Archives of Oral Biology 65 (2016) 72–76
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Effect of the association of maltodextrin and sucrose on the acidogenicity and adherence of cariogenic bacteria Clarissa Gewehr Steguesa , Rodrigo Alex Arthurb , Lina Naomi Hashizumeb,* a b
Faculty of Dentistry, Federal University of Rio Grande do Sul, Porto Alegre, RS CEP 90035-003, Brazil Department of Preventive and Social Dentistry, Faculty of Dentistry, Federal University of Rio Grande do Sul, Porto Alegre, RS CEP 90035-003, Brazil
A R T I C L E I N F O
A B S T R A C T
Article history: Received 14 September 2015 Received in revised form 11 November 2015 Accepted 27 January 2016
Objective: The aim was to investigate the effect of maltodextrin and sucrose association on the acidogenic and adherence profiles of cariogenic bacteria. Design: Streptococcus mutans (S. mutans) and Lactobacillus casei (L. casei) were cultivated in culture medium containing maltodextrin, sucrose, maltodextrin–sucrose mixture or glucose. Analyses of the acidogenicity and microbial adherence were conducted in triplicate for each microorganism and tested carbohydrate. Results: For L. casei, maltodextrin, sucrose and maltodextrin–sucrose mixture showed lower acidogenic potential compared to glucose. When the microorganism was S. mutans, sucrose and maltodextrin– sucrose mixture presented higher acidogenic potential compared to maltodextrin and glucose. Microbial adherence analysis revealed higher adherence for S. mutans in presence of sucrose and maltodextrin– sucrose mixture compared to maltodextrin and glucose. For L. casei, all the carbohydrates showed similar adherence percentages. Conclusion: The addition of maltodextrin to sucrose does not increase the cariogenicity of sucrose in terms of acidogenicity and adherence of the cariogenic bacteria. ã 2016 Elsevier Ltd. All rights reserved.
Keywords: Sugar Microbiology Dental caries
1. Introduction Dental caries is a biofilm-induced disease related to an imbalance on microbial composition of biofilms that occurs under frequent exposure to dietary carbohydrates (Marsh, 2003). These carbohydrates are metabolized by biofilm acid-lactic producer bacteria leading to a decrease of pH on microenvironments of dental biofilm and also on the surrounding aqueous phases of the oral cavity which favors the establishment of acid-tolerant bacteria, such as Streptococcus mutans (S. mutans) and Lactobacilli ssp., in detriment of acid-sensitive ones (Marsh, 2003; Takahashi & Nyvad, 2008). Besides tolerating low pH environments, these mentioned microorganisms are also acidogenic since they present cell membrane sugar-transporters and an intricate chain of glycolytic enzymes, which allows the conversion of dietary sugars onto short-chain organic acids, such as lactic acid (Bradshaw,
* Corresponding author at: Department of Preventive and Social Dentistry, Faculty of Dentistry, Federal University of Rio Grande do Sul, Ramiro Barcelos, 2492 Porto Alegre, RS, Brazil. Fax: +55 51 3308 5002. E-mail addresses: [email protected] (C.G. Stegues), [email protected] (R.A. Arthur), [email protected] (L.N. Hashizume). http://dx.doi.org/10.1016/j.archoralbio.2016.01.018 0003-9969/ ã 2016 Elsevier Ltd. All rights reserved.
McKee, & Marsh, 1989; Geddes, 1975). Several clinical studies have found a positive association between high counts of S. mutans and Lactobacilli ssp. and caries activity (Becker et al., 2002; Caufield, Li, & Dasanayake, 2005; Van Houte, 1994). Within the dietary carbohydrates, sucrose has been claimed as the most cariogenic one (Paes Leme, Koo, Bellato, Bedi, & Cury, 2006) since it induces several modifications on biofilm which become more thick, porous and well-adhered to dental surfaces (Carlsson & Egelberg, 1965; Dibdin & Shellis, 1988; Rölla, 1989). These structural changes are attributed to insoluble extracellular polysaccharides (EPS) synthesized by the biofilm microbiota only from sucrose as substrate (Koo, Xiao, & Klein, 2009; Newbrun, 1969). Besides that, it seems that the inorganic concentration of the biofilms is shifted in the presence of sucrose that reduces its calcium, inorganic phosphate and fluoride concentrations (Cury, Rebello, & Del Bel Cury, 1997; Cury, Rebelo, Del Bel Cury, Derbyshire, & Tabchoury, 2000). Altogether, these challenges induced by sucrose contribute to its higher cariogenicity in comparison with other dietary carbohydrates (Aires, Tabchoury, Del Bel Cury, & Cury, 2002; Aires et al., 2008; Cury et al., 2000; Ribeiro et al., 2005). Several industrialized products and food supplements contains sucrose associated or not with maltodextrins (De Mazer Papa et al.,
C.G. Stegues et al. / Archives of Oral Biology 65 (2016) 72–76
2010; Moynihan, Wright, & Walton, 1996). Maltodextrins are complex carbohydrates, obtained from partially hydrolyzed starch, composed of multiple a-D-glucose units linked by glycosidic linkages. They are obtained from partially-hydrolyzed starch and frequently used as sweeteners in industrialized food products. They are composed generally of glucose, maltose, maltotriose and glucose polymers, whose final concentration depending on the method and degree of hydrolysis of corn starch which is chemically defined as “dextrose equivalent” (DE) of this oligosaccharide, expressed as dextrose and calculated as a percentage of total dry mass. The higher the value of DE, the greater the amount of reducing sugars it will contain and the more readily the product will be metabolized by the oral bacteria. Maltodextrins are complex in nature and have a DE of lower than 20 (Moynihan, 1998). A previous study evaluated the possibility of maltodextrin fermentation by the microbiota of dental biofilms (Al-Khatib, Duggal & Toumba, 2001). The authors found that a rinse with distinct dextrose equivalent index maltodextrin-containing solutions reduced pH in the oral cavity to an extent that induced demineralization of the dentine. They also observed that the pH reduction on exposure to maltodextrin was lower than that produced by sucrose solution. Moreover, other study reported that the addition of maltodextrin or maltodextrin associated with other dextrins on cold-teas increased their acidogenic potential (Meyerowitz, Syrrakou & Raubertas, 1996). It is known that the cariogenic potential of sucrose is enhanced when it is associated with other carbohydrates. A previous study investigated the adhesion of mutans streptococci in the presence of starch hydrolysates. The authors reported that some bacteria displayed higher adhesion activities for the glucan made in the presence of the starch hydrolysates (Vacca-Smith, Venkitaraman, Quivey & Bowen, 1996). It is believed that EPS synthesized by the biofilm in the presence of both sucrose and starch are more insoluble than those synthesized only from sucrose. This may enhance the adhesiveness of the biofilm to dental surfaces making it more cariogenic (Duarte et al., 2008). However, little is known whether the addition of maltodextrin enhances the cariogenic potential of sucrose. Therefore the aim of this study was to investigate the effect of the association between these carbohydrates on the acidogenicity and adherence of cariogenic bacteria. 2. Materials and methods 2.1. Strains and source of carbohydrates S. mutans (UA159) and L. casei (ATCC 4646) were the tested strains. These were obtained from frozen stocks and grown on Brain Heart Infusion (BHI) agar (Kasvi, Curitiba, Brazil) and Rogosa SL agar (HI Media, Mumbai, India), respectively, at 37 C for 48 h. A loopful of colony growth was transferred to tubes containing BHI supplemented with 1% glucose and incubated at 37 C for 18 h. The carbohydrates used in this study were glucose, sucrose and maltodextrin (Sigma–Aldrich, Saint Louis, USA). The index of maltodextrin (DE = 13–17), chosen for the experiments, was based on the most frequent one found in industrialized food. 2.2. Analysis of acidogenic potential Acidogenic potential was determined according to protocol described in previous study (Arthur et al., 2013). Approximately 108 CFU/mL aliquots of individual cultures of S. mutans and L. casei were grown for 18 h in BHI broth. The cultures were centrifuged, and the pellets were resuspended in 50 mM KCl supplemented with 1 mM MgCl2. The pH of the solution was adjusted to 7.0 and
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glucose, maltodextrin, sucrose, or a sucrose–maltodextrin mixture was added to each tube at a final concentration of 0.1%. The decrease in pH was then assessed during 180 min using a glass electrode previously calibrated with pH standards. All individual values of pH at each time point and for each carbohydrate were plotted in a standardized way. All graphs had same length and width. They were then individually imported into UTHSCSA ImageTool software, version 3.0. The area under the curve (AUC) was manually delimitated by drawing tools. Spatial measurements were calibrated previously to the AUC delimitation. AUC was calculated considering drop in pH after 180 min and pH 3.0 as a cutoff point. In order to avoid discrepancies, all the analysis were done by the same operator and each delimitation was done three times for each tested condition. The acidogenicity was expressed as the AUC (cm2). Each analysis was performed in three distinct experiments. 2.3. Analysis of the microbial adherence The experiments for microbial adhesion in the presence of the glucose, maltodextrin, sucrose or a sucrose–maltodextrin mixture were carried out following the methodology adapted from previous study (Ma et al., 2013). An aliquot of 100 mL of 108 CFU/mL of S. mutans or L. casei suspension was inoculated into 3 mL of BHI containing 0.5% of glucose or 0.5% maltodextrin or 0.5% sucrose or 0.5% sucrose + 0.5% maltodextrin. The cultures were incubated for 18 h at a 30 angle, in a clean test tube (10 mm 75 mm), in an anaerobic atmosphere (95% N2, 5% CO2) at 37 C. After incubation, the tube (A) was rotated twice, and the detached bacterial cells were transferred to a second tube (B). After addition of 3 mL of a phosphate buffer (0.05 M PBS, pH 6.8) to tube A, the tube was rotated again. The released bacterial cells were transferred to a third tube (C). Tubes B and C were centrifuged at 3000 rpm for 15 min and the supernatants were discarded. Buffer solution was added (3 mL) to all tubes and the cells were dispersed by vortexing. The optical density (OD) at 540 nm was determined by spectrophotometry, and the percentages of microbial adherence in media containing the different carbohydrates were calculated according to the following formula: adherence percentage = ODa/(ODa + ODb + ODc) 100%. 2.4. Statistical analysis Analysis of variance (ANOVA) was used to determine the effect of each carbohydrate on the response variables. The assumption of equality of variances and normal distribution of errors were checked for all the response variables tested. One-way ANOVA, followed by Tukey test was used to compare the acidogenic potential of carbohydrates for S. mutans and to compare the percentage of adhered cells among each type of carbohydrate for both microorganisms. Kruskal–Wallis test was used to compare the acidogenic potential of carbohydrates for L. casei and to compare the values of pH at each time point for different carbohydrates. The t-test was used for a comparison of the acidogenic potential of glucose between both microorganisms, while Mann–Whitney test was used for a comparison between the other carbohydrates. Comparison of the percentage of adherent cells for each carbohydrate between S. mutans and L. casei was also performed using the t-test. The level of significance was set at 5%. The statistical analysis was carried out using BioEstat 5.3 (Instituto de Desenvolvimento Sustentável Mamirauá, Brazil). 3. Results Fig. 1 shows the typical pH fall curves for S. mutans (Fig. 1A) and L. casei (Fig. 1B) with respect to each carbohydrate. For S. mutans,
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Fig. 1. pH-fall curves of S. mutans (A) and L. casei (B) in response to each tested carbohydrate. (*) means statistical differences in relation to the other tested carbohydrates by Kruskal–Wallis test (p < 0.05).
the fermentation of glucose, sucrose, and maltodextrin–sucrose mixture produced similar pH fall curves, which were significantly lower than that for maltodextrin alone at 120 and 180 min (p < 0.05). The final pH values were also significantly lower in the presence of glucose, sucrose, and maltodextrin–sucrose mixture (3.65; 3.66 and 3.77, respectively) compared to that in the presence of maltodextrin alone (5.02). For L. casei, the fermentation of glucose produced significantly lower pH values compared to the other carbohydrates from 30 to 180 min, with a final pH of 3.46. Similar pH curves were obtained in the presence of maltodextrin, sucrose, and maltodextrin–sucrose mixture (6.18, 6.39 and 6.16, respectively). S. mutans showed lower AUC values in the presence of glucose, sucrose, and maltodextrin–sucrose mixture compared to maltodextrin (p < 0.05), which were not significantly different from each other. L. casei showed lower values of AUC in the presence of
glucose, compared with the other carbohydrates (p < 0.01), which had similar values. No significant difference was observed between the AUC values of S. mutans and L. casei in the presence of glucose. However, for all the other tested carbohydrates, S. mutans showed significantly lower AUC values in comparison with L. casei (p < 0.05) (Table 1). In terms of microbial adherence, the percentage of adherent cells of S. mutans was significantly lower in the presence of maltodextrin in comparison to the other carbohydrates (p < 0.01), which were mostly similar. On the other hand, no differences were observed among the tested carbohydrates for L. casei. S. mutans showed a significantly higher percentage of adherent cells than L. casei in the presence of glucose, sucrose, and maltodextrin–sucrose mixture (p < 0.001). In the presence of maltodextrin, there was no difference between these two microorganisms (Table 1).
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Table 1 Acidogenic potential (AUC; cm2) and percentage of adherent cells (%) of S. mutans and L. casei for each tested condition (mean SD). Carbohydrate
Glucose Sucrose Maltodextrin Maltodextrin + sucrose
AUC
Adherent cells (%)
S. mutans
L. casei
S. mutans
L. casei
33.2 4.2Ba* 34.9 4.5Ba** 74.2 4.8Aa** 33.6 3.5Ba**
29.3 4.6Ba* 112.9 14.7Ab** 117.4 4.8Ab** 105.6 9.6Ab**
30.6 0.05Ba* 56.7 0.1Aa* 6.9 0.05Ca* 56.3 0.05Aa*
7.3 0.04Ab* 7.0 0.03Ab* 7.3 0.03Aa* 9.1 0.03Ab*
Means followed by distinct upper-case superscript letters differ statistically among different carbohydrates within the same microorganism. Means followed by distinct lower-case letters differ statistically between microorganisms for each carbohydrate (*t-test; **Mann–Whitney test), p < 0.05.
4. Discussion Maltodextrin is a carbohydrate widely used in industrialized food products, which may be associated with sucrose in some of these products (De Mazer Papa et al., 2010). This study aimed to investigate whether the association between sucrose with maltodextrin contributes to enhance the cariogenic potential of sucrose through the assessment of acidogenicity and adherence of cariogenic bacteria. The data from the present study suggest that maltodextrin is slowly fermented by S. mutans and L. casei in comparison to glucose (Fig. 1A and B), which is represented by a higher AUC observed in the presence of maltodextrin than that found in the presence of glucose (Table 1). Glucose (a carbohydrate chosen as an experimental control for the tested assays) is a simple carbohydrate molecule that is readily available for glycolysis, whereas maltodextrin is a complex carbohydrate molecule that needs to be converted into simpler molecules before being used as a substrate in the glycolitic pathway. Maltodextrin is imported into the cytosol by cell membrane ABC-transporter via maltose-transport system (Boos & Shuman, 1998; Monedero, Yebra, Poncet, & Deutscher, 2008; Nakai et al., 2009; Sato, Okamoto-Shibayama, & Azuma, 2013; Tao, Sutcliffe, Russell, & Ferretti, 1993; Webb, Homer, & Hosie, 2007). After being taken up, maltodextrin is hydrolyzed by intracellular a-glucosidases into maltose which is converted later into glucose (Moller et al., 2012; Nakai et al., 2009; Schönert, Bruder, & Dahl, 1999). This might also explain the lower fermentation rate of maltodextrin in comparison to glucose within the same period. The lowest pH reached after maltodextrin fermentation by S. mutans and by L. casei was 5.02 and 6.18, respectively. This suggests that maltodextrin might be potentially cariogenic for exposed dentine, but not for enamel. At this point, it is important to discuss that the high acidogenic potential of S. mutans in the presence of the sucrose–maltodextrin mixture might be due to the fermentation of sucrose molecules present in the mixture. Sucrose is a disaccharide rapidly converted into monosaccharides, glucose and fructose, by the enzymatic activity of an exo-b-fructosidase (Burne, Schilling, Bowen, & Yasbin, 1987). It has been suggested that S. mutans metabolizes sucrose faster than other oral bacteria, since it possess at least three systems for sucrose uptake, which includes two phosphoenolpyruvate-dependent phosphotransferase systems (PTS) and a non-PTS system (Slee & Tanzer, 1982). This explains the similar fermentation rates for glucose, sucrose, and the sucrose–maltodextrin mixture found in these bacteria (Table 1). On the other hand, L. casei was shown to produce acids from sucrose slower than some cariogenic bacteria, including S. mutans (Hedberg, Hasslöf, Sjöström, Twetman & Stecksén-Blicks, 2008), which agrees with the current data (Table 1). Adherence ability was also investigated as the percentage of cells that remained adhered to glass surfaces after the removal of loosely adhered bacteria by washes with PBS. In this respect, the findings of the present study show that S. mutans present similar ability to adhere to a substrate in the presence of sucrose and
maltodextrin–sucrose mixture. Several experimental and clinical studies have demonstrated the role of sucrose in enhancing bacterial adhesion to tooth surface, which leads to biofilm formation (Rölla, 1989; Zero, 2004). It is well known that glucose residuals obtained after hydrolysis of sucrose are used to synthesize EPS by the enzymatic activity of glucosyltransferases (GTF) of S. mutans, mainly GTF-B (Bowen & Koo, 2011), which enhances the adhesiveness of these bacteria to each other and to the dental surface, contributing to an increase in the thickness of the biofilm (Tamesada, Kawabata, Fujiwara & Hamada, 2004). In addition, EPS induces some changes in the tridimensional structure of the biofilm matrix, which are related to the enhanced cariogenicity of the biofilm (Koo, Falsetta & Klein, 2013). Interestingly, the percentage of adherent cells in the presence of glucose was statistically lower compared with adherent cells in the presence of sucrose (Table 1). This could be explained by the fact that glucose fermentation does not lead to EPS synthesis. In this context, it has been suggested that the chemical composition of EPS might change when sucrose is associated with other carbohydrates. Duarte et al. (2008) showed an increase in the insolubility of the EPS synthesized, when sucrose is associated with starch. The authors suggested that it is due to higher GTF-B expression on association of those carbohydrates. Clinically, the increase in insolubility of EPS makes the biofilm more adhered to dental surfaces, which might explain its higher cariogenicity in comparison to sucrose only (Ribeiro et al., 2005). We were not able to determine whether the EPS synthesized on association of sucrose and maltodextrin also show changes in chemical composition, and if these changes also modify the matrix of the biofilm. Further studies will be designed to investigate these features. Additionally, it was shown that S. mutans possesses greater biofilm formation ability than L. casei even in the presence of glucose. This might be due the fact that Lactobacilli lack important surface adhesin molecules, which impairs their ability to grow as biofilms (Stamatova, Kari, Vladimirov & Meurman, 2009). This is in agreement with clinical studies showing that Lactobacilli are usually found on biofilms as secondary colonizer, or even on cavitated carious lesions (Beighton et al., 2004; Parisotto et al., 2010) that act as mechanic barriers favoring their colonization. Based on this in vitro study’s results, it can be concluded that the addition of maltodextrin to sucrose does not increase the cariogenicity of sucrose in terms of acidogenicity and adherence of the cariogenic bacteria. Conflict of interest There are no conflict of interest to declare. References Aires, C. P., Tabchoury, C. P., Del Bel Cury, A. A., & Cury, J. A. (2002). Effect of a lactose-containing sweetener on root dentine demineralization in situ. Caries Research, 36(3), 167–169.
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Contents lists available at ScienceDirect
Archives of Oral Biology journal homepage: www.elsevier.com/locate/aob
Effect of the association of maltodextrin and sucrose on the acidogenicity and adherence of cariogenic bacteria Clarissa Gewehr Steguesa , Rodrigo Alex Arthurb , Lina Naomi Hashizumeb,* a b
Faculty of Dentistry, Federal University of Rio Grande do Sul, Porto Alegre, RS CEP 90035-003, Brazil Department of Preventive and Social Dentistry, Faculty of Dentistry, Federal University of Rio Grande do Sul, Porto Alegre, RS CEP 90035-003, Brazil
A R T I C L E I N F O
A B S T R A C T
Article history: Received 14 September 2015 Received in revised form 11 November 2015 Accepted 27 January 2016
Objective: The aim was to investigate the effect of maltodextrin and sucrose association on the acidogenic and adherence profiles of cariogenic bacteria. Design: Streptococcus mutans (S. mutans) and Lactobacillus casei (L. casei) were cultivated in culture medium containing maltodextrin, sucrose, maltodextrin–sucrose mixture or glucose. Analyses of the acidogenicity and microbial adherence were conducted in triplicate for each microorganism and tested carbohydrate. Results: For L. casei, maltodextrin, sucrose and maltodextrin–sucrose mixture showed lower acidogenic potential compared to glucose. When the microorganism was S. mutans, sucrose and maltodextrin– sucrose mixture presented higher acidogenic potential compared to maltodextrin and glucose. Microbial adherence analysis revealed higher adherence for S. mutans in presence of sucrose and maltodextrin– sucrose mixture compared to maltodextrin and glucose. For L. casei, all the carbohydrates showed similar adherence percentages. Conclusion: The addition of maltodextrin to sucrose does not increase the cariogenicity of sucrose in terms of acidogenicity and adherence of the cariogenic bacteria. ã 2016 Elsevier Ltd. All rights reserved.
Keywords: Sugar Microbiology Dental caries
1. Introduction Dental caries is a biofilm-induced disease related to an imbalance on microbial composition of biofilms that occurs under frequent exposure to dietary carbohydrates (Marsh, 2003). These carbohydrates are metabolized by biofilm acid-lactic producer bacteria leading to a decrease of pH on microenvironments of dental biofilm and also on the surrounding aqueous phases of the oral cavity which favors the establishment of acid-tolerant bacteria, such as Streptococcus mutans (S. mutans) and Lactobacilli ssp., in detriment of acid-sensitive ones (Marsh, 2003; Takahashi & Nyvad, 2008). Besides tolerating low pH environments, these mentioned microorganisms are also acidogenic since they present cell membrane sugar-transporters and an intricate chain of glycolytic enzymes, which allows the conversion of dietary sugars onto short-chain organic acids, such as lactic acid (Bradshaw,
* Corresponding author at: Department of Preventive and Social Dentistry, Faculty of Dentistry, Federal University of Rio Grande do Sul, Ramiro Barcelos, 2492 Porto Alegre, RS, Brazil. Fax: +55 51 3308 5002. E-mail addresses: [email protected] (C.G. Stegues), [email protected] (R.A. Arthur), [email protected] (L.N. Hashizume). http://dx.doi.org/10.1016/j.archoralbio.2016.01.018 0003-9969/ ã 2016 Elsevier Ltd. All rights reserved.
McKee, & Marsh, 1989; Geddes, 1975). Several clinical studies have found a positive association between high counts of S. mutans and Lactobacilli ssp. and caries activity (Becker et al., 2002; Caufield, Li, & Dasanayake, 2005; Van Houte, 1994). Within the dietary carbohydrates, sucrose has been claimed as the most cariogenic one (Paes Leme, Koo, Bellato, Bedi, & Cury, 2006) since it induces several modifications on biofilm which become more thick, porous and well-adhered to dental surfaces (Carlsson & Egelberg, 1965; Dibdin & Shellis, 1988; Rölla, 1989). These structural changes are attributed to insoluble extracellular polysaccharides (EPS) synthesized by the biofilm microbiota only from sucrose as substrate (Koo, Xiao, & Klein, 2009; Newbrun, 1969). Besides that, it seems that the inorganic concentration of the biofilms is shifted in the presence of sucrose that reduces its calcium, inorganic phosphate and fluoride concentrations (Cury, Rebello, & Del Bel Cury, 1997; Cury, Rebelo, Del Bel Cury, Derbyshire, & Tabchoury, 2000). Altogether, these challenges induced by sucrose contribute to its higher cariogenicity in comparison with other dietary carbohydrates (Aires, Tabchoury, Del Bel Cury, & Cury, 2002; Aires et al., 2008; Cury et al., 2000; Ribeiro et al., 2005). Several industrialized products and food supplements contains sucrose associated or not with maltodextrins (De Mazer Papa et al.,
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2010; Moynihan, Wright, & Walton, 1996). Maltodextrins are complex carbohydrates, obtained from partially hydrolyzed starch, composed of multiple a-D-glucose units linked by glycosidic linkages. They are obtained from partially-hydrolyzed starch and frequently used as sweeteners in industrialized food products. They are composed generally of glucose, maltose, maltotriose and glucose polymers, whose final concentration depending on the method and degree of hydrolysis of corn starch which is chemically defined as “dextrose equivalent” (DE) of this oligosaccharide, expressed as dextrose and calculated as a percentage of total dry mass. The higher the value of DE, the greater the amount of reducing sugars it will contain and the more readily the product will be metabolized by the oral bacteria. Maltodextrins are complex in nature and have a DE of lower than 20 (Moynihan, 1998). A previous study evaluated the possibility of maltodextrin fermentation by the microbiota of dental biofilms (Al-Khatib, Duggal & Toumba, 2001). The authors found that a rinse with distinct dextrose equivalent index maltodextrin-containing solutions reduced pH in the oral cavity to an extent that induced demineralization of the dentine. They also observed that the pH reduction on exposure to maltodextrin was lower than that produced by sucrose solution. Moreover, other study reported that the addition of maltodextrin or maltodextrin associated with other dextrins on cold-teas increased their acidogenic potential (Meyerowitz, Syrrakou & Raubertas, 1996). It is known that the cariogenic potential of sucrose is enhanced when it is associated with other carbohydrates. A previous study investigated the adhesion of mutans streptococci in the presence of starch hydrolysates. The authors reported that some bacteria displayed higher adhesion activities for the glucan made in the presence of the starch hydrolysates (Vacca-Smith, Venkitaraman, Quivey & Bowen, 1996). It is believed that EPS synthesized by the biofilm in the presence of both sucrose and starch are more insoluble than those synthesized only from sucrose. This may enhance the adhesiveness of the biofilm to dental surfaces making it more cariogenic (Duarte et al., 2008). However, little is known whether the addition of maltodextrin enhances the cariogenic potential of sucrose. Therefore the aim of this study was to investigate the effect of the association between these carbohydrates on the acidogenicity and adherence of cariogenic bacteria. 2. Materials and methods 2.1. Strains and source of carbohydrates S. mutans (UA159) and L. casei (ATCC 4646) were the tested strains. These were obtained from frozen stocks and grown on Brain Heart Infusion (BHI) agar (Kasvi, Curitiba, Brazil) and Rogosa SL agar (HI Media, Mumbai, India), respectively, at 37 C for 48 h. A loopful of colony growth was transferred to tubes containing BHI supplemented with 1% glucose and incubated at 37 C for 18 h. The carbohydrates used in this study were glucose, sucrose and maltodextrin (Sigma–Aldrich, Saint Louis, USA). The index of maltodextrin (DE = 13–17), chosen for the experiments, was based on the most frequent one found in industrialized food. 2.2. Analysis of acidogenic potential Acidogenic potential was determined according to protocol described in previous study (Arthur et al., 2013). Approximately 108 CFU/mL aliquots of individual cultures of S. mutans and L. casei were grown for 18 h in BHI broth. The cultures were centrifuged, and the pellets were resuspended in 50 mM KCl supplemented with 1 mM MgCl2. The pH of the solution was adjusted to 7.0 and
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glucose, maltodextrin, sucrose, or a sucrose–maltodextrin mixture was added to each tube at a final concentration of 0.1%. The decrease in pH was then assessed during 180 min using a glass electrode previously calibrated with pH standards. All individual values of pH at each time point and for each carbohydrate were plotted in a standardized way. All graphs had same length and width. They were then individually imported into UTHSCSA ImageTool software, version 3.0. The area under the curve (AUC) was manually delimitated by drawing tools. Spatial measurements were calibrated previously to the AUC delimitation. AUC was calculated considering drop in pH after 180 min and pH 3.0 as a cutoff point. In order to avoid discrepancies, all the analysis were done by the same operator and each delimitation was done three times for each tested condition. The acidogenicity was expressed as the AUC (cm2). Each analysis was performed in three distinct experiments. 2.3. Analysis of the microbial adherence The experiments for microbial adhesion in the presence of the glucose, maltodextrin, sucrose or a sucrose–maltodextrin mixture were carried out following the methodology adapted from previous study (Ma et al., 2013). An aliquot of 100 mL of 108 CFU/mL of S. mutans or L. casei suspension was inoculated into 3 mL of BHI containing 0.5% of glucose or 0.5% maltodextrin or 0.5% sucrose or 0.5% sucrose + 0.5% maltodextrin. The cultures were incubated for 18 h at a 30 angle, in a clean test tube (10 mm 75 mm), in an anaerobic atmosphere (95% N2, 5% CO2) at 37 C. After incubation, the tube (A) was rotated twice, and the detached bacterial cells were transferred to a second tube (B). After addition of 3 mL of a phosphate buffer (0.05 M PBS, pH 6.8) to tube A, the tube was rotated again. The released bacterial cells were transferred to a third tube (C). Tubes B and C were centrifuged at 3000 rpm for 15 min and the supernatants were discarded. Buffer solution was added (3 mL) to all tubes and the cells were dispersed by vortexing. The optical density (OD) at 540 nm was determined by spectrophotometry, and the percentages of microbial adherence in media containing the different carbohydrates were calculated according to the following formula: adherence percentage = ODa/(ODa + ODb + ODc) 100%. 2.4. Statistical analysis Analysis of variance (ANOVA) was used to determine the effect of each carbohydrate on the response variables. The assumption of equality of variances and normal distribution of errors were checked for all the response variables tested. One-way ANOVA, followed by Tukey test was used to compare the acidogenic potential of carbohydrates for S. mutans and to compare the percentage of adhered cells among each type of carbohydrate for both microorganisms. Kruskal–Wallis test was used to compare the acidogenic potential of carbohydrates for L. casei and to compare the values of pH at each time point for different carbohydrates. The t-test was used for a comparison of the acidogenic potential of glucose between both microorganisms, while Mann–Whitney test was used for a comparison between the other carbohydrates. Comparison of the percentage of adherent cells for each carbohydrate between S. mutans and L. casei was also performed using the t-test. The level of significance was set at 5%. The statistical analysis was carried out using BioEstat 5.3 (Instituto de Desenvolvimento Sustentável Mamirauá, Brazil). 3. Results Fig. 1 shows the typical pH fall curves for S. mutans (Fig. 1A) and L. casei (Fig. 1B) with respect to each carbohydrate. For S. mutans,
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Fig. 1. pH-fall curves of S. mutans (A) and L. casei (B) in response to each tested carbohydrate. (*) means statistical differences in relation to the other tested carbohydrates by Kruskal–Wallis test (p < 0.05).
the fermentation of glucose, sucrose, and maltodextrin–sucrose mixture produced similar pH fall curves, which were significantly lower than that for maltodextrin alone at 120 and 180 min (p < 0.05). The final pH values were also significantly lower in the presence of glucose, sucrose, and maltodextrin–sucrose mixture (3.65; 3.66 and 3.77, respectively) compared to that in the presence of maltodextrin alone (5.02). For L. casei, the fermentation of glucose produced significantly lower pH values compared to the other carbohydrates from 30 to 180 min, with a final pH of 3.46. Similar pH curves were obtained in the presence of maltodextrin, sucrose, and maltodextrin–sucrose mixture (6.18, 6.39 and 6.16, respectively). S. mutans showed lower AUC values in the presence of glucose, sucrose, and maltodextrin–sucrose mixture compared to maltodextrin (p < 0.05), which were not significantly different from each other. L. casei showed lower values of AUC in the presence of
glucose, compared with the other carbohydrates (p < 0.01), which had similar values. No significant difference was observed between the AUC values of S. mutans and L. casei in the presence of glucose. However, for all the other tested carbohydrates, S. mutans showed significantly lower AUC values in comparison with L. casei (p < 0.05) (Table 1). In terms of microbial adherence, the percentage of adherent cells of S. mutans was significantly lower in the presence of maltodextrin in comparison to the other carbohydrates (p < 0.01), which were mostly similar. On the other hand, no differences were observed among the tested carbohydrates for L. casei. S. mutans showed a significantly higher percentage of adherent cells than L. casei in the presence of glucose, sucrose, and maltodextrin–sucrose mixture (p < 0.001). In the presence of maltodextrin, there was no difference between these two microorganisms (Table 1).
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Table 1 Acidogenic potential (AUC; cm2) and percentage of adherent cells (%) of S. mutans and L. casei for each tested condition (mean SD). Carbohydrate
Glucose Sucrose Maltodextrin Maltodextrin + sucrose
AUC
Adherent cells (%)
S. mutans
L. casei
S. mutans
L. casei
33.2 4.2Ba* 34.9 4.5Ba** 74.2 4.8Aa** 33.6 3.5Ba**
29.3 4.6Ba* 112.9 14.7Ab** 117.4 4.8Ab** 105.6 9.6Ab**
30.6 0.05Ba* 56.7 0.1Aa* 6.9 0.05Ca* 56.3 0.05Aa*
7.3 0.04Ab* 7.0 0.03Ab* 7.3 0.03Aa* 9.1 0.03Ab*
Means followed by distinct upper-case superscript letters differ statistically among different carbohydrates within the same microorganism. Means followed by distinct lower-case letters differ statistically between microorganisms for each carbohydrate (*t-test; **Mann–Whitney test), p < 0.05.
4. Discussion Maltodextrin is a carbohydrate widely used in industrialized food products, which may be associated with sucrose in some of these products (De Mazer Papa et al., 2010). This study aimed to investigate whether the association between sucrose with maltodextrin contributes to enhance the cariogenic potential of sucrose through the assessment of acidogenicity and adherence of cariogenic bacteria. The data from the present study suggest that maltodextrin is slowly fermented by S. mutans and L. casei in comparison to glucose (Fig. 1A and B), which is represented by a higher AUC observed in the presence of maltodextrin than that found in the presence of glucose (Table 1). Glucose (a carbohydrate chosen as an experimental control for the tested assays) is a simple carbohydrate molecule that is readily available for glycolysis, whereas maltodextrin is a complex carbohydrate molecule that needs to be converted into simpler molecules before being used as a substrate in the glycolitic pathway. Maltodextrin is imported into the cytosol by cell membrane ABC-transporter via maltose-transport system (Boos & Shuman, 1998; Monedero, Yebra, Poncet, & Deutscher, 2008; Nakai et al., 2009; Sato, Okamoto-Shibayama, & Azuma, 2013; Tao, Sutcliffe, Russell, & Ferretti, 1993; Webb, Homer, & Hosie, 2007). After being taken up, maltodextrin is hydrolyzed by intracellular a-glucosidases into maltose which is converted later into glucose (Moller et al., 2012; Nakai et al., 2009; Schönert, Bruder, & Dahl, 1999). This might also explain the lower fermentation rate of maltodextrin in comparison to glucose within the same period. The lowest pH reached after maltodextrin fermentation by S. mutans and by L. casei was 5.02 and 6.18, respectively. This suggests that maltodextrin might be potentially cariogenic for exposed dentine, but not for enamel. At this point, it is important to discuss that the high acidogenic potential of S. mutans in the presence of the sucrose–maltodextrin mixture might be due to the fermentation of sucrose molecules present in the mixture. Sucrose is a disaccharide rapidly converted into monosaccharides, glucose and fructose, by the enzymatic activity of an exo-b-fructosidase (Burne, Schilling, Bowen, & Yasbin, 1987). It has been suggested that S. mutans metabolizes sucrose faster than other oral bacteria, since it possess at least three systems for sucrose uptake, which includes two phosphoenolpyruvate-dependent phosphotransferase systems (PTS) and a non-PTS system (Slee & Tanzer, 1982). This explains the similar fermentation rates for glucose, sucrose, and the sucrose–maltodextrin mixture found in these bacteria (Table 1). On the other hand, L. casei was shown to produce acids from sucrose slower than some cariogenic bacteria, including S. mutans (Hedberg, Hasslöf, Sjöström, Twetman & Stecksén-Blicks, 2008), which agrees with the current data (Table 1). Adherence ability was also investigated as the percentage of cells that remained adhered to glass surfaces after the removal of loosely adhered bacteria by washes with PBS. In this respect, the findings of the present study show that S. mutans present similar ability to adhere to a substrate in the presence of sucrose and
maltodextrin–sucrose mixture. Several experimental and clinical studies have demonstrated the role of sucrose in enhancing bacterial adhesion to tooth surface, which leads to biofilm formation (Rölla, 1989; Zero, 2004). It is well known that glucose residuals obtained after hydrolysis of sucrose are used to synthesize EPS by the enzymatic activity of glucosyltransferases (GTF) of S. mutans, mainly GTF-B (Bowen & Koo, 2011), which enhances the adhesiveness of these bacteria to each other and to the dental surface, contributing to an increase in the thickness of the biofilm (Tamesada, Kawabata, Fujiwara & Hamada, 2004). In addition, EPS induces some changes in the tridimensional structure of the biofilm matrix, which are related to the enhanced cariogenicity of the biofilm (Koo, Falsetta & Klein, 2013). Interestingly, the percentage of adherent cells in the presence of glucose was statistically lower compared with adherent cells in the presence of sucrose (Table 1). This could be explained by the fact that glucose fermentation does not lead to EPS synthesis. In this context, it has been suggested that the chemical composition of EPS might change when sucrose is associated with other carbohydrates. Duarte et al. (2008) showed an increase in the insolubility of the EPS synthesized, when sucrose is associated with starch. The authors suggested that it is due to higher GTF-B expression on association of those carbohydrates. Clinically, the increase in insolubility of EPS makes the biofilm more adhered to dental surfaces, which might explain its higher cariogenicity in comparison to sucrose only (Ribeiro et al., 2005). We were not able to determine whether the EPS synthesized on association of sucrose and maltodextrin also show changes in chemical composition, and if these changes also modify the matrix of the biofilm. Further studies will be designed to investigate these features. Additionally, it was shown that S. mutans possesses greater biofilm formation ability than L. casei even in the presence of glucose. This might be due the fact that Lactobacilli lack important surface adhesin molecules, which impairs their ability to grow as biofilms (Stamatova, Kari, Vladimirov & Meurman, 2009). This is in agreement with clinical studies showing that Lactobacilli are usually found on biofilms as secondary colonizer, or even on cavitated carious lesions (Beighton et al., 2004; Parisotto et al., 2010) that act as mechanic barriers favoring their colonization. Based on this in vitro study’s results, it can be concluded that the addition of maltodextrin to sucrose does not increase the cariogenicity of sucrose in terms of acidogenicity and adherence of the cariogenic bacteria. Conflict of interest There are no conflict of interest to declare. References Aires, C. P., Tabchoury, C. P., Del Bel Cury, A. A., & Cury, J. A. (2002). Effect of a lactose-containing sweetener on root dentine demineralization in situ. Caries Research, 36(3), 167–169.
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