Appl Microbiol Biotechnol DOI 10.1007/s00253-015-6693-z
BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING
Comparison of inhibitory activity of bioactive molecules on the dextransucrase from Streptococcus mutans Choon Geun Lee 1 & Jae Kweon Park 1
Received: 23 March 2015 / Revised: 8 May 2015 / Accepted: 13 May 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract The effect of chitosan with different molecular weights and other natural substances on dextransucrase (DSase) activity from a representative oral pathogen Streptococcus mutans was elucidated. Among other bioactive substances, amino-monosaccharides such as glucosamine, mannosamine, and galactosamine exerted the enzyme inhibitory activity over 95 % of DSase. The specified hydrolysates derived from the hydrolysis of high molecular weight chitosan (HMWC) designated to CTSN, CTSN-P, CTSN-B, and CTSN-S with different molecular weights ranging from 3 to 8 kDa showed the similar inhibitory activity toward DSase. Also, the hyaluronic acid (MW 8.9 kDa), sulfated chitin, and amino-monosaccharides demonstrated the significant activity, CTSN, CTSN-P, CTSN-B, and CTSN-S are of potent bioactive substances that can be prepared in the cheapest way compared with other molecules tested available for antibacterial agent useful for human oral health. Keywords Dextransucrase . Streptococcus mutans . Antimicrobial activity . Oral health . Chitosan
Introduction Streptococcus mutans is a Gram-positive organism that is the principal etiological agent in the formation of dental cavities in humans (Li et al. 2014). Streptococcusis classified as a genus of spherical gram-positive bacteria
* Jae Kweon Park [email protected] 1
Department of Life Sciences, Gachon University, Seongnamdaero 1342, Seongnam-si, Gyeonggi-do 461-701, South Korea
belonging to the phylum Firmicutes and the lactic acid producing bacterial group. S. mutans, which is widely recognized as the main etiological pathogenic agent of dental cavities in humans (Liao et al. 2014; Liu et al. 2014; Moye et al. 2014). Not only bacterial infections but also conditions in the oral cavity are truly diverse and complex. Therefore, S. mutans must tolerate rapidly to harsh environmental fluctuations including exposure to various antimicrobial agents in order to survive in the oral cavity (Guo et al. 2014; Liao et al. 2014). Most of etiological agents of dental caries are associated with its ability to catabolize various sugars to form insoluble biofilm and produce lactic acid to generate the acid environment (Guo et al. 2014). Owing to these features concerted with its survivability, natural bioactive substances such as sugar-alcohol, antocyanine, and specific sugars have been focused as potent antimicrobial materials. It has been implicated that extracellular water-insoluble polysaccharide from dietary sugar produced by dextransucrase (DSase) and its adhesion to dental enamel are known to the most clinically important biological abilities of S. mutans (Chen et al. 2012; Decker et al. 2014). However, the mechanisms under which this cariogenic pathogen can survive or proliferate under in such environmental conditions are so far largely unknown. Dental caries produced from oral acidogenic bacteria is well defined as a dental biofilm-related oral disease associated with the increasing consumption of dietary sugars and/or fermentable carbohydrates. Along with frequent exposure to sugars such as glucose and sucrose, S. mutans producing dental biofilms can metabolize the sugars to organic acids. Significant persistence of S. mutans under the acidic environment encourages the proliferation of S. mutans including other acidogenic bacteria, resulting in their ability to survive at a low-pH condition. Upon changing to the low-pH condition, the initiation of the
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dental caries starts under the surface of teeth covered with biofilm (Chen et al. 2012; Decker et al. 2014). Therefore, the potential acidification of dental biofilms and later cavity formations can be decreased by eliminating the adherence of S. mutans to the surface of teeth or dental biofilms. In addition, individual susceptibility to disease varies by immunological response which has been proposed to confer protection or susceptibility to dental diseases. These mechanisms have yet to be fully elucidated but it seems that immunological tolerance to S. mutans at the mucosal surface may make individuals more prone to colonization with S. mutans (Moon et al. 2007; Zhao et al. 2006). For several decades, antimicrobial activity of natural substances toward S. mutans has extensively been investigated. Among the most of polysaccharides chitosan, a linear polysaccharide of β-1,4 linkage of glucosamine has been reported as a potent antimicrobial substance with different molecular weights on bacterial growth (Aam et al. 2010). Although the precise mechanisms are so far largely unknown, several mechanisms have been proposed for the antimicrobial activity by chitosan and its related molecules (Alencar de Queiroz et al. 2006; Alipour et al. 2009). For instance, one of the mechanisms proposed is the ability of chitosan to be attached to the cell wall of bacteria, which results in the reduction of bacterial metabolism (Busscher et al. 2008; Chen et al. 2008). Another mechanism is proposed that would inhibit activity of chitosan on the transcription of RNA by adsorption of chitosan to the DNA of bacteria (Chen et al. 2008), depending on the size of chitosan and its degree of deacetylation, and chemical features (Choi et al. 2001; Fernandes et al. 2008). Owing to these features, it has drawn attention as a potent antimicrobial agent specifically in human oral health (Sano et al. 1991; Sarasam et al. 2008; Zivanovic et al. 2004). However, despite the usefulness of chitosan, its low water-solubility due to its high degree of polymerization is a limiting factor for its application in various fields. Therefore, several studies have been focused on the relationship between the depolymerization of chitosan to reduce the molecular weight and its biological activities, which is readily soluble in water or diluted weak acidic solution (Jing et al. 2007; Kulikov et al. 2009; No et al. 2002). Although the various molecular weights of chitosans derived from the hydrolysis of high molecular weight chitosan (HMWC) are well known to have considerable antimicrobial activities against various pathogenic bacteria, there was little work reported on their biological activity against the periodontal pathogen S. mutans. Therefore, the aim of this study is to elucidate the antimicrobial activity of chitosans with different molecular weights and other bioactive substances against the representative periodontopathogen S. mutans. Our results can propose the potent utility of those molecules as an antimicrobial agent to support human health.
Materials and methods Materials A high molecular weight chitosan (HMWC, 95 % degree of deacetylation) with approximately 200 kDa was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Various sizes of different molecular weight chitosans were prepared in enzymatic digestion using chitosanase from Streptomyces griseus, which was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Bioactive substances including various monosaccharides and other polysaccharides such as alginate, β-glucan, and hyaluronic acid were also purchased from Sigma Chemical Co. (St. Louis, MO, USA). Other crude products were extracted from the marine algal sources. S. mutans Clarke (ATCC® 700610D-5™) was provided by Prof. Jae-Gyu Jeon (Chonbuk University, Korea). All other reagents were of the highest grade available. Preparation of chitosan hydrolysates In order to increase the potent possibility to get partial degradation of HMWC, the HMWC was dissolved in 2 % acetic acid to be 0.5, 1.0, and 2.0 g/0.1 L and subjected to partial depolymerization by using 0.2 U of chitosanase, respectively. Mixture was reacted with mixing at 700 rpm using magnetic bar for 2 h at room temperature to perform the partial digestion. Reactants were boiled for 5 min at 100 °C in order to denature chitosanase and also centrifuged for 10 min at 13, 000 rpm to remove denatured chitosanase and indigested material. Pre-chilled ethanol was used to precipitate hydrolysates derived from the hydrolysis of HMWC. The precipitates were recovered by centrifugation for 10 min at 13,000 rpm and washed with the ethanol until the pH value was same as the ethanol. Each hydrolysate after digestion of HMWC was further fractionated to remove low molecular weight chitosan using a small size of gel-filtration column chromatograph (Φ 1.0 cm × L 30 cm) packed with Bio Gel-P 4 (Bio-RAD, USA) gel, which was pre-equilibrated with a volatile buffer consisting of ammonia/formic acid (pH 7.5). The final products derived from the hydrolysis of HMWC with different concentration of 0.5, 1.0, and 2.0 g/0.1 L were lyophilized (IlShin Co. Ltd., Seoul Korea), designated to CTSN-S, CTSN-B, and CTSN-P respectively. CTSN is prepared as a mixture of CTSN-P/B/S, which were pooled together equally and used all for subsequent experiments. Instrumental analysis of MW of CTSNs To determine the average molecular weight of chitosan, each hydrolysate obtained from the hydrolysis of HMWC was applied to high-performance liquid chromatography (HPLC), using a PolySep-GFC-P3000 column (7.8 × 300 mm,
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Phenomenex, USA). For the detection, 10 μL of 1.0 % sample solution was injected into the system, eluted with water at a flow rate of 0.8 mL/min at 63.8 °C, and detected with ELSD (evaporative light scattering detector, Alltech). Pullulans (Sigma Chemical Co., St. Louis, MO, USA) with a wide range of molecular weights (5600∼2000 kDa) were used as standard molecular markers. The average MW of hydrolysates (CTSNs) of chitosan was calculated from the calibration curve and area ratio of major peaks. Determination of bacterial susceptibility S. mutans was incubated anaerobically in brain-heart infusion (BHI) broth consisting of 1 % sucrose at 37 °C for 3 days. In order to evaluate the susceptibility of S. mutans toward various dietary sugars and its metabolic condition, the assay was performed as follows: solutions containing various bioactive substances were each prepared in 1 % (w/v) distilled water or diluted acetic acid (0.1 %). Each solution prepared was added to BHI broth ranging from 0.1 to 0.5 % (v/v). The 10 L of inoculums of S. mutans pre-incubated in BHI broth was added in fresh broth with or without sample. Each broth was then further incubated at 37 °C for 12 h anaerobically. For antimicrobial activity, Staphylococcus aureus cultured in LB medium was also used as a comparative microorganism. Susceptibility of S. mutans toward those materials was determined by measuring the growth rate at 600 nm. Inhibitory effect on the growth of S. mutans S. mutans were cultured in the presence or absence of bioactive materials under the optimum condition to assign the metabolic ability for the formation of biofilms. This is known as the main factor causing dental caries. Since S. mutans can effectively use dietary sucrose to rapidly synthesize insoluble and some soluble extra cellular polysaccharides (EPSs) using dextransucrase (DSase), EPS production was quantitatively estimated by phenol-sulfuric assay (Kumar et al. 1988; Rasouli et al. 2014). Briefly, the aliquots taken from each sample at time intervals of 3, 6, 9, and 12 h were subjected to the method of using glucose as the standard material. With this in regard, several chitosans with different molecular weights and other comparative materials were tested to estimate their effectiveness as DSase inhibitor. For determination of influence of bioactive materials on DSase, enzyme activities were determined by the quantitative analysis of EPS produced by DSase, as described above. In order to determine the cytotoxicity, RAW264.7 murine macrophages cells (5 × 104 cells/well) were suspended in complete RPMI1640 medium, and the proliferation of the cells was assessed in micro-culture tetrazolium viability assay using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), using a multi-detection microplate reader
(Molecular Devices, Sunnyvale,CA, USA) as described the previous study (Park et al. 2011). All experiments for the MTT assay were performed in triplicates. Statistical analysis Representative data demonstrated in this study were expressed as the means of ±SD from at least three separate experiments, unless otherwise indicated. The statistical significance of the differences between mean values obtained from each experiment of less than 0.05 (as p value) was considered.
Results Determination of inhibitory concentration (IC50) of chitosan with different molecular weight Average molecular weights of chitosans designated to CTSN, CTSN-P, CTSN-B, and CTSN-S obtained from the partial hydrolysis of HMWC and CTSN made of CTSN-P/B/S as a mixture were determined in HPLC analysis to be ranging from 3.0 to 8.0 kDa, respectively (Table 1). Till a decade ago, antibacterial applications of chitosan hydrolysates with different molecular weights or deacetylation degree were widely studied to investigate their biological activities. However, due to shortcomings of the preparation of chitosan hydrolysates having the specific sizes, there has been a surge in interest for enzymatic partial digestion of chitosan under various mild or extreme conditions to yield high (Table 1). Since all types of chitosan hydrolysates (CTSNs) could effectively inhibit the growth of bacterial cell growth toward both PWG and F1 strains isolated as antibiotic-resistant bacteria from porcine sperm fluid (data not shown), each hydrolysate was applied as a potent antimicrobial agent toward various pathogenic microorganisms. IC50 for each compound was determined based on bacterial cell growth, as shown in Table 1. All compounds have showed similar activity toward both bacterial strains tested in this study. These results are correlated to the previous studies that demonstrated the relationship between Table 1 activity
Effect of molecular weight of chitosan on antimicrobial
Sample
Average Yield IC50 (μg/mL) MW (kDa) (%) Streptococcus MRSA (Staphylococcus mutans aureus CCARM 3230)
CTSN CTSN-P CTSN-B CTSN-S
6.650 7.783 5.452 3.510
26.7 38.4 26.8 14.8
150 200 190 170
230 220 240 210
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efficacy and the significance of the size of molecules. Based on these results, we suggest that average molecular weight of chitosan on antimicrobial activity is an important factor, as early studies demonstrated the significance of the size of molecule (Busscher et al. 2008; Fernandes et al. 2008; Jing et al. 2007). Measurement of DSase To address the preliminary antibacterial mechanism of bioactive substances against S. mutans, production of EPS was quantitatively elucidated. As shown in Fig. 1, DSase activity was estimated in the ability for EPS production which gradually increased in a time-dependent manner. The results indicated that S. mutans can efficiently metabolize sucrose for the proliferation of bacterial cells and utilize its products for the expression of protein associated in sugar transferase, called DSase. As many reports demonstrated, DSase is a critically important enzyme involved in the glucose transfer to make insoluble EPS, also known as a biofilm (Lee et al. 2015; Liao et al. 2014). Upon determination of enzyme activity using a culture supernatant containing crude DSase, we found that EPS was proportionally synthesized in a time-dependent manner corresponding to the DSase activity (Fig. 1). Inhibitory activity of monosaccharides on DSase Among the most several polymers, chitosan exhibited a considerable antimicrobial activity against S. mutans depending
Fig. 1 Determination of DSase activity. S. mutans was incubated in brain heart-infusion (BHI) broth consisting of 1 % starch and 1 % sucrose, respectively. The incubation was performed at 37 °C for 3 days in CO2 incubator. Culture was centrifuged at 13,000 rpm for 10 min to collect the supernatant containing active DSase in crude. Enzyme activity of DSase was estimated to measure the amount of EPS in the reaction mixture consisting of 1 % sucrose in 25 mM phosphate buffer (pH 7.0) time dependently. EPS produced by DSase was obtained by ethanolic precipitation, and amount of the EPS was determined by phenolsulfuric acid method (Kumar et al. 1988; Rasouli et al. 2014)
on the size of molecule. It has not yet been clearly understood; however, it is hypothesized that protonation on amino groups at C-2 on chitosan backbones is one of the most important factors in electrostatic interaction between the polycationic structure and the predominantly anionic components of the microorganism’s cell wall (Sarasam et al. 2008; Virga et al. 2003). On the other hand, degree of deacetylation of chitosan is also known to be a critical factor determining the capability of biological function. Herein, we tested the inhibitory effect of monosaccharides on DSase activity. As seen in Fig. 2, amino sugars such as glucosamine, mannosamine, and galactosamine showed strong inhibitory activity on DSase activity. No inhibitory activity of neutral sugars on DSase was observed; instead of this, the activity tended to increase. These results were correspondingly similar to our previous result, which demonstrated that amino sugars inhibited bacterial cell growth during the culture in vitro (Jung et al. 2013). Although inhibitory mechanisms by those amino sugars for DSase which were produced from S. mutans were not completely understood, these results made us to suggest that S. mutans may not able to metabolize such amino sugar as carbon and nitrogen sources because of their high sensitivity on electrostatic interaction with some uncertain component of S. mutans. Effect of molecular weight on DSase It is well defined that chitosan and its hydrolysates produced by several ways have showed significant antibacterial activity depending on the size and degree of deacetylation (Busscher et al. 2008; Choi et al. 2001; Fernandes et al. 2008; No et al. 2002). In order to address more precise mechanism how those molecules inhibit bacterial cell growth, we strived to elucidate the function of highly polymerized chitosan and other potent agents. As seen in Table 1, average molecular weight of hydrolysates called CTSNs prepared in this study was determined by HPLC, obtained successfully from the hydrolysis of HMWC. As seen in Fig. 3, various sizes of chitosans (CTSN, CTSN-P, CTSN-B, and CTSN-S) prepared as low molecular weight chitosan showed also significant inhibitory effect on DSase activity. These results suggest that exposure of DSase from S. mutans to the CTSNseries prepared in this study may rapidly be rendered by them or has much higher affinity toward them than glucose used as control. Based on these results, we found that amino sugars and its high polymerized polysaccharides are potent inhibitor for DSase, which plays a critical role in the formation of biofilm. Although it is not able to explain the exact mechanism of the antimicrobial activity of CTSNs toward S. mutans yet, our current investigation is apparently important point to explain the functions of CTSNs how polycationic polymer can interact to form polyelectrolyte complex with DSase. Based on these
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Fig. 2 Inhibitory effect of various monosaccharides on DSase activity. Enzyme activity of DSase was estimated to measure the amount of extracellular polysaccharide (EPS) in the reaction mixture consisting of 1 % sucrose in the presence of various monosaccharides (1 % each, respectively) in 25 mM phosphate buffer (pH 7.0). EPS produced by DSase was obtained by ethanolic precipitation, and amount of the EPS
was determined by phenol-sulfuric acid method, as describe in Fig. 1. Distilled water (DW) was used as negative control, herein no bacterial cells were provided into the culture. Mean values were estimated to be with ±SD expressed with error bars from three separate experiments (p < 0.05)
results, we suggest that inhibitory effect of series of CTSNs on DSase activity or growth rate attenuates the metabolic capacity by blocking of nutrient permeation through cell wall associated with the expression of
DSase. Therefore, inhibition of either cell growth (Fig. 3b) or DSase activity should be a crucial point to prevent the biofilm formation from S. mutans. Here, a question arose how glucosamine can inhibit the DSase
Fig. 3 Inhibitory effect of chitosan hydrolysates on DSase activity. Enzyme activity of DSase was estimated to measure the amount of EPS in the reaction mixture consisting of 1 % sucrose in the presence of various size of chitosans (CTSNs, 1 % each, respectively) in 25 mM acetate buffer (pH 4.5) (a). EPS produced by DSase was obtained by ethanolic precipitation and amount of the EPS was determined after precipitated the chitosan by bring pH to pH 9.0 using 1 M NaOH. Distilled water (DW) was used as negative control without bacterial cells. The morphology of bacterial cells was observed under 400 magnification using a microscope (Olympus CKX41, Hicksville, NY, USA) in the presence or absence of chitosans (b). Tested samples were asfollows: a, control; b, CTSN-P; c, CTSN-b; d, CTSN-S; e, CTSN
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activity. We are not able to answer clearly, but we hypothesize that it may be possibly placed on the active or biding site of DSase, which is not able to bind or interact with sucrose for further process. Inhibitory effect of bioactive substances on DSase Dental caries to be the single most common biofilmdependent oral infectious disease resulted from the interaction specific bacteria like S. mutans because it can synthesize EPS from dietary sucrose using DSase that adsorb to surface of teeth. This fact demonstrated that the adsorption of bacterial cells on surface and the formation of highly structured cell clusters must be associated with the ability of an organism to survive (Decker et al. 2014; Kubota et al. 2005), which are key factors causing a dental caries. Upon investigating using CTSNs, the inhibitory effects of other bioactive substances on DSase to elucidate biological functions that compare with the CTSNs were also performed. As shown in Fig. 4, obviously, hyaluronic acid (MW estimated to be 8.9 kDa) showed significant inhibitory activity on DSase, whereas other polysaccharides tested in this study showed slightly lower ability toward DSase than CTSNs. No significant activity difference between CTSNs and hyaluronic acid used with specific size of molecular weight was observed, indicating that not only molecular weight of bioactive materials and their electrostatics are critical factors on the inhibition of acidogenic properties of S. mutans. On the other hand, both extracts from Curcuma and Artemisa species were tested separately in this study to sort out other candidates useful with CTSNs as a co-effector. In the preliminary test, we found that these extracts showed highly significant antioxidant activities (unpublished data) can be used for oral hygiene in general in Asian countries. However, less is known about the biological activities of the extracts from Curcuma and Artemisa toward S. mutans. It is worthy to investigate the functional activity regarding the Fig. 4 Inhibitory effect of bioactive substances on DSase activity. The reaction was carried out in same condition aforementioned in Fig. 3. Enzyme activity of DSase was directly estimated in time dependently without ethanol precipitation by phenol-sulfuric acid method. Distilled water (DW) was used as negative control without bacterial cells. Mean values were estimated to be with ±SD expressed with error bars from three separate experiments (p < 0.05)
relationships between antioxidant and antimicrobial activities of them. Therefore, in the present study, we tried to get valuable information by using several kinds of natural biomaterials including various kinds of mono- and polysaccharides and plants extracts that can be used for oral hygiene in general in Asian countries. As shown in Fig. 5, water-soluble extract of Curcuma showed significant inhibitory activity; however, Artemisa’s extract did not show any significant inhibitory activity on DSase. These results are correlated to the antimicrobial activity of Artemisa’s extract, not showed any significant effect toward S. mutans by seeing the growth rate. Based on these result, Curcuma’s water-soluble extract can be used as a potent co-effector with CTSNs to handle the protection of biofilm formation by DSase in S. mutans as for oral hygiene. Overall, the mechanism associated with biofilm synthesis and aciduric properties of S. mutans must be considered with many types of glucosyltransferase and fructosylase released from S. mutans. Furthermore, phylogenetically different types of S. mutans form structurally and metabolically distinctive biofilms depend on type of sugars used in extracellular polysaccharides synthesis by S. mutans. Based on these results, we suggest that some specificity and size of sugars might be involved in the inhibition of enzyme activity of DSase from S. mutans, not necessary of bioactive materials having strong antioxidant activities. To confirm the absence of toxicity for the application of CTSN-series as pharmaceutical material for human oral health, we tested the cytotoxicity of CTSN toward RAW264.7 cells. As shown in Fig. 6, no significantly detectable level of cytotoxicity of CTSN on the RAW264.7 cells at the concentrations ranging from 1 to 1000 μg/mL was observed. The results clearly demonstrated that the oral application of CTSNs may be effective in alleviating or preventing the oral disease caused by S. mutans in vivo. Taken collectively, our data described here suggest that CTSN with specified size of molecular weight can be used as pharmaceutical material for oral health for human.
Appl Microbiol Biotechnol Fig. 5 Inhibitory effect of watersoluble extracts from Curcuma and Artemisa on DSase. The reaction was carried out in same condition aforementioned in Fig. 1. Mean values were estimated to be with ±SD expressed with error bars from three separate experiments (p < 0.05). Distilled water (DW) was used as negative control without bacterial cells
Discussion The antibacterial application of chitosan with different molecular weight is an attractive alternative to investigate the unique biological tool. In this context, we demonstrated the importance of (1) the assessment of inhibitory properties of CTSNs toward DSase and (2) the comparison of activity of natural bioactive compounds. We also demonstrated the properties of CTSN with different molecular weights against the specific enzyme DSase produced as one of representative pathogenic agents from S. mutans to elucidate the antimicrobial activity for oral health for human. An
Fig. 6 Cytotoxicity of chitosans. Murine macrophage RAW264.7 cells were suspended in complete RPMI1640 medium. RAW264.7 cells were seeded at a density of 5 × 104 cells in 100 μL per well and incubated in a 96-well microplate for 24 h. Cells were treated with different concentrations of the CTSN prepared with serial dilution ranging from 1 to 1000 μg/mL, while the negative control was treated with only the medium. The proliferation of the cells was assessed using the 3-[4,5-
emphasis of antimicrobial activity of the series of CTSN was made to include S. mutans and S. aureus that were resistant to commercial antibiotics (Table 1). Especially, S. mutans can tightly adhere to the surface of teeth. Owing to the potential acidification of dental biofilms, protection or susceptibility to dental diseases caused by S. mutans associated with individual susceptibility to disease varies by immunological response has been emphasized to overcome dental diseases. These mechanisms have yet to be fully elucidated but it seems that immunological tolerance to S. mutans, an important issue that must be assessed is the evaluation of the potential antimicrobial
dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) microculture tetrazolium viability assay [36]. A total of 20 μL of MTT solution (5 mg/mL) was added to each well and incubated further for 4 h at 37 °C. After reaction was done, the supernatant was removed and 200 μL of dimethyl sulfoxide (DMSO) was added into each well. Absorbance was measured at 595 nm using a microplate reader
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activity of natural compounds. Herewith, we have focused on the molecular weight of chitosan and other bioactive compounds. As the result, CTSN having average molecular weight about 6.6 kDa markedly inhibited the enzyme function of DSase from S. mutans was characterized as the potential compound look very promising in this regard. Biochemical analysis to elucidate effect of CTSNs and other bioactive substances on DSase known as a major causing dental caries of S. mutans revealed that aminomonosaccharides such as glucosamine, mannosamine, and galactosamine, a specified hydrolysate of chitosan designated to CTSNs (MW 3∼8 kDa), and hyaluronic acid remarkably exerted the growth inhibition over 95 % of DSase (Figs. 2 and 4). Our report also confirms that S. aureus is less susceptible in vitro to the series of CTSN than S. mutans (Table 1). Obviously, it remains to be addressed whether this behavior reveals that S. aureus has overall less susceptibility to the series of CTSNs, or is rather a consequence related with the lower toxicity of the compounds. These observations are enforced by several demonstration of in vitro antimicrobial activity of chitosan depending on the molecular weight, and its degree of deacetylation to other bacterial strains, as earlier study demonstrated (Choi et al. 2001; Fernandes et al. 2008). For several decades, various applications of HMWC and its hydrolysates with different molecular weights or deacetylation degree were widely focused to investigate their biological activities including antimicrobial (Moon et al. 2007; No et al. 2002) and immunostimulating (Moon et al. 2007; Park et al. 2011) activities. However, due to shortcomings of the preparation of chitosan hydrolysates (CTSNs), various studies were performed to establish the best conditions for the high-yield production of the specific size of hydrolysates. In particular, all sizes of CTSNs tested in this study could effectively inhibit the growth of bacterial cells which were isolated as antibiotic-resistant bacteria from porcine sperm fluid (data not shown). These CTNSs could be applied as a potent antimicrobial agent toward various pathogenic microorganisms. Conclusively, these results suggest that especially CTSNs ranging from 3 to 8 kDa are of the potent natural material that can be prepared in the cheapest way compared with other molecules tested in this study as a potential ingredient useful for antibacterial component against oral diseases.
Acknowledgments This study was supported by Regional Innovation System (RIS, No. R0001090) funded from the Ministry of Knowledge Economy for Gachon University Bio Health Solution, and partly supported by grants from the grant at Gachon University. Conflict of interest The authors declare that they have no competing interests.
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Appl Microbiol Biotechnol Liao S, Klein MI, Heim KP, Fan Y, Bitoun JP, Ahn SJ, Burne RA, Koo H, Jeannine Brady L, Wen ZT (2014) Streptococcus mutans extracellular DNA is upregulated during growth in biofilms, actively released via membrane vesicles, and influenced by components of the protein secretion machinery. J Bacteriol 196:2355–2366. doi: 10.1128/JB.01493-14 Liu Y, Chu L, Wu F, Guo L, Li M, Wang Y, Wu L (2014) Influence of pH on inhibition of Streptococcus mutans by Streptococcus oligofermentans. Eur J Oral Sci 122:57–61. doi:10.1111/eos.12102 Moon JS, Kim HK, Koo HC, Joo YS, Nam HM, Park YH, Kang MI (2007) The antibacterial and immunostimulative effect of chitosanoligosaccharides against infection by staphylococcus aureus isolated from bovine mastitis. Appl Microbiol Biotechnol 75:989–998. doi: 10.1007/s00253-007-0898-8 Moye ZD, Zeng L, Burne RA (2014) Fueling the caries process: carbohydrate metabolism and gene regulation by Streptococcus mutans. J Oral Microbiol 6. doi:10.3402/jom.v6.24878 24878 No HK, Park NY, Lee SH, Meyers SP (2002) Antibacterial activity of chitosans and chitosan oligomers with different molecular weights. Int J Food Microbiol 74:65–72 Park JK, Kim ZH, Lee CG, Synytsya A, Jo HS, Kim SW, Park JW, Park YI (2011) Characterization and immunostimulating activity of a
water-soluble polysaccharide isolated from Haematococcus lacustris. Biotechnol Bioprocess Eng 16:1090–1098 Rasouli M, Ostovar-Ravari A, Shokri-afra H (2014) Characterization and improvement of phenol-sulfuric acid microassay for glucose-based glycogen. Eur Rev Med Pharmacol Sci 18:2020–2024 Sano H, Matsukubo T, Shibasaki K, Itoi H, Takaesu Y (1991) Inhibition of adsorption of oral Streptococci to saliva treated hydroxyapatite by chitin derivatives. Bull Tokyo Dent Coll 32:9–17 Sarasam AR, Brown P, Khajotia SS, Dmytryk JJ, Madihally SV (2008) Antibacterial activity of chitosan-based matrices on oral pathogens. J Mater Sci Mater Med 19:1083–1090. doi:10.1007/s10856-007-3072-z Virga C, Landa C, Beltramo D, Ausar F, Dorronsoro ST (2003) Low and high molecular weight chitosans interactions with Streptococcus mutans: an in vitro study. Acta Odontol Latinoam 16:9-16 Zhao H, Wu B, Wu H, Su L, Pang J, Yang T, Liu Y (2006) Protective immunity in rats by intranasal immunization with Streptococcus mutans glucan-binding protein D encapsulated into chitosancoated poly(lactic-co-glycolic acid) microspheres. Biotechnol Lett 28:1299–1304. doi:10.1007/s10529-006-9086-7 Zivanovic S, Basurto CC, Chi S, Davidson PM, Weiss J (2004) Molecular weight of chitosan influences antimicrobial activity in oil-in-water emulsions. J Food Prot 67:952–959
BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING
Comparison of inhibitory activity of bioactive molecules on the dextransucrase from Streptococcus mutans Choon Geun Lee 1 & Jae Kweon Park 1
Received: 23 March 2015 / Revised: 8 May 2015 / Accepted: 13 May 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract The effect of chitosan with different molecular weights and other natural substances on dextransucrase (DSase) activity from a representative oral pathogen Streptococcus mutans was elucidated. Among other bioactive substances, amino-monosaccharides such as glucosamine, mannosamine, and galactosamine exerted the enzyme inhibitory activity over 95 % of DSase. The specified hydrolysates derived from the hydrolysis of high molecular weight chitosan (HMWC) designated to CTSN, CTSN-P, CTSN-B, and CTSN-S with different molecular weights ranging from 3 to 8 kDa showed the similar inhibitory activity toward DSase. Also, the hyaluronic acid (MW 8.9 kDa), sulfated chitin, and amino-monosaccharides demonstrated the significant activity, CTSN, CTSN-P, CTSN-B, and CTSN-S are of potent bioactive substances that can be prepared in the cheapest way compared with other molecules tested available for antibacterial agent useful for human oral health. Keywords Dextransucrase . Streptococcus mutans . Antimicrobial activity . Oral health . Chitosan
Introduction Streptococcus mutans is a Gram-positive organism that is the principal etiological agent in the formation of dental cavities in humans (Li et al. 2014). Streptococcusis classified as a genus of spherical gram-positive bacteria
* Jae Kweon Park [email protected] 1
Department of Life Sciences, Gachon University, Seongnamdaero 1342, Seongnam-si, Gyeonggi-do 461-701, South Korea
belonging to the phylum Firmicutes and the lactic acid producing bacterial group. S. mutans, which is widely recognized as the main etiological pathogenic agent of dental cavities in humans (Liao et al. 2014; Liu et al. 2014; Moye et al. 2014). Not only bacterial infections but also conditions in the oral cavity are truly diverse and complex. Therefore, S. mutans must tolerate rapidly to harsh environmental fluctuations including exposure to various antimicrobial agents in order to survive in the oral cavity (Guo et al. 2014; Liao et al. 2014). Most of etiological agents of dental caries are associated with its ability to catabolize various sugars to form insoluble biofilm and produce lactic acid to generate the acid environment (Guo et al. 2014). Owing to these features concerted with its survivability, natural bioactive substances such as sugar-alcohol, antocyanine, and specific sugars have been focused as potent antimicrobial materials. It has been implicated that extracellular water-insoluble polysaccharide from dietary sugar produced by dextransucrase (DSase) and its adhesion to dental enamel are known to the most clinically important biological abilities of S. mutans (Chen et al. 2012; Decker et al. 2014). However, the mechanisms under which this cariogenic pathogen can survive or proliferate under in such environmental conditions are so far largely unknown. Dental caries produced from oral acidogenic bacteria is well defined as a dental biofilm-related oral disease associated with the increasing consumption of dietary sugars and/or fermentable carbohydrates. Along with frequent exposure to sugars such as glucose and sucrose, S. mutans producing dental biofilms can metabolize the sugars to organic acids. Significant persistence of S. mutans under the acidic environment encourages the proliferation of S. mutans including other acidogenic bacteria, resulting in their ability to survive at a low-pH condition. Upon changing to the low-pH condition, the initiation of the
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dental caries starts under the surface of teeth covered with biofilm (Chen et al. 2012; Decker et al. 2014). Therefore, the potential acidification of dental biofilms and later cavity formations can be decreased by eliminating the adherence of S. mutans to the surface of teeth or dental biofilms. In addition, individual susceptibility to disease varies by immunological response which has been proposed to confer protection or susceptibility to dental diseases. These mechanisms have yet to be fully elucidated but it seems that immunological tolerance to S. mutans at the mucosal surface may make individuals more prone to colonization with S. mutans (Moon et al. 2007; Zhao et al. 2006). For several decades, antimicrobial activity of natural substances toward S. mutans has extensively been investigated. Among the most of polysaccharides chitosan, a linear polysaccharide of β-1,4 linkage of glucosamine has been reported as a potent antimicrobial substance with different molecular weights on bacterial growth (Aam et al. 2010). Although the precise mechanisms are so far largely unknown, several mechanisms have been proposed for the antimicrobial activity by chitosan and its related molecules (Alencar de Queiroz et al. 2006; Alipour et al. 2009). For instance, one of the mechanisms proposed is the ability of chitosan to be attached to the cell wall of bacteria, which results in the reduction of bacterial metabolism (Busscher et al. 2008; Chen et al. 2008). Another mechanism is proposed that would inhibit activity of chitosan on the transcription of RNA by adsorption of chitosan to the DNA of bacteria (Chen et al. 2008), depending on the size of chitosan and its degree of deacetylation, and chemical features (Choi et al. 2001; Fernandes et al. 2008). Owing to these features, it has drawn attention as a potent antimicrobial agent specifically in human oral health (Sano et al. 1991; Sarasam et al. 2008; Zivanovic et al. 2004). However, despite the usefulness of chitosan, its low water-solubility due to its high degree of polymerization is a limiting factor for its application in various fields. Therefore, several studies have been focused on the relationship between the depolymerization of chitosan to reduce the molecular weight and its biological activities, which is readily soluble in water or diluted weak acidic solution (Jing et al. 2007; Kulikov et al. 2009; No et al. 2002). Although the various molecular weights of chitosans derived from the hydrolysis of high molecular weight chitosan (HMWC) are well known to have considerable antimicrobial activities against various pathogenic bacteria, there was little work reported on their biological activity against the periodontal pathogen S. mutans. Therefore, the aim of this study is to elucidate the antimicrobial activity of chitosans with different molecular weights and other bioactive substances against the representative periodontopathogen S. mutans. Our results can propose the potent utility of those molecules as an antimicrobial agent to support human health.
Materials and methods Materials A high molecular weight chitosan (HMWC, 95 % degree of deacetylation) with approximately 200 kDa was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Various sizes of different molecular weight chitosans were prepared in enzymatic digestion using chitosanase from Streptomyces griseus, which was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Bioactive substances including various monosaccharides and other polysaccharides such as alginate, β-glucan, and hyaluronic acid were also purchased from Sigma Chemical Co. (St. Louis, MO, USA). Other crude products were extracted from the marine algal sources. S. mutans Clarke (ATCC® 700610D-5™) was provided by Prof. Jae-Gyu Jeon (Chonbuk University, Korea). All other reagents were of the highest grade available. Preparation of chitosan hydrolysates In order to increase the potent possibility to get partial degradation of HMWC, the HMWC was dissolved in 2 % acetic acid to be 0.5, 1.0, and 2.0 g/0.1 L and subjected to partial depolymerization by using 0.2 U of chitosanase, respectively. Mixture was reacted with mixing at 700 rpm using magnetic bar for 2 h at room temperature to perform the partial digestion. Reactants were boiled for 5 min at 100 °C in order to denature chitosanase and also centrifuged for 10 min at 13, 000 rpm to remove denatured chitosanase and indigested material. Pre-chilled ethanol was used to precipitate hydrolysates derived from the hydrolysis of HMWC. The precipitates were recovered by centrifugation for 10 min at 13,000 rpm and washed with the ethanol until the pH value was same as the ethanol. Each hydrolysate after digestion of HMWC was further fractionated to remove low molecular weight chitosan using a small size of gel-filtration column chromatograph (Φ 1.0 cm × L 30 cm) packed with Bio Gel-P 4 (Bio-RAD, USA) gel, which was pre-equilibrated with a volatile buffer consisting of ammonia/formic acid (pH 7.5). The final products derived from the hydrolysis of HMWC with different concentration of 0.5, 1.0, and 2.0 g/0.1 L were lyophilized (IlShin Co. Ltd., Seoul Korea), designated to CTSN-S, CTSN-B, and CTSN-P respectively. CTSN is prepared as a mixture of CTSN-P/B/S, which were pooled together equally and used all for subsequent experiments. Instrumental analysis of MW of CTSNs To determine the average molecular weight of chitosan, each hydrolysate obtained from the hydrolysis of HMWC was applied to high-performance liquid chromatography (HPLC), using a PolySep-GFC-P3000 column (7.8 × 300 mm,
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Phenomenex, USA). For the detection, 10 μL of 1.0 % sample solution was injected into the system, eluted with water at a flow rate of 0.8 mL/min at 63.8 °C, and detected with ELSD (evaporative light scattering detector, Alltech). Pullulans (Sigma Chemical Co., St. Louis, MO, USA) with a wide range of molecular weights (5600∼2000 kDa) were used as standard molecular markers. The average MW of hydrolysates (CTSNs) of chitosan was calculated from the calibration curve and area ratio of major peaks. Determination of bacterial susceptibility S. mutans was incubated anaerobically in brain-heart infusion (BHI) broth consisting of 1 % sucrose at 37 °C for 3 days. In order to evaluate the susceptibility of S. mutans toward various dietary sugars and its metabolic condition, the assay was performed as follows: solutions containing various bioactive substances were each prepared in 1 % (w/v) distilled water or diluted acetic acid (0.1 %). Each solution prepared was added to BHI broth ranging from 0.1 to 0.5 % (v/v). The 10 L of inoculums of S. mutans pre-incubated in BHI broth was added in fresh broth with or without sample. Each broth was then further incubated at 37 °C for 12 h anaerobically. For antimicrobial activity, Staphylococcus aureus cultured in LB medium was also used as a comparative microorganism. Susceptibility of S. mutans toward those materials was determined by measuring the growth rate at 600 nm. Inhibitory effect on the growth of S. mutans S. mutans were cultured in the presence or absence of bioactive materials under the optimum condition to assign the metabolic ability for the formation of biofilms. This is known as the main factor causing dental caries. Since S. mutans can effectively use dietary sucrose to rapidly synthesize insoluble and some soluble extra cellular polysaccharides (EPSs) using dextransucrase (DSase), EPS production was quantitatively estimated by phenol-sulfuric assay (Kumar et al. 1988; Rasouli et al. 2014). Briefly, the aliquots taken from each sample at time intervals of 3, 6, 9, and 12 h were subjected to the method of using glucose as the standard material. With this in regard, several chitosans with different molecular weights and other comparative materials were tested to estimate their effectiveness as DSase inhibitor. For determination of influence of bioactive materials on DSase, enzyme activities were determined by the quantitative analysis of EPS produced by DSase, as described above. In order to determine the cytotoxicity, RAW264.7 murine macrophages cells (5 × 104 cells/well) were suspended in complete RPMI1640 medium, and the proliferation of the cells was assessed in micro-culture tetrazolium viability assay using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), using a multi-detection microplate reader
(Molecular Devices, Sunnyvale,CA, USA) as described the previous study (Park et al. 2011). All experiments for the MTT assay were performed in triplicates. Statistical analysis Representative data demonstrated in this study were expressed as the means of ±SD from at least three separate experiments, unless otherwise indicated. The statistical significance of the differences between mean values obtained from each experiment of less than 0.05 (as p value) was considered.
Results Determination of inhibitory concentration (IC50) of chitosan with different molecular weight Average molecular weights of chitosans designated to CTSN, CTSN-P, CTSN-B, and CTSN-S obtained from the partial hydrolysis of HMWC and CTSN made of CTSN-P/B/S as a mixture were determined in HPLC analysis to be ranging from 3.0 to 8.0 kDa, respectively (Table 1). Till a decade ago, antibacterial applications of chitosan hydrolysates with different molecular weights or deacetylation degree were widely studied to investigate their biological activities. However, due to shortcomings of the preparation of chitosan hydrolysates having the specific sizes, there has been a surge in interest for enzymatic partial digestion of chitosan under various mild or extreme conditions to yield high (Table 1). Since all types of chitosan hydrolysates (CTSNs) could effectively inhibit the growth of bacterial cell growth toward both PWG and F1 strains isolated as antibiotic-resistant bacteria from porcine sperm fluid (data not shown), each hydrolysate was applied as a potent antimicrobial agent toward various pathogenic microorganisms. IC50 for each compound was determined based on bacterial cell growth, as shown in Table 1. All compounds have showed similar activity toward both bacterial strains tested in this study. These results are correlated to the previous studies that demonstrated the relationship between Table 1 activity
Effect of molecular weight of chitosan on antimicrobial
Sample
Average Yield IC50 (μg/mL) MW (kDa) (%) Streptococcus MRSA (Staphylococcus mutans aureus CCARM 3230)
CTSN CTSN-P CTSN-B CTSN-S
6.650 7.783 5.452 3.510
26.7 38.4 26.8 14.8
150 200 190 170
230 220 240 210
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efficacy and the significance of the size of molecules. Based on these results, we suggest that average molecular weight of chitosan on antimicrobial activity is an important factor, as early studies demonstrated the significance of the size of molecule (Busscher et al. 2008; Fernandes et al. 2008; Jing et al. 2007). Measurement of DSase To address the preliminary antibacterial mechanism of bioactive substances against S. mutans, production of EPS was quantitatively elucidated. As shown in Fig. 1, DSase activity was estimated in the ability for EPS production which gradually increased in a time-dependent manner. The results indicated that S. mutans can efficiently metabolize sucrose for the proliferation of bacterial cells and utilize its products for the expression of protein associated in sugar transferase, called DSase. As many reports demonstrated, DSase is a critically important enzyme involved in the glucose transfer to make insoluble EPS, also known as a biofilm (Lee et al. 2015; Liao et al. 2014). Upon determination of enzyme activity using a culture supernatant containing crude DSase, we found that EPS was proportionally synthesized in a time-dependent manner corresponding to the DSase activity (Fig. 1). Inhibitory activity of monosaccharides on DSase Among the most several polymers, chitosan exhibited a considerable antimicrobial activity against S. mutans depending
Fig. 1 Determination of DSase activity. S. mutans was incubated in brain heart-infusion (BHI) broth consisting of 1 % starch and 1 % sucrose, respectively. The incubation was performed at 37 °C for 3 days in CO2 incubator. Culture was centrifuged at 13,000 rpm for 10 min to collect the supernatant containing active DSase in crude. Enzyme activity of DSase was estimated to measure the amount of EPS in the reaction mixture consisting of 1 % sucrose in 25 mM phosphate buffer (pH 7.0) time dependently. EPS produced by DSase was obtained by ethanolic precipitation, and amount of the EPS was determined by phenolsulfuric acid method (Kumar et al. 1988; Rasouli et al. 2014)
on the size of molecule. It has not yet been clearly understood; however, it is hypothesized that protonation on amino groups at C-2 on chitosan backbones is one of the most important factors in electrostatic interaction between the polycationic structure and the predominantly anionic components of the microorganism’s cell wall (Sarasam et al. 2008; Virga et al. 2003). On the other hand, degree of deacetylation of chitosan is also known to be a critical factor determining the capability of biological function. Herein, we tested the inhibitory effect of monosaccharides on DSase activity. As seen in Fig. 2, amino sugars such as glucosamine, mannosamine, and galactosamine showed strong inhibitory activity on DSase activity. No inhibitory activity of neutral sugars on DSase was observed; instead of this, the activity tended to increase. These results were correspondingly similar to our previous result, which demonstrated that amino sugars inhibited bacterial cell growth during the culture in vitro (Jung et al. 2013). Although inhibitory mechanisms by those amino sugars for DSase which were produced from S. mutans were not completely understood, these results made us to suggest that S. mutans may not able to metabolize such amino sugar as carbon and nitrogen sources because of their high sensitivity on electrostatic interaction with some uncertain component of S. mutans. Effect of molecular weight on DSase It is well defined that chitosan and its hydrolysates produced by several ways have showed significant antibacterial activity depending on the size and degree of deacetylation (Busscher et al. 2008; Choi et al. 2001; Fernandes et al. 2008; No et al. 2002). In order to address more precise mechanism how those molecules inhibit bacterial cell growth, we strived to elucidate the function of highly polymerized chitosan and other potent agents. As seen in Table 1, average molecular weight of hydrolysates called CTSNs prepared in this study was determined by HPLC, obtained successfully from the hydrolysis of HMWC. As seen in Fig. 3, various sizes of chitosans (CTSN, CTSN-P, CTSN-B, and CTSN-S) prepared as low molecular weight chitosan showed also significant inhibitory effect on DSase activity. These results suggest that exposure of DSase from S. mutans to the CTSNseries prepared in this study may rapidly be rendered by them or has much higher affinity toward them than glucose used as control. Based on these results, we found that amino sugars and its high polymerized polysaccharides are potent inhibitor for DSase, which plays a critical role in the formation of biofilm. Although it is not able to explain the exact mechanism of the antimicrobial activity of CTSNs toward S. mutans yet, our current investigation is apparently important point to explain the functions of CTSNs how polycationic polymer can interact to form polyelectrolyte complex with DSase. Based on these
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Fig. 2 Inhibitory effect of various monosaccharides on DSase activity. Enzyme activity of DSase was estimated to measure the amount of extracellular polysaccharide (EPS) in the reaction mixture consisting of 1 % sucrose in the presence of various monosaccharides (1 % each, respectively) in 25 mM phosphate buffer (pH 7.0). EPS produced by DSase was obtained by ethanolic precipitation, and amount of the EPS
was determined by phenol-sulfuric acid method, as describe in Fig. 1. Distilled water (DW) was used as negative control, herein no bacterial cells were provided into the culture. Mean values were estimated to be with ±SD expressed with error bars from three separate experiments (p < 0.05)
results, we suggest that inhibitory effect of series of CTSNs on DSase activity or growth rate attenuates the metabolic capacity by blocking of nutrient permeation through cell wall associated with the expression of
DSase. Therefore, inhibition of either cell growth (Fig. 3b) or DSase activity should be a crucial point to prevent the biofilm formation from S. mutans. Here, a question arose how glucosamine can inhibit the DSase
Fig. 3 Inhibitory effect of chitosan hydrolysates on DSase activity. Enzyme activity of DSase was estimated to measure the amount of EPS in the reaction mixture consisting of 1 % sucrose in the presence of various size of chitosans (CTSNs, 1 % each, respectively) in 25 mM acetate buffer (pH 4.5) (a). EPS produced by DSase was obtained by ethanolic precipitation and amount of the EPS was determined after precipitated the chitosan by bring pH to pH 9.0 using 1 M NaOH. Distilled water (DW) was used as negative control without bacterial cells. The morphology of bacterial cells was observed under 400 magnification using a microscope (Olympus CKX41, Hicksville, NY, USA) in the presence or absence of chitosans (b). Tested samples were asfollows: a, control; b, CTSN-P; c, CTSN-b; d, CTSN-S; e, CTSN
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activity. We are not able to answer clearly, but we hypothesize that it may be possibly placed on the active or biding site of DSase, which is not able to bind or interact with sucrose for further process. Inhibitory effect of bioactive substances on DSase Dental caries to be the single most common biofilmdependent oral infectious disease resulted from the interaction specific bacteria like S. mutans because it can synthesize EPS from dietary sucrose using DSase that adsorb to surface of teeth. This fact demonstrated that the adsorption of bacterial cells on surface and the formation of highly structured cell clusters must be associated with the ability of an organism to survive (Decker et al. 2014; Kubota et al. 2005), which are key factors causing a dental caries. Upon investigating using CTSNs, the inhibitory effects of other bioactive substances on DSase to elucidate biological functions that compare with the CTSNs were also performed. As shown in Fig. 4, obviously, hyaluronic acid (MW estimated to be 8.9 kDa) showed significant inhibitory activity on DSase, whereas other polysaccharides tested in this study showed slightly lower ability toward DSase than CTSNs. No significant activity difference between CTSNs and hyaluronic acid used with specific size of molecular weight was observed, indicating that not only molecular weight of bioactive materials and their electrostatics are critical factors on the inhibition of acidogenic properties of S. mutans. On the other hand, both extracts from Curcuma and Artemisa species were tested separately in this study to sort out other candidates useful with CTSNs as a co-effector. In the preliminary test, we found that these extracts showed highly significant antioxidant activities (unpublished data) can be used for oral hygiene in general in Asian countries. However, less is known about the biological activities of the extracts from Curcuma and Artemisa toward S. mutans. It is worthy to investigate the functional activity regarding the Fig. 4 Inhibitory effect of bioactive substances on DSase activity. The reaction was carried out in same condition aforementioned in Fig. 3. Enzyme activity of DSase was directly estimated in time dependently without ethanol precipitation by phenol-sulfuric acid method. Distilled water (DW) was used as negative control without bacterial cells. Mean values were estimated to be with ±SD expressed with error bars from three separate experiments (p < 0.05)
relationships between antioxidant and antimicrobial activities of them. Therefore, in the present study, we tried to get valuable information by using several kinds of natural biomaterials including various kinds of mono- and polysaccharides and plants extracts that can be used for oral hygiene in general in Asian countries. As shown in Fig. 5, water-soluble extract of Curcuma showed significant inhibitory activity; however, Artemisa’s extract did not show any significant inhibitory activity on DSase. These results are correlated to the antimicrobial activity of Artemisa’s extract, not showed any significant effect toward S. mutans by seeing the growth rate. Based on these result, Curcuma’s water-soluble extract can be used as a potent co-effector with CTSNs to handle the protection of biofilm formation by DSase in S. mutans as for oral hygiene. Overall, the mechanism associated with biofilm synthesis and aciduric properties of S. mutans must be considered with many types of glucosyltransferase and fructosylase released from S. mutans. Furthermore, phylogenetically different types of S. mutans form structurally and metabolically distinctive biofilms depend on type of sugars used in extracellular polysaccharides synthesis by S. mutans. Based on these results, we suggest that some specificity and size of sugars might be involved in the inhibition of enzyme activity of DSase from S. mutans, not necessary of bioactive materials having strong antioxidant activities. To confirm the absence of toxicity for the application of CTSN-series as pharmaceutical material for human oral health, we tested the cytotoxicity of CTSN toward RAW264.7 cells. As shown in Fig. 6, no significantly detectable level of cytotoxicity of CTSN on the RAW264.7 cells at the concentrations ranging from 1 to 1000 μg/mL was observed. The results clearly demonstrated that the oral application of CTSNs may be effective in alleviating or preventing the oral disease caused by S. mutans in vivo. Taken collectively, our data described here suggest that CTSN with specified size of molecular weight can be used as pharmaceutical material for oral health for human.
Appl Microbiol Biotechnol Fig. 5 Inhibitory effect of watersoluble extracts from Curcuma and Artemisa on DSase. The reaction was carried out in same condition aforementioned in Fig. 1. Mean values were estimated to be with ±SD expressed with error bars from three separate experiments (p < 0.05). Distilled water (DW) was used as negative control without bacterial cells
Discussion The antibacterial application of chitosan with different molecular weight is an attractive alternative to investigate the unique biological tool. In this context, we demonstrated the importance of (1) the assessment of inhibitory properties of CTSNs toward DSase and (2) the comparison of activity of natural bioactive compounds. We also demonstrated the properties of CTSN with different molecular weights against the specific enzyme DSase produced as one of representative pathogenic agents from S. mutans to elucidate the antimicrobial activity for oral health for human. An
Fig. 6 Cytotoxicity of chitosans. Murine macrophage RAW264.7 cells were suspended in complete RPMI1640 medium. RAW264.7 cells were seeded at a density of 5 × 104 cells in 100 μL per well and incubated in a 96-well microplate for 24 h. Cells were treated with different concentrations of the CTSN prepared with serial dilution ranging from 1 to 1000 μg/mL, while the negative control was treated with only the medium. The proliferation of the cells was assessed using the 3-[4,5-
emphasis of antimicrobial activity of the series of CTSN was made to include S. mutans and S. aureus that were resistant to commercial antibiotics (Table 1). Especially, S. mutans can tightly adhere to the surface of teeth. Owing to the potential acidification of dental biofilms, protection or susceptibility to dental diseases caused by S. mutans associated with individual susceptibility to disease varies by immunological response has been emphasized to overcome dental diseases. These mechanisms have yet to be fully elucidated but it seems that immunological tolerance to S. mutans, an important issue that must be assessed is the evaluation of the potential antimicrobial
dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) microculture tetrazolium viability assay [36]. A total of 20 μL of MTT solution (5 mg/mL) was added to each well and incubated further for 4 h at 37 °C. After reaction was done, the supernatant was removed and 200 μL of dimethyl sulfoxide (DMSO) was added into each well. Absorbance was measured at 595 nm using a microplate reader
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activity of natural compounds. Herewith, we have focused on the molecular weight of chitosan and other bioactive compounds. As the result, CTSN having average molecular weight about 6.6 kDa markedly inhibited the enzyme function of DSase from S. mutans was characterized as the potential compound look very promising in this regard. Biochemical analysis to elucidate effect of CTSNs and other bioactive substances on DSase known as a major causing dental caries of S. mutans revealed that aminomonosaccharides such as glucosamine, mannosamine, and galactosamine, a specified hydrolysate of chitosan designated to CTSNs (MW 3∼8 kDa), and hyaluronic acid remarkably exerted the growth inhibition over 95 % of DSase (Figs. 2 and 4). Our report also confirms that S. aureus is less susceptible in vitro to the series of CTSN than S. mutans (Table 1). Obviously, it remains to be addressed whether this behavior reveals that S. aureus has overall less susceptibility to the series of CTSNs, or is rather a consequence related with the lower toxicity of the compounds. These observations are enforced by several demonstration of in vitro antimicrobial activity of chitosan depending on the molecular weight, and its degree of deacetylation to other bacterial strains, as earlier study demonstrated (Choi et al. 2001; Fernandes et al. 2008). For several decades, various applications of HMWC and its hydrolysates with different molecular weights or deacetylation degree were widely focused to investigate their biological activities including antimicrobial (Moon et al. 2007; No et al. 2002) and immunostimulating (Moon et al. 2007; Park et al. 2011) activities. However, due to shortcomings of the preparation of chitosan hydrolysates (CTSNs), various studies were performed to establish the best conditions for the high-yield production of the specific size of hydrolysates. In particular, all sizes of CTSNs tested in this study could effectively inhibit the growth of bacterial cells which were isolated as antibiotic-resistant bacteria from porcine sperm fluid (data not shown). These CTNSs could be applied as a potent antimicrobial agent toward various pathogenic microorganisms. Conclusively, these results suggest that especially CTSNs ranging from 3 to 8 kDa are of the potent natural material that can be prepared in the cheapest way compared with other molecules tested in this study as a potential ingredient useful for antibacterial component against oral diseases.
Acknowledgments This study was supported by Regional Innovation System (RIS, No. R0001090) funded from the Ministry of Knowledge Economy for Gachon University Bio Health Solution, and partly supported by grants from the grant at Gachon University. Conflict of interest The authors declare that they have no competing interests.
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