PO 4 solution (pH 4.2) at a flow rate of 100 µl/min. The absorbance at 210 nm was monitored.

The LC-MS analysis of the purified product
An HPLC-purified sample was analyzed using an Ultimate 3000 UHPLC System (Thermo Fisher Scientific, Inc., Waltham, MA, USA), and a Shim-pack VP-ODS column (150 mm × φ4.6 mm, Shimadzu, Tokyo, Japan). Gradient elution was performed using 10-50% acetonitrile with 0.05% formic acid. An MS analysis was performed with a Mass Spectrometry Q Exaction TM Quadrupole/Orbitrap hybrid mass spectrometer (Thermo Fisher Scientific). Ionization was performed using the ESI method and detected in the positive mode.

Screening of the probiotic strains
As shown in Table 1, two strains of lactobacilli out of 50 strains of lactic acid bacteria exhibited strong antibacterial activity against P. gingivalis: L. plantarum 122 (8 Units) and L. fermentum ALAL020 (16 Units). Other strains of the same species, showed only 2 Units. Therefore, the following analysis of the antibacterial constituents was performed with these two strains.
Fractionation of the L. plantarum 122 and L. fermentum ALAL020 culture supernatants.
The culture supernatant of L. plantarum 122 was separated on a Sephadex G-25 column into four fractions by absorbance at 260 nm; a substantial amount of carbohydrates was contained in fractions 3 and 4 ( Figure 1A). The culture supernatant of L. fermentum ALAL020 was separated into five fractions, however, the carbohydrate level was very low in comparison to L. plantarum 122 (Figure 2A).

The antibacterial activity of the L. plantarum 122 fractions
An antibacterial assay was performed. At the maximum dilution, there were 4 Units each in fractions 3 and 4 ( Figure 1B). The concentrations of sodium lactate in fractions 3 and 4 were 23.4% (w/w) and 41.7% (w/w), respectively ( Figure 1B). When the antibacterial activity of sodium lactate against P. gingivalis was measured in advance, the MIC of sodium lactate was 2.0% (20 mg/ml). The sodium lactate concentrations of fractions 3 and 4 were higher than the MIC of sodium lactate. The antibacterial activity of the L. fermentum ALAL020 fractions As shown in Figure 2B, fraction 3 showed the highest antibacterial activity of 1.82 SLU. The concentration of sodium lactate in fraction 3 was 1.3% (w/w) ( Figure 2B), which was less than the MIC of sodium lactate (2.0%). This result indicated that L. fermentum ALAL020 might have some active substances other than sodium lactate. Therefore, the fractions were purified and the following analyses were performed.
Four volumes of acetone were added to the fraction 3 solution and the antibacterial activity of the acetone supernatant and precipitate was determined, revealing that the antibacterial activity of the acetone supernatant was as strong as 8.0 SLU ( Figure 2C). The culture supernatant was then subjected to acetone precipitation for further experiments and the supernatant was separated using a Sephadex G-25 column. One peak fraction was obtained by this procedure ( Figure  3A). This peak fraction was subsequently separated in the ODS (C18) column by reverse-phase HPLC and divided into eight fractions: H1-  Figure 3B). H8-1 showed the strongest antibacterial activity against P. gingivalis (5.0 SLU), followed by H8-2 (3.1 SLU) ( Figure 3C). The LC-MS analysis of the antibacterial constituents of L. fermentum ALAL020 An amino acid analysis of H8-1 and H8-2 was performed, however, no apparent peak was detected (data not shown). We therefore performed an LC-MS analysis. H8-1 and H8-2 were subjected to precision mass spectrometry. The molecular weights of both were estimated to be 226.131 Da ( Figure 4A, 4B), with a molecular formula of C 11 H 18 O 3 N 2 .

Discussion
P. gingivalis is the most important periodontal pathogen [17][18][19] because it is frequently isolated from patients with chronic periodontal disease. Vivekananda et al. [13] reported that L. reuteri DSM 17938 and L. reuteri ATCC PTA 5289, which we also used in our screening study, reduced the number of P. gingivalis in the oral cavity. In addition, Ishikawa et al. [20] reported that L. salivarius LS1 reduced the number of P. gingivalis cells in vitro. In the present study, L. plantarum 122 and L. fermentum ALAL020 showed stronger antibacterial activities than these other lactobacilli at a neutral pH.  The sterilization or antibacterial substances produced by lactic acid bacteria include organic acids such as lactic and acetic acids, hydrogen peroxide and bacteriocins [21][22][23]. Kang et al. [24] reported that L. reuteri showed antibacterial activity against oral pathogens including P. gingivalis, but it was decreased when the pH was neutralized. Thus, they concluded that the antibacterial substance of L. reuteri was lactic acid. Furthermore, Takahashi et al. [25] reported that P. gingivalis proliferation was inhibited at a pH ≤ 6.5. However, it is possible that low pH conditions in the oral cavity may induce dental caries or hypersensitivity. In this study, the antibacterial test against P. gingivalis was performed after the pH neutralized. Despite the neutral pH of the L. plantarum 122 and L. fermentum ALAL020 culture supernatants, both strains strongly inhibited the growth of P. gingivalis. These results indicate that sodium lactate, a neutralized form of lactic acid, also has antibacterial properties. This result is in line with the findings of Matsuoka et al. [26]. The antibacterial fraction of L. plantarum 122 contained more than 2% sodium lactate (MIC of sodium lactate against P. gingivalis). The presence of sodium lactate strongly contributed to the antibacterial activity of L. plantarum 122.
As for L. fermentum ALAL020, strong antibacterial activity was observed in the ethyl acetate or acetone supernatant of fraction 3 ( Figure 2B, 2C), but the concentration of sodium lactate was only 1.3% ( Figure 2B). Therefore, fraction 3 was divided in HPLC ( Figure 3B) to obtain purified active fractions and analyzed by LC-MS (Figure 4). In L. fermentum, bacteriocin-like substances have been reported [27][28][29].
However, the antibacterial substances obtained from H8-1 and H8-2 in the present study showed a different molecular weight (226.131 Da, Figure 4) from bacteriocin-like proteins and the following composition formula: C 11 H 18 O 3 N 2 . The substances from H8-1 and H8-2 showed different behaviors in the LC, and were divided into two proximal peaks; we speculate that the substance was a structural isomer. An amino acid analysis revealed that this substance did not contain amino acids, and it was confirmed with FT-IR (data not shown). Reuterin, an antibacterial substance in L. reuteri (also known as 3hydroxypropionaldehyde; molecular weight, 74 Da; composition formula, C 3 H 6 O 2 ) also has a low molecular weight and does not contain amino acids [30,31]. Because the molecular weight and formula of our isolated substance were different from those of reuterin, the isolated substance may be a novel antibacterial substance. In our preliminary experiment, L. fermentum ALAL020 was less effective against Prevotella intermedia or Fusobacterium nucleatum (date not shown), but highly effective against P. gingivalis. These results indicate the possibility that it may specifically target P. gingivalis. According to these results, it is necessary to analyze the mechanisms that underlie its antibacterial activity.
In conclusion, an antibacterial substance which exhibited activity against the periodontal pathogen P. gingivalis was isolated and identified from L. plantarum 122 and L. fermentum ALAL020. It was hypothesized that the major antibacterial substance produced by L. plantarum 122 was lactic acid. The antibacterial substance produced by L. fermentum ALAL020 we purified was a novel low molecular substance active in neutral pH. This substance might be useful in dental treatment and for the prevention of periodontal diseases. A further structural analysis of this novel substance is necessary to elucidate its mechanisms before its use in the clinical setting.