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Antibacterial and anticavity activity of probiotic Lactobacillus plantarum 200661 isolated from fermented foods against Streptococcus mutans
LWT - Food Science and Technology xxx (xxxx) xxxx
Contents lists available at ScienceDirect
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Antibacterial and anticavity activity of probiotic Lactobacillus plantarum 200661 isolated from fermented foods against Streptococcus mutans Sung-Min Lim, Na-Kyoung Lee, Hyun-Dong Paik∗ Department of Food Science and Biotechnology of Animal Resources, Konkuk University, Seoul, 05029, Republic of Korea
A R T I C LE I N FO
A B S T R A C T
Keywords: Probiotics Streptococcus mutans Anticavity activity Antimicrobial effect Antibiofilm effect
This study investigated the characteristics of Lactobacillus strains as an oral probiotic. Lactobacillus delbrueckii 200170 and L. plantarum 200661 were isolated from raw crab marinated in soy sauce and fermented tomato kimchi, respectively. L. rhamnosus GG was used as control. L. rhamnosus GG and L. plantarum 200661 survived in gastrointestinal conditions. They could stably produce enzymes and transfer antibiotic resistance. The antibacterial activity of L. rhamnosus GG, L. delbrueckii 200170, and L. plantarum 200661 inhibited three Streptococcus mutans strains. L. plantarum 200661 showed the highest antibacterial activity and inhibited S. mutans biofilm formation by 71.8–94.79%. L. plantarum 200661 inhibited glucan formation by 58.4%. L. plantarum 200661 could also inhibit the expression levels of biofilm formation and glucan related genes in S. mutans strains. Therefore, L. plantarum 200661 can be used as a biofilm inhibitor and as an oral probiotic in functional foods.
1. Introduction
Rosalen, Cury, Park, & Bowen, 2002). Recent studies have shown that probiotics play a positive role in oral health. Lactobacillus rhamnosus GG, Lactobacillus plantarum, and Lactobacillus reuteri inhibited biofilm formation by S. mutans (Amez, López, Devesa, Montero, & Salas, 2017; Söderling, Marttinen, & Haukioja, 2011). Chewing gum containing probiotics and xylitol reportedly have preventive effects against plague and gingival score (Kaur et al., 2018). Lactobacillus casei, L. plantarum ST-III, and Lactobacillus paracasei LPC27 could reduce cell growth and biofilm formation of S. mutans (Lin, Chen, Tu, Wang, & Chen, 2017). The antibacterial components produced by Lactobacillus sp. included bacteriocin or bacteriocin-like substances, lactic acid, and hydrogen peroxide (Shanker & Federle, 2017). Most of reports focused antimicrobial and anticavity activity. Therefore, this study aimed to confirm the probiotic potential having oral therapy. Lactic acid bacteria (LAB) isolated from fermented foods was investigated probiotic characteristics included acid and bile salt tolerance. In addition, oral therapy was determined using antimicrobial, anticavity, and antibiofilm activities against S. mutans.
Probiotics are live bacteria that can modulate the intestinal microflora when ingested in adequate amounts by the host (Lee, Kim, Han, Eom, & Paik, 2014). The characterization of probiotics has shown their immune modulatory, anticancer, antioxidant, and antimicrobial effects (Lee et al., 2015; Yang et al., 2019). Probiotics can act as carriers in various functional foods, such as yogurt, cheese, cookies, and chewing gum (Kaur, Nekkanti, Madiyal, & Choudhary, 2018; Taha et al., 2017). Dental caries is an infectious disease and a public health problem among children (Coqueiro, Bonviin, Raizel, Tirapegui, & Rogero, 2018). Cariogenic bacteria such as Streptococcus mutans, Streptococcus sobrinus, and Lactobacillus sp. play important roles in the pathogenesis of caries (Balakrishnan, Simmonds, & Tagg, 2000). Among the various bacteria producing biofilms, S. mutans is the main species responsible for causing the carious lesions. S. mutans produces glucosyltransferases (GTFs), which can synthesize intracellular polysaccharides (IPS) as well as extracellular polysaccharides (EPS). Among the EPS, water-insoluble glucans mediate the initial stage of adherence of oral bacteria onto the tooth surfaces and facilitate the formation of mature dental plaque (Beloin & Ghigo, 2005; Leme, Koo, Bellato, Bedi, & Cury, 2006). Although various antibacterial compounds such as chlorhexidine, ampicillin, spiramycin, and vancomycin have been effective in preventing dental caries, they can cause unexpected side effects (Koo,
∗
2. Materials and methods 2.1. Bacterial strains and sample preparation L. delbrueckii 200170 and L. plantarum 200661 were isolated using
Corresponding author. E-mail address: [email protected] (H.-D. Paik).
https://doi.org/10.1016/j.lwt.2019.108840 Received 7 August 2019; Received in revised form 6 November 2019; Accepted 10 November 2019 0023-6438/ © 2019 Published by Elsevier Ltd.
Please cite this article as: Sung-Min Lim, Na-Kyoung Lee and Hyun-Dong Paik, LWT - Food Science and Technology, https://doi.org/10.1016/j.lwt.2019.108840
LWT - Food Science and Technology xxx (xxxx) xxxx
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Table 1 Probiotic characteristics of LAB strains. Characteristics Initial cell count (log CFU/mL) Gastric conditions (log CFU/mL) Bile salt (log CFU/mL) β-Glucuronidase Adhesion ability (%)
L. rhamnosus GG
L. delbrueckii 200170
a
7.45 5.42 6.47 ND 1.34
8.70 ± 0.15 8.61 ± 0.02a 8.78 ± 0.09a ND 6.3 ± 1.21b
b
± 0.02 ± 0.09c ± 0.21c ± 0.35c
L. plantarum 200661 7.67 ± 0.08b 7.61 ± 0.02b 8.12 ± 0.02b ND 13.24 ± 1.63a
The results are represented as mean ± standard deviation. ND, not detected. a-c Values with different letters in the same row column are significantly different (p < 0.05). Table 2 Antimicrobial activity of the LAB cell free supernatant against S. mutans strains. Oral pathogens
S. mutans KCTC 5124 S. mutans KCTC 5458 S. mutans KCTC 5316
Minimum inhibitory concentration (%) L. rhamnosus GG
L. delbrueckii 200170
L. plantarum 200661
12.5 ± 0.0b 25.0 ± 0.0b 25.0 ± 0.0b
6.25 ± 0.0a 25.0 ± 0.0b 25.0 ± 0.0b
6.25 ± 0.0a 12.5 ± 0.0a 12.5 ± 0.0a
Data are represented as the mean ± standard deviation of triplicate experiments. Sodium ampicillin was used as control and it was serially diluted from 500 μg/mL to 7.8 μg/mL. All S. mutans strains were effectively inhibited at 7.8 μg/mL. a-b Values with different letters in the same row are significantly different (p < 0.05).
Lactobacillus selective medium (BD Biosciences, Franklin Lakes, NJ, USA) from raw crab marinated in soy sauce and fermented tomato kimchi, respectively. These strains identified 16S rRNA sequencing. Lactobacillus rhamnosus GG (Cell Biotech., Ltd., Gimpo, Korea) was obtained commercially. Lactobacillus strains were propagated and maintained in lactobacilli MRS medium (BD Biosciences) at 37 °C. They were cultured in MRS broth for 24 h at 37 °C and centrifuged (12,000×g for 10 min at 4 °C) to obtain the supernatant, which was filtered and used as samples. Streptococcus mutans KCTC 5124, KCTC 5458, and KCTC 5316 were obtained from the Korean Collection for Type Culture (Daejeon, Korea). S. mutans strains were cultured in BHI broth (BD Biosciences) for 24 h at 37 °C and used as oral pathogens.
Adhesion ability (%) = {adhered cell no. (CFU)/initial cell no. (CFU)} × 100 The antibiotic resistance of LAB strains was determined according to Clinical and Laboratory Standards Institute (CLSI) guideline by using the disc diffusion method. One-hundred microliter of each LAB stains were spread on MRS agar and then placed paper discs containing antibiotics. The inhibition zone was measured after incubation at 37 °C for 24 h. 2.3. Antimicrobial effect of LAB against S. mutans strains The minimum inhibitory concentration (MIC) of samples was measured against S. mutans using 96 well plates. Filter-sterilized LAB supernatants were serial diluted by two-fold using BHI broth ranging from 100% to 0.78%. Then, S. mutans (1 × 105 CFU/mL) and diluted samples were added into each well. The lowest sample concentration that inhibited 99% of the inoculum was considered as the MIC. Sodium ampicillin used as control was diluted from 500 μg/mL to 7.8 μg/mL.
2.2. Probiotic characterization of LAB The artificial gastric acid and bile acid consisted of 0.3% pepsin in pH 2.5 and 3% oxgall in pH 7, respectively. Overnight cultures of each LAB strains were resuspended in artificial gastric acid and bile acid conditions and incubated for 3 h and for 24 h at 37 °C, respectively, to confirm the tolerance. The viable cells were counted by dilution and plating on MRS medium after each reaction.
2.4. Measurement of biofilm formation using crystal violet staining Biofilm formation was assayed using the method described by Wu et al. (2015) with modifications. S. mutans (1 × 106 CFU/mL) was cultured at 37 °C for 24 h in the presence or absence of sample in BHI broth supplemented with 3% sucrose in a 24 well plate, and the sample was inoculated into each well. After incubation, the planktonic bacteria were removed by gentle washing with distilled water, and biofilms were stained with 0.1% crystal violet solution for 10 min at room temperature. The plates were rinsed with distilled water and then, the adhered dye was dissolved with acid-alcohol solutions. The absorbance was measured at 540 nm and biofilm inhibition rate was calculated as
Survival rate (%) = {cell no. after reaction (CFU)/initial cell no. (CFU)} × 100 Enzyme production was measured using API ZYM kit (BioMerieux, Lyon, France). Each LAB strain was suspended in saline and inoculated into each cupule. The inoculated strips were incubated at 37 °C for 4 h. One drop of each ZYM A and ZYM B reagent was serially added into each cupule and the results were measured based on the intensity of the color. To measure the adhesion ability to intestinal epithelial cells, HT29 cells were plated at 2 × 105 cells/well in 24-well plate and incubated at 37 °C for 24 h. Approximately 1 × 108 CFUs/well of LAB strain was added to the each well with HT-29 cells monolayer. The plate was incubated at 37 °C for 2 h in 5% CO2 incubator and non-adherent cells were removed by four washes with PBS. Further, 1 mL of 0.1% Triton X-100 was added to each well, and the cells were harvested. The samples were diluted ten-fold, spread onto solid plates, and incubated at 37 °C for 24 h. Adhesion ability was calculated as
Biofilm inhibition rate (%) = {1- (absorbance of sample)/(absorbance of control)} × 100
2.5. Inhibition of water-insoluble glucan synthesis The inhibition of water-insoluble glucan was evaluated using the method previously described by Koo et al. (2002). 2
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Fig. 1. Inhibitory effect of the culture medium and supernatant of LAB strains on S. mutans biofilm formation. (A) S. mutans KCTC 5124, (B) S. mutans KCTC 5458, (C) S. mutans KCTC 5316. The different superscript letters on each bar in the same LAB strain represent significant difference between values (p < 0.05). □, 100 μL; , 200 μL; , 500 μL; ■, 1000 μL.
supernatant, and 800 μL of 62.5 mM potassium phosphate buffer (pH 6.5) with 12.5 g of sucrose and 0.25 g of sodium azide was used. This mixture was incubated at 37 °C for 30 h. After incubation, the unsolidified sample was removed, the contents adhering to the tube wall were washed with sterile water and dispersed by sonication. The total amount of water-insoluble glucan was measured at an absorbance of
To obtain the crude enzyme, S. mutans was incubated in BHI broth at 37 °C for 24 h, centrifuged at 4 °C for 15 min and filtered using a 0.2 μm membrane filter. Then, the cell-free supernatants were extracted using Amicon ultra centrifugal filters (MWCO 30 kDa, Millipore, USA) and upper retentate was used as the crude enzyme sample. A reaction mixture containing 20 μL of crude enzyme, 180 μL of LAB 3
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glucuronidase, shown using the API ZYM kit. The adhesion ability of LAB to intestinal cells reportedly influences its colonization in the host, immunomodulatory effects, and prevention of pathogen infection (Garcia-Cayuela et al., 2014). L. plantarum 200661 shows higher adhesion ability (13.24%) compared to L. rhamnosus GG (6.3%). This shows that L. plantarum 200661 can attach and colonize in intestinal epithelial cells better than commercial strains. While the LAB strains were sensitive to ampicillin, tetracycline, chloramphenicol, and doxycycline, they were resistant to gentamycin, kanamycin, streptomycin, and ciprofloxacin (data not shown). These results suggest that L. plantarum 200661 strain was stable in gastric conditions, is antibiotic resistant, and does not produce any hazardous enzymes.
Table 3 Inhibitory effect of supernatant of lactic acid bacteria on the synthesis of waterinsoluble glucan by the GTFs from S. mutans. Oral pathogens
S. mutans KCTC 5124 S. mutans KCTC 5458 S. mutans KCTC 5316
GTFs inhibition (%) L. rhamnosus GG
L. plantarum 200661
37.24 ± 2.25a 26.42 ± 2.13a 15.38 ± 6.61a
47.03 ± 2.57b 24.81 ± 0.82a 26.24 ± 2.59b
Data are represented as the mean ± standard deviation of triplicate experiments. a-b Values with different letters in the same row are significantly different (p < 0.05).
3.2. Antimicrobial activity against S. mutans
550 nm and the GTFs inhibition rate was calculated as
The virulence of S. mutans is due to their ability to survive in acidic pH and produce biofilms (Krzyściak, Jurczak, Kościelniak, Bystrowska, & Skalniak, 2014). The antimicrobial activities of LAB were investigated against S. mutans strains (Table 2). Sodium ampicillin was used as control and its MIC was < 7.8 μg/mL for all S. mutans. The MIC of L. rhamnosus GG were 12.5%, 25%, and 25% against S. mutans KCTC 5124, KCTC 5458, and KCTC 5316, respectively. The MIC of L. plantarum 200661 were 6.25%, 12.5%, and 12.5%, respectively. L. plantarum 200661 showed good antimicrobial activity against all three S. mutans strains. These effects could be due to the antimicrobial substances produced including organic acids, bacteriocin, and biosurfactants (Lin et al., 2017).
GTFs inhibition rate (%) = {1- (absorbance of treated sample)/(absorbance of control)} × 100
2.6. RNA extraction and semi-quantitative real-time PCR S. mutans (1 × 105 CFU/mL) was grown in 10 mL of BHI broth at 37 °C for 24 h and then, 1 mL of LAB supernatant was added into it followed by anaerobic incubation at 37 °C for 12 h. RNA extraction was performed using Trizol® Max™ bacterial RNA isolation kit (Thermo Fisher Scientific, Waltham, MA, USA), according to manufacturer's protocol. The RNA quality was quantified using Multiscan GO (Thermo Fisher Scientific). The cDNA was synthesized using Revertaid first strand cDNA synthesis kit (Thermo Fisher Scientific). Semi-quantitative real-time PCR was performed using the SYBR Green PCR Master Mix in the PikoReal 96 system (Thermo Fisher Scientific). The reaction mixture contained SYBR Green master mix, primer, cDNA, and RNase free water. Further, 20 μL of the mixture was amplified as follows: 95 °C for 10 min as initial denaturation, following by 40 cycles of 95 °C for 15 s as denaturation and 60 °C for 45 s as annealing and extension. The analysis was done using the melt curve analysis method to determine the reaction. The primer used for this was sucrose phosphorylase gene (gtfA), GTF B gene (gtfB), GTF-I gene (gtfD), fructosyltransferase gene (ftf), histidine kinase two-component regulatory system gene (vicR), competence-stimulating system gene (comDE), and biofilm-regulation protein gene (brpA) (Shemesh, Tam, & Steinberg, 2007; Steinberg, Moreinos, Featherstone, Shemesh, & Feuerstein, 2008).
3.3. Inhibition of biofilm formation While both the culture medium and the supernatant of LAB could inhibit the S. mutans biofilm formation, the supernatant showed higher inhibitory effects (Fig. 1). L. rhamnosus GG inhibited the S. mutans biofilm formation by over 50% at 200 μL while L. delbrueckii 200170 inhibited it at 1000 μL. L. plantarum 200661 inhibited the same by over 60% at 100 μL, making it the strongest inhibitor. Therefore, it was confirmed that L. plantarum 200661 could inhibit the S. mutans biofilm formation at low doses. This mechanism has been reported to be due to the co-aggregation with LAB resulting in physical interference (Wu et al., 2015) and induction of EPS production (Ahn, Baik, Park, Yun, & Han, 2018). 3.4. Inhibition of synthesis of water insoluble glucan
3. Results and discussion
Glucan is an intermediate between S. mutans and tooth surfaces, and it also promotes the cell to cell adhesion, structural integrity and stability of biofilms (Leme et al., 2006). The LAB supernatants could inhibit water-insoluble glucan synthesis by the crude GTFs from S. mutans (Table 3). L. rhamnosus GG inhibited the water insoluble glucan formation in S. mutans KCTC 5124, KCTC 5458, and KCTC 5316 by 37.24%, 26.42%, and 15.38%, respectively. L. plantarum 200661 was more effective than L. rhamnosus GG on S. mutans KCTC 5124 and KCTC 5316. This result showed that the supernatant of L. plantarum 200661 can effectively inhibit GTFs.
3.1. Probiotic characterization of the LAB strains
3.5. Expression of genes involved in the virulence of S. mutans
Probiotic strains should be tolerant toward intestinal conditions in order to survive in intestinal cells. Under gastric conditions, the survival rate of L. rhamnosus GG, L. delbrueckii 200170, and L. plantarum 200661 were 81.28%, 0.93%, and 87.1%, respectively (Table 1). However, in the presence of bile acids, the survival rate of L. rhamnosus GG and L. plantarum 200661 was > 100% except for L. delbrueckii 200170 (10.47%). Enzyme production was important in use of probiotics, βglucuronidase could be induced cancer substances in liver and colon (Son et al., 2018). All three LAB strains did not produce β-
To evaluate the effect of L. rhamnosus GG and L. plantarum 200661 supernatants on S. mutans, the expression of the genes involved in the virulence of S. mutans were investigated (Fig. 2). The representative genes involved in glucan formation are gtfA, gtfB, and gtfD, and in fructation formation was ftf (Shemesh et al., 2007). In addition, the signal transduction system was controlled by the comDE, vicR, relA, and brpA genes. L. rhamnosus GG and L. plantarum 200661 down-regulated the genes responsible for EPS formation in all S. mutans strains except for brpA in
2.7. Statistical analysis All the tested data are represented as mean and standard deviation of three replicates. One-way analysis of variance (ANOVA) was used to determine the significant differences. The mean values were used for the Duncan's multiple range test to perform post hoc verification (p < 0.05).
4
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Fig. 2. Relative gene expression associated with biofilm formation in S. mutans strains as determined by RT-PCR. (A) S. mutans KCTC 5124, (B) S. mutans KCTC 5458, (C) S. mutans KCTC 5316. The different superscript letters on each bar in the same gene represent significant difference between values (p < 0.05). □, L. rhamnosus GG; ■, L. plantarum 200661.
inhibitory effects on the gene expression depends on the species of S. mutans. Moreover, the LAB supernatant showed lower inhibitory effect on the expression of genes involved in EPS formation than the signal transduction genes. Therefore, these results suggest that disrupting the synthesis of insoluble glucans can reduce the biofilm formation, which
S. mutans KCTC 5124 and relA in S. mutans KCTC 5316. In S. mutans KCTC 5458, L. rhamnosus GG and L. plantarum 200661 down-regulated all genes compared to control (< 1-fold change). In addition, L. plantarum 200661 compared to L. rhamnosus GG showed significantly lower gene expression in S. mutans KCTC 5316. The difference in the
5
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can influence the pathogenesis. In S. mutans, the biosurfactants produced by L. casei inhibited the expression of ftf and gtfB genes, while L. acidophilus inhibited the gtfB and gtfC gene expressions (Savabi et al., 2014; Tahmourespour, Salehi, & Kermanshahi, 2011).
trial. Journal of International Oral Health, 10, 237–243. Koo, H., Rosalen, P. L., Cury, J. A., Park, Y. K., & Bowen, W. H. (2002). Effects of compounds found in propolis on Streptococcus mutans growth and on glucosyltransferase activity. Antimicrobial Agents and Chemotherapy, 46, 1302–1309. Krzyściak, W., Jurczak, A., Kościelniak, D., Bystrowska, B., & Skalniak, A. (2014). The virulence of Streptococcus mutans and the ability to form biofilms. European Journal of Clinical Microbiology & Infectious Diseases, 33, 499–515. Lee, N. K., Kim, S. Y., Han, K. J., Eom, S. J., & Paik, H. D. (2014). Probiotic potential of Lactobacillus strains with anti-allergic effects from kimchi for yogurt starters. LWT – Food Science and Technology, 58, 130–134. Lee, N. K., Son, S. H., Jeon, E. B., Jung, G. H., Lee, J. Y., & Paik, H. D. (2015). The prophylactic effect of probiotic Bacillus polyfermenticus KU3 against cancer cells. Journal of Functional Foods, 14, 513–518. Leme, A. P., Koo, H., Bellato, C. M., Bedi, G., & Cury, J. A. (2006). The role of sucrose in cariogenic dental biofilm formation—new insight. Journal of Dental Research, 85, 878–887. Lin, X., Chen, X., Tu, Y., Wang, S., & Chen, H. (2017). Effect of probiotic lactobacilli on the growth of Streptococcus mutans and multispecies biofilms isolated from children with active caries. Medical Science Monitor, 23, 4175–4781. Savabi, O., Kazemi, M., Kamali, S., Salehi, A. R., Eslami, G., Tahmourespour, A., et al. (2014). Effects of biosurfactant produced by Lactobacillus casei on gtfB, gtfC, and ftf gene expression level in S. mutans by real-time RT-PCR. Advanced Biomedical Research, 3, 231. Shanker, E., & Federle, M. (2017). Quorum sensing regulation of competence and bacteriocins in Streptococcus pneumoniae and mutans. Genes, 8, 15. Shemesh, M., Tam, A., & Steinberg, D. (2007). Differential gene expression profiling of Streptococcus mutans cultured under biofilm and planktonic conditions. Microbiology, 153, 1307–1317. Söderling, E. M., Marttinen, A. M., & Haukioja, A. L. (2011). Probiotic lactobacilli interfere with Streptococcus mutans biofilm formation in vitro. Current Microbiology, 62, 618–622. Son, S. H., Jeon, H. L., Yang, S. J., Sim, M. H., Kim, Y. J., Lee, N. K., et al. (2018). Probiotic lactic acid bacteria isolated from traditional Korean fermented foods based on β-glucosidase activity. Food Science and Biotechnology, 27, 123–129. Steinberg, D., Moreinos, D., Featherstone, J., Shemesh, M., & Feuerstein, O. (2008). Genetic and physiological effects of noncoherent visible light combined with hydrogen peroxide on Streptococcus mutans in biofilm. Antimicrobial Agents and Chemotherapy, 52, 2626–2631. Taha, S., El Abd, M., De Gobba, C., Abdel-Hamid, M., Khalil, E., & Hassan, D. (2017). Antioxidant and antibacterial activities of bioactive peptides in buffalo's yoghurt fermented with different starter cultures. Food Science and Biotechnology, 26, 1325–1332. Tahmourespour, A., Salehi, R., & Kermanshahi, R. K. (2011). Lactobacillus acidophilusderived biosurfactant effect on gtfB and gtfC expression level in Streptococcus mutans biofilm cells. Brazilian Journal of Microbiology, 42, 330–339. Wu, C. C., Lin, C. T., Wu, C. Y., Peng, W. S., Lee, M. J., & Tsai, Y. C. (2015). Inhibitory effect of Lactobacillus salivarius on Streptococcus mutans biofilm formation. Molecular Oral Microbiology, 30, 16–26. Yang, S. J., Lee, J. E., Lim, S. M., Kim, Y. J., Lee, N. K., & Paik, H. D. (2019). Antioxidant and immune-enhancing effects of probiotic Lactobacillus plantarum 200655 isolated from kimchi. Food Science and Biotechnology, 28, 491–499.
4. Conclusions L. plantarum 200661 is stable under gastric conditions and can adhere to intestinal walls. This strain was safe in enzyme production and antibiotic resistance. This strain also exerts antibacterial and antibiofilm activity against S. mutans by inhibiting the formation of water insoluble glucan and by downregulating the expression of related genes. Therefore, L. plantarum 200661 can be used as a probiotic as well as an inhibitor of biofilm formation of several oral pathogen strains in functional foods. Declaration of competing interest All authors declare no conflict of interest. Acknowledgements This paper was supported by Konkuk University in 2016. References Ahn, K. B., Baik, J. E., Park, O. J., Yun, C. H., & Han, S. H. (2018). Lactobacillus plantarum lipoteichoic acid inhibits biofilm formation of Streptococcus mutans. PLoS One, 13, e0192694. Amez, M. S., López, J. L., Devesa, A. E., Montero, R. A., & Salas, E. J. (2017). Probiotics and oral health: A systematic review. Medicina Oral, Patología Oral Y Cirugía Bucal, 22, 19. Balakrishnan, M., Simmonds, R. S., & Tagg, J. R. (2000). Dental caries is a preventable infectious disease. Australian Dental Journal, 45, 235–245. Beloin, C., & Ghigo, J. M. (2005). Finding gene-expression patterns in bacterial biofilms. Trends in Microbiology, 13, 16–19. Coqueiro, A. Y., Bonviin, A., Raizel, R., Tirapegui, J., & Rogero, M. M. (2018). Probiotic supplementation in dental caries: Is it possible to replace conventional treatment? Nutrire, 43, 6. Garcia-Cayuela, T., Korany, A. M., Bustos, I., de Cardinanos, G. L. P., Requena, T., Pelaez, C., et al. (2014). Adhesion abilities of dairy Lactobacillus plantarum strains showing an aggregation phenotype. Food Research International, 57, 44–50. Kaur, K., Nekkanti, S., Madiyal, M., & Choudhary, P. (2018). Effect of chewing gums containing probiotics and xylitol on oral health in children: A randomized controlled
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Contents lists available at ScienceDirect
LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt
Antibacterial and anticavity activity of probiotic Lactobacillus plantarum 200661 isolated from fermented foods against Streptococcus mutans Sung-Min Lim, Na-Kyoung Lee, Hyun-Dong Paik∗ Department of Food Science and Biotechnology of Animal Resources, Konkuk University, Seoul, 05029, Republic of Korea
A R T I C LE I N FO
A B S T R A C T
Keywords: Probiotics Streptococcus mutans Anticavity activity Antimicrobial effect Antibiofilm effect
This study investigated the characteristics of Lactobacillus strains as an oral probiotic. Lactobacillus delbrueckii 200170 and L. plantarum 200661 were isolated from raw crab marinated in soy sauce and fermented tomato kimchi, respectively. L. rhamnosus GG was used as control. L. rhamnosus GG and L. plantarum 200661 survived in gastrointestinal conditions. They could stably produce enzymes and transfer antibiotic resistance. The antibacterial activity of L. rhamnosus GG, L. delbrueckii 200170, and L. plantarum 200661 inhibited three Streptococcus mutans strains. L. plantarum 200661 showed the highest antibacterial activity and inhibited S. mutans biofilm formation by 71.8–94.79%. L. plantarum 200661 inhibited glucan formation by 58.4%. L. plantarum 200661 could also inhibit the expression levels of biofilm formation and glucan related genes in S. mutans strains. Therefore, L. plantarum 200661 can be used as a biofilm inhibitor and as an oral probiotic in functional foods.
1. Introduction
Rosalen, Cury, Park, & Bowen, 2002). Recent studies have shown that probiotics play a positive role in oral health. Lactobacillus rhamnosus GG, Lactobacillus plantarum, and Lactobacillus reuteri inhibited biofilm formation by S. mutans (Amez, López, Devesa, Montero, & Salas, 2017; Söderling, Marttinen, & Haukioja, 2011). Chewing gum containing probiotics and xylitol reportedly have preventive effects against plague and gingival score (Kaur et al., 2018). Lactobacillus casei, L. plantarum ST-III, and Lactobacillus paracasei LPC27 could reduce cell growth and biofilm formation of S. mutans (Lin, Chen, Tu, Wang, & Chen, 2017). The antibacterial components produced by Lactobacillus sp. included bacteriocin or bacteriocin-like substances, lactic acid, and hydrogen peroxide (Shanker & Federle, 2017). Most of reports focused antimicrobial and anticavity activity. Therefore, this study aimed to confirm the probiotic potential having oral therapy. Lactic acid bacteria (LAB) isolated from fermented foods was investigated probiotic characteristics included acid and bile salt tolerance. In addition, oral therapy was determined using antimicrobial, anticavity, and antibiofilm activities against S. mutans.
Probiotics are live bacteria that can modulate the intestinal microflora when ingested in adequate amounts by the host (Lee, Kim, Han, Eom, & Paik, 2014). The characterization of probiotics has shown their immune modulatory, anticancer, antioxidant, and antimicrobial effects (Lee et al., 2015; Yang et al., 2019). Probiotics can act as carriers in various functional foods, such as yogurt, cheese, cookies, and chewing gum (Kaur, Nekkanti, Madiyal, & Choudhary, 2018; Taha et al., 2017). Dental caries is an infectious disease and a public health problem among children (Coqueiro, Bonviin, Raizel, Tirapegui, & Rogero, 2018). Cariogenic bacteria such as Streptococcus mutans, Streptococcus sobrinus, and Lactobacillus sp. play important roles in the pathogenesis of caries (Balakrishnan, Simmonds, & Tagg, 2000). Among the various bacteria producing biofilms, S. mutans is the main species responsible for causing the carious lesions. S. mutans produces glucosyltransferases (GTFs), which can synthesize intracellular polysaccharides (IPS) as well as extracellular polysaccharides (EPS). Among the EPS, water-insoluble glucans mediate the initial stage of adherence of oral bacteria onto the tooth surfaces and facilitate the formation of mature dental plaque (Beloin & Ghigo, 2005; Leme, Koo, Bellato, Bedi, & Cury, 2006). Although various antibacterial compounds such as chlorhexidine, ampicillin, spiramycin, and vancomycin have been effective in preventing dental caries, they can cause unexpected side effects (Koo,
∗
2. Materials and methods 2.1. Bacterial strains and sample preparation L. delbrueckii 200170 and L. plantarum 200661 were isolated using
Corresponding author. E-mail address: [email protected] (H.-D. Paik).
https://doi.org/10.1016/j.lwt.2019.108840 Received 7 August 2019; Received in revised form 6 November 2019; Accepted 10 November 2019 0023-6438/ © 2019 Published by Elsevier Ltd.
Please cite this article as: Sung-Min Lim, Na-Kyoung Lee and Hyun-Dong Paik, LWT - Food Science and Technology, https://doi.org/10.1016/j.lwt.2019.108840
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Table 1 Probiotic characteristics of LAB strains. Characteristics Initial cell count (log CFU/mL) Gastric conditions (log CFU/mL) Bile salt (log CFU/mL) β-Glucuronidase Adhesion ability (%)
L. rhamnosus GG
L. delbrueckii 200170
a
7.45 5.42 6.47 ND 1.34
8.70 ± 0.15 8.61 ± 0.02a 8.78 ± 0.09a ND 6.3 ± 1.21b
b
± 0.02 ± 0.09c ± 0.21c ± 0.35c
L. plantarum 200661 7.67 ± 0.08b 7.61 ± 0.02b 8.12 ± 0.02b ND 13.24 ± 1.63a
The results are represented as mean ± standard deviation. ND, not detected. a-c Values with different letters in the same row column are significantly different (p < 0.05). Table 2 Antimicrobial activity of the LAB cell free supernatant against S. mutans strains. Oral pathogens
S. mutans KCTC 5124 S. mutans KCTC 5458 S. mutans KCTC 5316
Minimum inhibitory concentration (%) L. rhamnosus GG
L. delbrueckii 200170
L. plantarum 200661
12.5 ± 0.0b 25.0 ± 0.0b 25.0 ± 0.0b
6.25 ± 0.0a 25.0 ± 0.0b 25.0 ± 0.0b
6.25 ± 0.0a 12.5 ± 0.0a 12.5 ± 0.0a
Data are represented as the mean ± standard deviation of triplicate experiments. Sodium ampicillin was used as control and it was serially diluted from 500 μg/mL to 7.8 μg/mL. All S. mutans strains were effectively inhibited at 7.8 μg/mL. a-b Values with different letters in the same row are significantly different (p < 0.05).
Lactobacillus selective medium (BD Biosciences, Franklin Lakes, NJ, USA) from raw crab marinated in soy sauce and fermented tomato kimchi, respectively. These strains identified 16S rRNA sequencing. Lactobacillus rhamnosus GG (Cell Biotech., Ltd., Gimpo, Korea) was obtained commercially. Lactobacillus strains were propagated and maintained in lactobacilli MRS medium (BD Biosciences) at 37 °C. They were cultured in MRS broth for 24 h at 37 °C and centrifuged (12,000×g for 10 min at 4 °C) to obtain the supernatant, which was filtered and used as samples. Streptococcus mutans KCTC 5124, KCTC 5458, and KCTC 5316 were obtained from the Korean Collection for Type Culture (Daejeon, Korea). S. mutans strains were cultured in BHI broth (BD Biosciences) for 24 h at 37 °C and used as oral pathogens.
Adhesion ability (%) = {adhered cell no. (CFU)/initial cell no. (CFU)} × 100 The antibiotic resistance of LAB strains was determined according to Clinical and Laboratory Standards Institute (CLSI) guideline by using the disc diffusion method. One-hundred microliter of each LAB stains were spread on MRS agar and then placed paper discs containing antibiotics. The inhibition zone was measured after incubation at 37 °C for 24 h. 2.3. Antimicrobial effect of LAB against S. mutans strains The minimum inhibitory concentration (MIC) of samples was measured against S. mutans using 96 well plates. Filter-sterilized LAB supernatants were serial diluted by two-fold using BHI broth ranging from 100% to 0.78%. Then, S. mutans (1 × 105 CFU/mL) and diluted samples were added into each well. The lowest sample concentration that inhibited 99% of the inoculum was considered as the MIC. Sodium ampicillin used as control was diluted from 500 μg/mL to 7.8 μg/mL.
2.2. Probiotic characterization of LAB The artificial gastric acid and bile acid consisted of 0.3% pepsin in pH 2.5 and 3% oxgall in pH 7, respectively. Overnight cultures of each LAB strains were resuspended in artificial gastric acid and bile acid conditions and incubated for 3 h and for 24 h at 37 °C, respectively, to confirm the tolerance. The viable cells were counted by dilution and plating on MRS medium after each reaction.
2.4. Measurement of biofilm formation using crystal violet staining Biofilm formation was assayed using the method described by Wu et al. (2015) with modifications. S. mutans (1 × 106 CFU/mL) was cultured at 37 °C for 24 h in the presence or absence of sample in BHI broth supplemented with 3% sucrose in a 24 well plate, and the sample was inoculated into each well. After incubation, the planktonic bacteria were removed by gentle washing with distilled water, and biofilms were stained with 0.1% crystal violet solution for 10 min at room temperature. The plates were rinsed with distilled water and then, the adhered dye was dissolved with acid-alcohol solutions. The absorbance was measured at 540 nm and biofilm inhibition rate was calculated as
Survival rate (%) = {cell no. after reaction (CFU)/initial cell no. (CFU)} × 100 Enzyme production was measured using API ZYM kit (BioMerieux, Lyon, France). Each LAB strain was suspended in saline and inoculated into each cupule. The inoculated strips were incubated at 37 °C for 4 h. One drop of each ZYM A and ZYM B reagent was serially added into each cupule and the results were measured based on the intensity of the color. To measure the adhesion ability to intestinal epithelial cells, HT29 cells were plated at 2 × 105 cells/well in 24-well plate and incubated at 37 °C for 24 h. Approximately 1 × 108 CFUs/well of LAB strain was added to the each well with HT-29 cells monolayer. The plate was incubated at 37 °C for 2 h in 5% CO2 incubator and non-adherent cells were removed by four washes with PBS. Further, 1 mL of 0.1% Triton X-100 was added to each well, and the cells were harvested. The samples were diluted ten-fold, spread onto solid plates, and incubated at 37 °C for 24 h. Adhesion ability was calculated as
Biofilm inhibition rate (%) = {1- (absorbance of sample)/(absorbance of control)} × 100
2.5. Inhibition of water-insoluble glucan synthesis The inhibition of water-insoluble glucan was evaluated using the method previously described by Koo et al. (2002). 2
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Fig. 1. Inhibitory effect of the culture medium and supernatant of LAB strains on S. mutans biofilm formation. (A) S. mutans KCTC 5124, (B) S. mutans KCTC 5458, (C) S. mutans KCTC 5316. The different superscript letters on each bar in the same LAB strain represent significant difference between values (p < 0.05). □, 100 μL; , 200 μL; , 500 μL; ■, 1000 μL.
supernatant, and 800 μL of 62.5 mM potassium phosphate buffer (pH 6.5) with 12.5 g of sucrose and 0.25 g of sodium azide was used. This mixture was incubated at 37 °C for 30 h. After incubation, the unsolidified sample was removed, the contents adhering to the tube wall were washed with sterile water and dispersed by sonication. The total amount of water-insoluble glucan was measured at an absorbance of
To obtain the crude enzyme, S. mutans was incubated in BHI broth at 37 °C for 24 h, centrifuged at 4 °C for 15 min and filtered using a 0.2 μm membrane filter. Then, the cell-free supernatants were extracted using Amicon ultra centrifugal filters (MWCO 30 kDa, Millipore, USA) and upper retentate was used as the crude enzyme sample. A reaction mixture containing 20 μL of crude enzyme, 180 μL of LAB 3
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glucuronidase, shown using the API ZYM kit. The adhesion ability of LAB to intestinal cells reportedly influences its colonization in the host, immunomodulatory effects, and prevention of pathogen infection (Garcia-Cayuela et al., 2014). L. plantarum 200661 shows higher adhesion ability (13.24%) compared to L. rhamnosus GG (6.3%). This shows that L. plantarum 200661 can attach and colonize in intestinal epithelial cells better than commercial strains. While the LAB strains were sensitive to ampicillin, tetracycline, chloramphenicol, and doxycycline, they were resistant to gentamycin, kanamycin, streptomycin, and ciprofloxacin (data not shown). These results suggest that L. plantarum 200661 strain was stable in gastric conditions, is antibiotic resistant, and does not produce any hazardous enzymes.
Table 3 Inhibitory effect of supernatant of lactic acid bacteria on the synthesis of waterinsoluble glucan by the GTFs from S. mutans. Oral pathogens
S. mutans KCTC 5124 S. mutans KCTC 5458 S. mutans KCTC 5316
GTFs inhibition (%) L. rhamnosus GG
L. plantarum 200661
37.24 ± 2.25a 26.42 ± 2.13a 15.38 ± 6.61a
47.03 ± 2.57b 24.81 ± 0.82a 26.24 ± 2.59b
Data are represented as the mean ± standard deviation of triplicate experiments. a-b Values with different letters in the same row are significantly different (p < 0.05).
3.2. Antimicrobial activity against S. mutans
550 nm and the GTFs inhibition rate was calculated as
The virulence of S. mutans is due to their ability to survive in acidic pH and produce biofilms (Krzyściak, Jurczak, Kościelniak, Bystrowska, & Skalniak, 2014). The antimicrobial activities of LAB were investigated against S. mutans strains (Table 2). Sodium ampicillin was used as control and its MIC was < 7.8 μg/mL for all S. mutans. The MIC of L. rhamnosus GG were 12.5%, 25%, and 25% against S. mutans KCTC 5124, KCTC 5458, and KCTC 5316, respectively. The MIC of L. plantarum 200661 were 6.25%, 12.5%, and 12.5%, respectively. L. plantarum 200661 showed good antimicrobial activity against all three S. mutans strains. These effects could be due to the antimicrobial substances produced including organic acids, bacteriocin, and biosurfactants (Lin et al., 2017).
GTFs inhibition rate (%) = {1- (absorbance of treated sample)/(absorbance of control)} × 100
2.6. RNA extraction and semi-quantitative real-time PCR S. mutans (1 × 105 CFU/mL) was grown in 10 mL of BHI broth at 37 °C for 24 h and then, 1 mL of LAB supernatant was added into it followed by anaerobic incubation at 37 °C for 12 h. RNA extraction was performed using Trizol® Max™ bacterial RNA isolation kit (Thermo Fisher Scientific, Waltham, MA, USA), according to manufacturer's protocol. The RNA quality was quantified using Multiscan GO (Thermo Fisher Scientific). The cDNA was synthesized using Revertaid first strand cDNA synthesis kit (Thermo Fisher Scientific). Semi-quantitative real-time PCR was performed using the SYBR Green PCR Master Mix in the PikoReal 96 system (Thermo Fisher Scientific). The reaction mixture contained SYBR Green master mix, primer, cDNA, and RNase free water. Further, 20 μL of the mixture was amplified as follows: 95 °C for 10 min as initial denaturation, following by 40 cycles of 95 °C for 15 s as denaturation and 60 °C for 45 s as annealing and extension. The analysis was done using the melt curve analysis method to determine the reaction. The primer used for this was sucrose phosphorylase gene (gtfA), GTF B gene (gtfB), GTF-I gene (gtfD), fructosyltransferase gene (ftf), histidine kinase two-component regulatory system gene (vicR), competence-stimulating system gene (comDE), and biofilm-regulation protein gene (brpA) (Shemesh, Tam, & Steinberg, 2007; Steinberg, Moreinos, Featherstone, Shemesh, & Feuerstein, 2008).
3.3. Inhibition of biofilm formation While both the culture medium and the supernatant of LAB could inhibit the S. mutans biofilm formation, the supernatant showed higher inhibitory effects (Fig. 1). L. rhamnosus GG inhibited the S. mutans biofilm formation by over 50% at 200 μL while L. delbrueckii 200170 inhibited it at 1000 μL. L. plantarum 200661 inhibited the same by over 60% at 100 μL, making it the strongest inhibitor. Therefore, it was confirmed that L. plantarum 200661 could inhibit the S. mutans biofilm formation at low doses. This mechanism has been reported to be due to the co-aggregation with LAB resulting in physical interference (Wu et al., 2015) and induction of EPS production (Ahn, Baik, Park, Yun, & Han, 2018). 3.4. Inhibition of synthesis of water insoluble glucan
3. Results and discussion
Glucan is an intermediate between S. mutans and tooth surfaces, and it also promotes the cell to cell adhesion, structural integrity and stability of biofilms (Leme et al., 2006). The LAB supernatants could inhibit water-insoluble glucan synthesis by the crude GTFs from S. mutans (Table 3). L. rhamnosus GG inhibited the water insoluble glucan formation in S. mutans KCTC 5124, KCTC 5458, and KCTC 5316 by 37.24%, 26.42%, and 15.38%, respectively. L. plantarum 200661 was more effective than L. rhamnosus GG on S. mutans KCTC 5124 and KCTC 5316. This result showed that the supernatant of L. plantarum 200661 can effectively inhibit GTFs.
3.1. Probiotic characterization of the LAB strains
3.5. Expression of genes involved in the virulence of S. mutans
Probiotic strains should be tolerant toward intestinal conditions in order to survive in intestinal cells. Under gastric conditions, the survival rate of L. rhamnosus GG, L. delbrueckii 200170, and L. plantarum 200661 were 81.28%, 0.93%, and 87.1%, respectively (Table 1). However, in the presence of bile acids, the survival rate of L. rhamnosus GG and L. plantarum 200661 was > 100% except for L. delbrueckii 200170 (10.47%). Enzyme production was important in use of probiotics, βglucuronidase could be induced cancer substances in liver and colon (Son et al., 2018). All three LAB strains did not produce β-
To evaluate the effect of L. rhamnosus GG and L. plantarum 200661 supernatants on S. mutans, the expression of the genes involved in the virulence of S. mutans were investigated (Fig. 2). The representative genes involved in glucan formation are gtfA, gtfB, and gtfD, and in fructation formation was ftf (Shemesh et al., 2007). In addition, the signal transduction system was controlled by the comDE, vicR, relA, and brpA genes. L. rhamnosus GG and L. plantarum 200661 down-regulated the genes responsible for EPS formation in all S. mutans strains except for brpA in
2.7. Statistical analysis All the tested data are represented as mean and standard deviation of three replicates. One-way analysis of variance (ANOVA) was used to determine the significant differences. The mean values were used for the Duncan's multiple range test to perform post hoc verification (p < 0.05).
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Fig. 2. Relative gene expression associated with biofilm formation in S. mutans strains as determined by RT-PCR. (A) S. mutans KCTC 5124, (B) S. mutans KCTC 5458, (C) S. mutans KCTC 5316. The different superscript letters on each bar in the same gene represent significant difference between values (p < 0.05). □, L. rhamnosus GG; ■, L. plantarum 200661.
inhibitory effects on the gene expression depends on the species of S. mutans. Moreover, the LAB supernatant showed lower inhibitory effect on the expression of genes involved in EPS formation than the signal transduction genes. Therefore, these results suggest that disrupting the synthesis of insoluble glucans can reduce the biofilm formation, which
S. mutans KCTC 5124 and relA in S. mutans KCTC 5316. In S. mutans KCTC 5458, L. rhamnosus GG and L. plantarum 200661 down-regulated all genes compared to control (< 1-fold change). In addition, L. plantarum 200661 compared to L. rhamnosus GG showed significantly lower gene expression in S. mutans KCTC 5316. The difference in the
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can influence the pathogenesis. In S. mutans, the biosurfactants produced by L. casei inhibited the expression of ftf and gtfB genes, while L. acidophilus inhibited the gtfB and gtfC gene expressions (Savabi et al., 2014; Tahmourespour, Salehi, & Kermanshahi, 2011).
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4. Conclusions L. plantarum 200661 is stable under gastric conditions and can adhere to intestinal walls. This strain was safe in enzyme production and antibiotic resistance. This strain also exerts antibacterial and antibiofilm activity against S. mutans by inhibiting the formation of water insoluble glucan and by downregulating the expression of related genes. Therefore, L. plantarum 200661 can be used as a probiotic as well as an inhibitor of biofilm formation of several oral pathogen strains in functional foods. Declaration of competing interest All authors declare no conflict of interest. Acknowledgements This paper was supported by Konkuk University in 2016. References Ahn, K. B., Baik, J. E., Park, O. J., Yun, C. H., & Han, S. H. (2018). Lactobacillus plantarum lipoteichoic acid inhibits biofilm formation of Streptococcus mutans. PLoS One, 13, e0192694. Amez, M. S., López, J. L., Devesa, A. E., Montero, R. A., & Salas, E. J. (2017). Probiotics and oral health: A systematic review. Medicina Oral, Patología Oral Y Cirugía Bucal, 22, 19. Balakrishnan, M., Simmonds, R. S., & Tagg, J. R. (2000). Dental caries is a preventable infectious disease. Australian Dental Journal, 45, 235–245. Beloin, C., & Ghigo, J. M. (2005). Finding gene-expression patterns in bacterial biofilms. Trends in Microbiology, 13, 16–19. Coqueiro, A. Y., Bonviin, A., Raizel, R., Tirapegui, J., & Rogero, M. M. (2018). Probiotic supplementation in dental caries: Is it possible to replace conventional treatment? Nutrire, 43, 6. Garcia-Cayuela, T., Korany, A. M., Bustos, I., de Cardinanos, G. L. P., Requena, T., Pelaez, C., et al. (2014). Adhesion abilities of dairy Lactobacillus plantarum strains showing an aggregation phenotype. Food Research International, 57, 44–50. Kaur, K., Nekkanti, S., Madiyal, M., & Choudhary, P. (2018). Effect of chewing gums containing probiotics and xylitol on oral health in children: A randomized controlled
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