Turkish Journal of Biochemistry – Türk Biyokimya Dergisi; 2016; 41 (4): 269-274

Biochemistry Research Article – 19577

Chun ping Xu, Ying Zeng, Hongqian Shentu, Aijing Zhao, Duobin Mao* Effects of cigarette smoke condensate on the production and characterization of exopolysaccharides by

Sigara dumanı kondensatının Streptococcus mutans tarafından üretilen eksopolisakkaritlerin üretimi ve karakterizasyonu üzerine etkisi

DOI 10.1515/tjb-2016-0039 Conclusion: This study revealed that CSC was directly able Received October 2, 2015; accepted February 12, 2016; to affect molecular mass and structural conformation of published online August 1, 2016 EPS from S. mutans. The molecular characterization of EPS would become an indicator in certain pathological Abstract: Objective: Streptococcus mutans is regarded as the disorders. major agent causing dental caries. It has been well docu- mented that cigarette smoke affects the growth of S. mutans. Keywords: Exopolysaccharides, Cigarette smoke conden- This study investigate the effect of cigarette smoke conden- sate, Streptococcus mutans, Acetic acid sate (CSC) on the production and characterization of exo- polysaccharides (EPS) produced by S. mutans ATCC 35668. Özet: Amaç: Streptococcus mutans diş çürüklerine neden Methods: Cigarettes of Shanhua brand were used to olduğu bilinen en önemli ajandır. Sigara tüketiminin S. prepare the CSC. S. mutans was cultured in MSB media mutans üremesine neden olduğu literatürde detaylı olarak with the addition of CSC under anaerobic condition. bildirilmiştir. Bu çalışmada sigara dumanı kondensatının Furthermore, the EPS fraction was isolated and purified (CSC) S. mutans (ATCC 35668) eksopolisakkaritlerinin by gel filtration chro-matography on Sepharose CL-6B. (EPS) üretimi ve karakterizasyonu araştırılmıştır. The molecular characterization of EPS was analyzed by GC-MS, FT-IR and size exclusion chromatography/multi- Metod: CSC üretimi için Shanhua tütünü sigaraları kul- angle laser light scattering (SEC/MALLS) system. lanılmıştır. S. mutans kültürü MSB ortamında anerobik koşullarda CSC eklenmesi ile sağlanmıştır. Daha sonra Results: The results showed that CSC at tested concentra- EPS fraksiyonu izole edilmiş ve Sepharose CL-6B jel tions could significantly increase the growth of S. mutans filtrasyonu ile saflaştırılmıştır. EPS nin molekül yapısı and acetic acid production, compared with the control. GC-MS, FT-IR and boyut ayırıcı dışlama kromatografisi / The CSC was not found to affect carbohydrate composi- multi-angle laser light scattering (SEC/MALLS) sistemi ile tion of the EPS, but the molecular mass of EPS decreased karakterize edilmiştir. from 3.04×104 g/mol (without CSC) to 2.75×104 (with CSC). The SEC/MALLS also revealed the molecular conforma- Bulgular: Elde edilen sonuçlar, CSC test edilen konsant- tion of EPS changed from flexible coil to globular shape in rasyonlarda S. mutans üremesi ile asetik asit artışını, aqueous solution. kontrole göre, istatistiksel olarak anlamlı düzeyde artır-

Ying Zeng: College of Food and Biological Engineering, Zhengzhou *Corresponding author: Duobin Mao: College of Food and University of Light Industry, Zhengzhou, Henan 450002, People’s Biological Engineering, Zhengzhou University of Light Industry, Republic of China, e-mail: [email protected] Zhengzhou, Henan 450002, People’s Republic of China, e-mail: Hongqian Shentu: Technical Center of China Tobacco Chuanyu [email protected] Industrial Co. Ltd, Chengdu, Sichuan 610066, P.r. China, Chun ping Xu: College of Food and Biological Engineering, e-mail: [email protected] Zhengzhou University of Light Industry, Zhengzhou, Henan 450002, Aijing Zhao: College of Life Science, Henan Normal University, People’s Republic of China, e-mail: [email protected] Xinxiang, Henan, 453007, P.r. China, e-mail: [email protected] 270 Chun ping Xu et al.: Effect of cigarette smoke condensate dığını göstermiştir. CSC, EPS üzerinde karbohidrat yapı- concerning the influence of CSC on the molecular char- sını değiştirmemiştir, ancak EPS’nin moleküler kütlesinin acterization of exopolysaccharides (EPS) produced by S. CSC yokluğunda 3.04×104 g/mol iken CSC eklendiğinde mutans. In this study, the influence of CSC on the pro- düştüğü saptanmıştır (2.75×104 g/mol). SEC/MALLS analizi duction and molecular characterization of EPS produced de, sulu ortamda EPS esnek sarmal yapısının yuvarlak form S. mutans were investigated. (globüler) yapıya dönüştüğünü göstermiştir.

Sonuç: Bu çalışma CSC nin S. mutans tarafından üretilen EPS’lerin moleküler kütlesi ve yapısal biçimi üzerinde doğrudan etkisi olduğunu ortaya çıkarmıştır. Buna göre 2 Materials and Methods EPS nin moleküler karakterizasyonu bazı patolojik rahat- sızlıkların tanısında kullanılabilecek bir ayırtaç olabilir. 2.1 Cigarette smoke condensate Anahtar Kelimeler: Eksopolisakkaritler, Sigara dumanı The volatile products in the CSC were collected by kondensatı, Streptococcus mutans, Asetik asit washing the smoke from the complete burn of 5 ciga- rettes (brand: Shanhua) containing 11 mg of tar, 1.1 mg of nicotine, and 15 mg of carbon monoxide per ciga- 1 Introduction rette (Zhengzhou tobacco cigarette Co. Ltd, China). The preparation procedure of CSC was the same as our pre- Streptococcus mutans is one of important members vious report [14]. Dilutions were made in complete cell of the oral in the human oral cavity [1]. Dental culture medium as 1:20, 2:20 and 3:20 (v/v) using the caries remains the most prevalent chronic disease [2]. thawed aliquots. The initiation of efficient strategies for the management of this problem calls for the meticulous assessment of the factors affecting caries procedure. S. mutans is con- 2.2 Microorganisms sidered an important agent in the formation of human dental caries [3]. Streptococcus mutans ATCC 35668 was obtained from the Cigarette smoking is one important etiologic factor American Type Culture Collection (Manassas, USA) used with a documented effect on oral cavity microbial ecosys- throughout this study. The stock organism was stored in tems [4]. Cigarette smoke is a complex mixture of chemi- TSB (Mikrobiologie; Merck, Germany) containing 50% cals produced by the burning of tobacco and its additives glycerol at −80oC. The seed culture was grown in a 250 [5,6]. Tobacco smoking has important effects on the oral mL flask containing 50 mL tryptic soy broth (TSB) at 37oC cavity [7]. Zappacosta et al. demonstrated that cigarette for 18 h anaerobically (80%N2, 10%CO2 and 10%H2) using smoke could impair the salivary function, which has an the Gas Pak culture system (BBL, Cockeysville, MD) [15]. important protective role against dental caries [8]. Zonuz Cell culture experiments were performed in a 500 mL et al. reported that the growth of S. mutans is accelerated Erlenmeyer flask containing 200 mL of cell medium (TSB in the vicinity of cigarette smoke condensate (CSC) [9]. medium added the CSC filtrates as mentioned above) Baboni et al. reported that exposure of both S. mutans after inoculating with 10% (v/v) of seed culture. and Candida albicans to CSC increased the adherence of these oral pathogens to orthodontic material in the setting of increased biofilm formation [10]. Bagaitkar et 2.3 Cell growth, acetic acid and EPS production al. also observed increased biofilm formation following exposure of another oral pathogen, Porphyromonas gin- Bacterial growth was estimated by colony counting on givalis, to CSC [11]. Mutepe et al. investigated that expo- the Mitis Salivarius–Bacitracin (MSB) agar. Plates were sure to very low concentrations of CSC resulted in aug- incubated anaerobically for 72 h at 37°C. The EPS iso- mentation of biofilm formation and attenuation of the lation methods was the same as previous works [14]. pore-forming activity of pneumolysin [12]. The concentration of EPS was determined by the phe- S. mutans is a key contributor to the formation of the nol-sulfuric acid method [16]. Acetic acid content was exopolysaccharide (EPS) matrix in dental . Caries determined using a modified hydroxyl biphenyl assay development in the presence of S. mutans is associated according to the procedure of the carbazole-sulfuric acid with EPS [13]. However, no data are currently available method [17]. Chun ping Xu et al.: Effect of cigarette smoke condensate 271

2.4 Purification and molecular characteriza- (without CSC) were carried out and the results were tion of EPS showed in Figure 1. The results indicated that after 72 h, cell number was increased significantly at the ratio of The EPS purification procedure was the same as previous 1:20 (CSC: cell medium) (v/v), then decreased a little bit works [14]. Protein and total sugar content concentration (but still significantly higher than the control) at increas- was determined according to our previous report [18]. For ing concentrations of CSC. This result was consist with the measurements of composition and the finding by Zonuz et al. [9], who reported that the cig- FT-IR Spectroscopy of EPS, the methods were the same as arette smoke enhances the growth of S. mutans and S. previous works [18]. sanguis in vitro. Furthermore, EPS and acetic acid yields The molecular weights of EPS were estimated by SEC presented an increase tendency, though not all differ- coupled with MALLS Dawn DSP detector (Wyatt Technol- ences are statistically significant. These results proved ogy, Santa Babara, CA) and a Refractive Index (RI) detec- that cigarette smoke accelerated the secretion of EPS and tor (Optilab rEX, Wyatt Technology, Santa Barbara, CA). acetic acid. It has been reported that the EPS produced The methods to determine molecular weights and molecu- by S. mutans contributed to the cariogenic potential of lar properties were the same as previous works [19]. dental biofilms and their resistance to mea- sures [20]. S. mutans plays a major role in metabolizing in MSB medium to using the 2.5 Statistical analyses glucansucrase [21]. Acetic acid can increase the acidity of the oral cavity and promote the reproduction of many All the data were shown in means±SD within signif- harmful [22]. The increase of EPS and acetic acid icance p<0.05 after subjecting to an analysis of vari- production clearly indicates that CSC may facilitate the ance (ANOVA) and processed with SPSS 11.0 (SPSS Inc., formation of oral flora and augment the biofilm forma- Chicago, IL, USA). tion [10], which can explain why the flora of smokers harbors more potential pathogens as compared with the oral flora of non-smokers [23,24]. 3 Results and Discussion 3.2 Effect of CSC on the structural properties 3.1 Effect of CSC on cell growth, EPS and of EPS acetic acid production The EPS from submerged culture of S. mutans in the pres- To evaluate the effect of CSC on the growth of S. mutans, ence or absence of CSC were obtained upon precipitation a comparison between the experimental groups (with with ethanol. After gel filtration chromatography on Sep- different amount of addition of CSC) and controls harose CL-6B, only one fraction was co-eluted from both

6.0 100 16 (a) b (b) (c) c bc c c 5.5 c 14 a 90 b b b 5.0 a 12 a 4.5 80

log cfu/g 10 4.0 70 8 3.5 Exopolysaccharides (mg/L) Exopolysaccharides Lactic acid production (mg/L) production acid Lactic 3.0 60 6 Control 1 2 3 Control 1 2 3 Control 1 2 3 Group

Figure 1: Effect of CSC on cell concentration (a), expolysaccharides (b) and acetic acid (c) production by S. mutans obtained in PTYG agar medium. Control means medium without CSC. Group 1, 2 and 3 means radios of CSC and medium are 1:20, 2:20 and 3:20 (v/v), respectively. a, b, c Statistical analysis at 95% confidence level with same letters in the same figure indicating no significant difference. 272 Chun ping Xu et al.: Effect of cigarette smoke condensate

3.5 7 (a) (b) 3.0 6 Absorbance at 480 nm 2.5 5

2.0 4

1.5 3

Absorbance at 280 nm at Absorbance 1.0 2

0.5 1

0.0 0 0 10 20 30 40 50 60 10 20 30 40 50 60 Fraction No. (5 ml/tube)

Figure 2: Elution profiles of the EPS from submerged culture of S. mutans in the presence or absence of cigarette smoke condensate (CSC) in Sepharose CL-6B chromatography. Eluate was analyzed by measuring the absorbance at 490 nm for carbohydrate (●) and the absorbance at 280 nm for protein (○). (A) without CSC, (B) with CSC.

EPSs as shown in both Figure 2a and 2b. Both fractions Table 1: Carbohydrate composition in the purified EPS produced show the presence of protein peaks. from submerged culture of S. mutans with and without cigarette Quantitative determination of monosaccharide smoke condensate (CSC). composition pattern by GC analysis suggested that Carbohydrate Culture without CSC Culture with CSC there was no significance between cultures growth in composition (%) the presence and absence of CSC and , mannose and galactose were major in both EPS Mannose 33.2±1.7 34.2±1.9 Galactose 17.8±1.5 16.7±1.4 fractions (Table 1). Xylose 2.4±0.7 2.6±0.3 The IR spectra of both EPS from the culture in the Glucose 43.3±1.6 44.2±2.7 presence or absence of CSC were similar, which indicated Ribose 3.3±0.8 2.3±0.5 that CSC would not change the functional groups (Figure 3). Both samples exhibited a broad stretching intense characteristic band at ca. 3280 cm-1 for the hydroxyl group ence of molecular weight distribution on the functional and a weak C–H band at ca. 2940 cm-1. The band at ca. properties of polysaccharides. Results indicated generally 1634–1641 cm-1 was due to the bound water [25]. A signals higher values of polydispersity ratio as compared to EPS at 1535–1541 cm-1 corresponding to amino groups was from culture without CSC, indicating that of also observed. Bands at ca. 1027–1080 cm-1 were from the EPS from culture with CSC are remarkably more dispersed stretch vibration of C-O [26]. in aqueous solutions [14]. The RMS radii for four peaks ranged from 20.1 to 70.1 nm with no clear trends. One of the important advantages of MALLS detection 3.3 Effect of CSC on molecular properties of lies in an additional possibility to determine dimensions EPS of dissolved in terms of gyration radius from the angular dependence of scattered light [16]. The gross con- The molecular properties of EPS were obtained using the formation of EPS in aqueous solution (e.g. random coil, SEC/MALLS system and are summarized in Table 2. The compact sphere, stiff rod, or combinations of them) could average molar mass weight (Mw) of EPS generated in the be identified from the double logarithmic plot of RMS absence of CSC (3.04×104 g/mol) was larger in compar- radius vs. molecular mass of EPS according to the follow- ison to that observed for EPS produced in the presence ing equations: 5 3 of CSC (2.75×10 g/mol). Polydispersity values (Mw/Mn), Spheres: ri ∝ Mi→log ri = k+1/3log Mi 3 as an index of the width of molecular mass distribution, Random coils: ri ∝ Mi→log ri = k+1/2log Mi 3 are important due to the relevance and significant influ- Rigid rods : ri ∝ Mi→log ri = k+log Mi Chun ping Xu et al.: Effect of cigarette smoke condensate 273

in aqueous solution. In summary, all results from molec- ular properties indicated that CSC influenced the molec- ular size and structural conformation of the polysaccha- ride rather than the carbohydrate components of the EPS fraction. However, how the EPS altered by CSC affects the biofilm formation need to further investigation.

4 Conclusion

Cell growth and metabolites production by S. mutans have been found to be influenced by the presence of CSC, 4000 3000 2000 1000 Wavelength (cm-1) essentially leading to growth promotion and increase in EPS and acetic acid contents. These findings may explain the persistence of the oral pathogen and will pave the Figure 3: The FT-IR spectra of the EPS fractions (Fr-I and Fr-II) produ- ced by submerged culture of S. mutans in the presence or absence way for future investigations along the negative influence of cigarette smoke condensate (CSC). (A) Fr-I, (B) Fr-II. of tobacco and smoke-related condensates in microor- ganisms. This study revealed that CSC was directly able

Table 2: Relevant molecular parameters and the double logarithmic to affect molecular mass and structural conformation of plots of root mean square radius vs molecular mass of EPS produced EPS from S. mutans. The molecular characterization of by submerged S. mutans with and without cigarette smoke conden- EPS would become an indicator in certain pathological sate (CSC) in MALLS analysis. disorders. In order to understand how the molecules in CSC affect the oral flora, further work on the relationship Parameters EPS from culture EPS from culture between the main toxic compositions in CSC and oral flora without CSC with CSC (error %) (error %) is in progress in our laboratory.

-1 3 3 Mn (g mol ) 4.814×10 (1) 4.165×10 (1) -1 4 4 Acknowledgements: The authors acknowledge the finan- Mw (g mol ) 3.040×10 (2) 2.753×10 (2) -1 6 7 cial support from the International Science & Technology Mz (g mol ) 3.766×10 (3) 1.315×10 (3)

Mw/Mn 6.301 (2) 6.610 (2) Cooperation Program of Henan Province (144300510061).

Rn (nm) 19.0 (1.1) 20.1 (1) Conflict of interest: None declared.

Rw (nm) 22.3 (1) 22.4 (1.1)

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