Progress 12/01/05 to 11/30/09
Outputs
OUTPUTS: Grapes are rich in polyphenols, which are known to possess health benefits and bioactivity against Streptococcus mutans, the primary microorganism responsible for dental caries. However, the phenolic content is variable depending on grape variety, and may be modified during vinification. We examined the chemical composition and biological activity of phenolic extracts prepared from several wine grape varieties and their fermented by-product of winemaking (pomace) on many of the biological aspects involved in dental biofilm formation and caries development using in vitro and in vivo models. Grape phenolic extracts were obtained from Vitis vinifera varieties Cabernet Franc and Pinot Noir, and Vitis interspecific hybrid varieties Baco Noir and NoiretTM. Polyphenols were extracted and fractionated into anthocyanin, neutral, polymeric, oligomeric and monomeric fractions. Extracts were analyzed for total phenolic, anthocyanin and flavan-3-ol content, and for individual phenolic composition by HPLC. The anthocyanin and flavan-3-ols content were highly variable depending on grape variety and type of extract. The flavan-3-ol content ranged from 48 to 76 g CE/100g. The polymeric fraction had the most yield (4.8 g extract/100g fresh weight). Nevertheless, all grape phenolic extracts, except anthocyanin fraction, remarkably inhibited glucosyltransferases (Gtfs) B and C (70-85% inhibition) at concentrations as low as 62.5 g/ml. Furthermore, the glycolytic pH-drop by S. mutans cells was inhibited by the grape extracts without affecting the bacterial viability; an effect that can be attributed to partial inhibition of F-ATPase activity (30-65% inhibition at 125 g/ml). The biological activity of fermented pomace was either as effective as or significantly better than whole fruit grape extracts. Bioassay-guided fractionation of the pomace extract yielded a highly bioactive extract containing a mixture of proanthocyanidins (PACs) and flavonols based on its abilities to inhibit (i) glucan synthesis by surface adsorbed Gtfs, (ii) acid production and acid tolerance of S. mutans, and (iii) formation of biofilms by S. mutans on the apatitic surface. Biofilms treated with PACs+flavonols reduced the biovolume of exopolysaccharides (EPS) across the biofilm depth, especially in the middle and outer layers of the biofilms as determined by confocal imaging and computational analysis. This observation is consistent with reduction of biomass of the treated-biofilms, which was proportional to that of extracellular insoluble polysaccharides content in the biofilm matrix (up to 50% reduction). The data agrees well with the effective inhibition of streptococcal-derived Gtfs B and C by the bioactive extract, indicating that disruption of insoluble glucan synthesis is one of the mechanisms of action against biofilms formation. Moreover, myricetin (a flavonol found in the bioactive extract) reduced the gtfB mRNA levels as determined by real-time PCR. Furthermore, the PACs+flavonols-enriched fraction, alone or in combination with fluoride, exhibited cariostatic effect in vivo using our rodent model; PACs and flavonols appears to be the active constituents. PARTICIPANTS: Partner organization: Cornell University, Department of Food Science and Technology. TARGET AUDIENCES: Food scientists and dentists. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Dental caries is the single most prevalent and costly biofilm-related oral infectious diseases in US and worldwide. Red wine grapes are promising sources of active polyphenolic extracts for oral disease prevention. Red wine grapes are rich in polyphenols, which are known to possess health benefits and antimicrobial activity against Streptococcus mutans, the primary microorganism responsible for dental caries. Wine grapes are richer in polyphenols than table grapes but demand higher prices. Pomace, a waste product from the winemaking process, could be used as a source of polyphenols if the biological activity persists after processing. Our objective was to evaluate the phenolic composition of selected New York wine grape varieties and their pomace in relation to their bioactivity against virulence attributes of S. mutans associated with development of biofilms related to dental caries. The biological activity of phenolic extracts from fermented pomace was either as effective as or significantly better than that from whole fruit grape extracts. Fractionation of the main phenolic extracts resulted in distinct biological activity. Further studies related to inhibition of biofilm formation in vitro and dental caries development in vivo (rodent model) indicated that phenolic grape extracts composed mainly of flavan-3-ols (proanthocyanidins) and flavonols are effective and promising natural anti-biofilm agents, which could be used as novel adjunctive anti-caries chemotherapy. Therefore, the flavan-3-ol and flavonol content appears to be the best indicator for bioactivity of the grape extracts against pathogenic (oral) biofilms. The outcome of our studies also determined the feasibility of using grape polyphenols for commercially viable applications in oral health care and food industries. One of the potential applications is the production of a natural product displaying anti-biofilm/anti-caries properties from a current wine byproduct (waste), which is readily/widely available and economically feasible. These findings have been disseminated through technical presentations at professional meetings and through peer-reviewed scientific publications.
Publications
- Koo H, Jeon JG. 2009. Naturally occurring molecules as alternative therapeutic agents against cariogenic biofilms. Adv Dent Res, 21:63-68.
- Thimothe, J. 2009. Influence of red wine grape phenolic extracts on glucosyltransferase activity, acidogenicity and biofilm formation of Streptococcus mutans. Ph.D. Dissertation, Cornell University.
- Bonsi. I. A. 2009. Effect of processing on phenolic composition of wine grapes and their products, and production and characterization of grape phenolic extracts with biological activity against Streptococcus mutans and Alicyclobacillus acidoterrestris. Ph.D. Dissertation, Cornell University.
- Jeon J.G., Klein M.I., Xiao J., Gregoire S., Rosalen P.L., Koo H. 2009. Influences of naturally occurring agents in combination with fluoride on gene expression and structural organization of Streptococcus mutans in biofilms. BMC Microbiol, 9:228-238.
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Progress 12/01/07 to 11/30/08
Outputs
OUTPUTS: In our previous progress report we determined the chemical composition and biological activity of phenolic extracts prepared from several red wine grape varieties and their fermented by-product of winemaking (pomace) on some of the virulence properties of Streptococcus mutans involved in cariogenic biofilm formation. The anthocyanins and flavan-3-ols content were highly variable depending on grape variety (Cabernet Franc, Pinot Noir, Baco Noir and Noiret) and type of extract (whole fruit vs. fermented pomace). Nevertheless all grape phenolic extracts remarkably inhibited glucosyltransferases B and C (70-85% inhibition at 62.5 g/ml, P<0.01). Furthermore, glycolysis by S. mutans cells was inhibited by the grape extracts without affecting the bacterial viability; an effect that can be attributed to partial inhibition of F-ATPase activity. The biological activity of fermented pomace was either as effective as or significantly better than whole fruit extracts. The results showed that grape phenolic extracts, especially from pomace, are highly effective against specific virulence traits of S. mutans despite major differences in their anthocyanins content; pomace from Cabernet Franc provided the most biologically active extract. Furthermore, topical applications of pomace extracts significantly reduced the formation and accumulation of S. mutans biofilms using our well-established saliva-coated HA biofilm model (P<0.05). The reduction of the biomass of the treated-biofilms was proportional to that of extracellular insoluble polysaccharides content in the biofilm matrix (up to 50% reduction). This observation is consistent with the effective inhibition of GTFs by pomace extracts, suggesting that disruption of insoluble glucan synthesis is one of the mechanisms by which the bioactive polyphenols reduced biofilm formation. The elucidation of the particular mechanisms of action of the grape (pomace) extracts necessitates the identification of the specific bioactive compounds, and to evaluate their effectiveness in vivo. Thus, we designed a stepwise strategy using a combination of analytical technologies for isolation of polyphenolic flavonoids and in vitro assays based on anti-biofilm effects of the grape extracts. We have selected Cabernet Franc pomace extract (CFPE) as our source of extraction of flavonoids based on our previous biological and chemical data. Initially, we examined the influence of anthocyanins-, and proanthocyanidins (PACs) +flavonols-rich fractions of CFPE on S. mutans biofilm formation acidogenicity. The PACs+flavonols-rich fraction was highly effective against S. mutans virulence based on its abilities to inhibit: (1) glucan synthesis by surface absorbed GTFs B and C, and (2) acidogenic and aciduric properties of S. mutans; and the effects were comparable to CFPE. In contrast, anthocyanins-rich fraction was devoid of any significant biological activity. The results of this study suggest that flavonols and PACs are the putative active classes of polyphenolic flavonoids in CFPE. In parallel, we have developed a novel methodology to extract RNA from S. mutans biofilms for the gene expression studies using grape extracts. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Red wine grapes and pomace are promising sources of active polyphenolic extracts for oral disease prevention. Further research will determine the specific fractions or compounds responsible for the biological activity and their putative mechanisms of action. In addition, the outcome of our studies will determine the feasibility of using these extracts for commercially viable applications in our oral health care and food industries.
Publications
- Thimothe J, Bonsi IA, Padilla-Zakour OI, Koo H. 2007 Chemical characterization of red wine grapes (Vitis vinifera and Vitis interspecific hybrids) and pomace phenolic extracts and their biological activity against Streptococcus mutans. J Argic Food Chem 55:10200-7, 2007
- Cury JA, Koo H. 2007 Extraction and purification of total RNA from Streptococcus mutans biofilms. Anal biochem 365:208-14,2007
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Progress 12/01/06 to 11/30/07
Outputs
OUTPUTS: In our previous report, we have shown that phenolic extracts from fermented grape pomace was as effective as or significantly better than whole fruit grape extracts in disrupting specific virulence traits of Streptococcus mutans, the main microbial culprit associated with biofilm-related oral infectious diseases (e.g. dental caries). We also identified pomace from Cabernet Franc as the most biologically active extract, which effectively reduced the formation and the amounts of exopolysaccharide matrix of S. mutans biofilms. The elucidation of the particular mechanisms of action of the grape (pomace) extracts necessitates the identification of the specific bioactive compounds, and further evaluation of their effectiveness in vivo. Thus, we designed a stepwise strategy using a combination of analytical technologies for isolation of polyphenolic flavonoids and in vitro assays based on determination of anti-biofilm activity. Bioassay-guided purification of the pomace extract yielded a bioactive fraction containing a mixture of proanthocyanidins (PACs) and flavonols; PACs and flavonols-fraction effectively inhibited (i) glucan synthesis by surface adsorbed Gtfs, (ii) acid production and acid tolerance of S. mutans cells, and (iii) formation and accumulation of S. mutans biofilms. In contrast, anthocyanins-rich fraction was devoid of any significant biological activity. The results of this study suggest that flavonols and PACs are the putative active classes of polyphenolic flavonoids in the pomace. We also examined in more detail how the flavonols and PACs in combination affect the structural organization of S. mutans biofilms using confocal imaging. Biofilms treated with PACs+flavonols reduced the biovolume of extracellular polysaccharides (EPS) across the biofilm depth, especially in the middle and outer layers (close to the fluid phase) of the biofilm. The resulting structure-architecture of the biofilms matrix would be less stable and more susceptible to the inimical environmental influences (e.g. antimicrobial agents). This observation is consistent with reduction of biomass and polysaccharides content in the biofilms treated with PACs and flavonols as determined using biochemical assays. The disruptive effects on EPS are likely associated with inhibition of glucan synthesis by bacterial-derived Gtfs. The data collected thus far indicate that proanthocyanidins and flavonols in combination may exhibit cariostatic effect in vivo. We are currently assessing the ability of phenolic extract of pomace and its bioactive components (PACs+flavonols) in reducing S. mutans infection and development of dental caries in vivo using our well established rodent model. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Dental caries is the single most prevalent and costly biofilm-related oral infectious diseases in US and worldwide. Red wine grapes and pomace are promising sources of active polyphenolic extracts for oral disease prevention. The final step of our research project will determine whether proanthocyanidins and flavonols from fermented pomace exhibit cariostatic effects in vivo. The outcome of our studies will determine the feasibility of using grape polyphenols for commercially viable applications in oral health care and food industries. One of the potential applications is the production of natural anti-biofilm/anti-caries product from a current wine byproduct (waste), which is readily/widely available and economically feasible.
Publications
- Bonsi, I.A., Thimothe, J., Koo. H., Worobo, R.W., and Padilla-Zakour, O.I. 2008. Pinot noir grape polyphenolic extracts: Chemical composition and antimicrobial effects against Streptococcus mutans and Alicyclobacillus acidoterrestris. Institute of Food Technologists Annual Meeting & Food Expo, New Orleans, La. Abstract 056-01.
- Thimothe J, Bonsi IA, Padilla-Zakour OI, Koo H. 2007. Chemical characterization of red wine grape (Vitis vinifera and Vitis interspecific hybrids) and pomace phenolic extracts and their biological activity against Streptococcus mutans. J Agric Food Chem 55:10200-7.
- Cury JA, Koo H. Extraction and purification of total RNA from Streptococcus mutans biofilms. 2007. Anal Biochem 365:208-14.
- Thimothe, J. 2009. Influence of red wine grape phenolic extracts on glucosyltransferase activity, acidogenicity and biofilm formation of Streptococcus mutans. Ph.D. Dissertation, Cornell University.
- Bonsi. I. A. 2009. Effect of processing on phenolic composition of wine grapes and their products, and production and characterization of grape phenolic extracts with biological activity against Streptococcus mutans and Alicyclobacillus acidoterrestris. Ph.D. Dissertation, Cornell University.
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Progress 12/01/05 to 11/30/06
Outputs
Grapes are rich in polyphenols, which are known to possess health benefits and antimicrobial activity against Streptococcus mutans, the primary microorganism responsible for dental caries. Wine grapes are richer in polyphenols than table grapes but demand higher prices. Pomace, a waste product from the winemaking process, could be used as a source of polyphenols if antimicrobial activity persists after processing. Our objective was to evaluate the phenolic composition of selected New York wine grape varieties and their pomace in relation to their activity against the virulence traits of S. mutans involved in biofilm formation. Nine wine grapes and their pomace, obtained after grapes were crushed, fermented on skins and pressed, were freeze-dried and ground. Polyphenols were extracted by solvent extraction and analyzed for total phenolic and anthocyanin contents. Four red varieties were selected for extract preparation based on composition: Cabernet Franc, Pinot Noir,
Baco Noir and Noiret. Polyphenolic extracts were prepared by solid phase extraction using C18 SepPak cartridges, and freeze-dried to obtain a powdered extract. Total phenolic, total anthocyanin and flavan-3-ol contents were measured in the powders and expressed as grams gallic acid equivalents (g GAE/100 g), malvidin equivalents (g ME/100 g), and catechin equivalents (g CE/100 g). Phenolic profiles were analyzed by HPLC. Pinot Noir and Cabernet Franc pomace had the highest concentration of total phenols while Noiret grapes had the highest content of anthocyanins. Chemical composition of powdered extracts showed significant differences due to variety and processing. The highest anthocyanin content (22.7 g ME/100 g) was measured in Noiret grape extract. In contrast, Pinot Noir pomace and grapes had the highest polyphenolic (61.8 g GAE) and flavan-3-ol (56.7 g CE) content per 100 gram extract which corresponded to high activity against virulence traits of S. mutans. The ethanolic
extracts of grape prepared from whole fruit and pomace were examined on glucan synthesis by glucosyltransferases B and C (GTF B and C) and on acidogenicity of Streptococcus mutans cells in vitro. The effects of grape extracts on GTFs and F-ATPase activities and on glycolytic pH-drop by S. mutans were examined. GTF activity was measured by the incorporation of [14C] glucose from labeled sucrose into glucans. F-ATPase activity was assessed in terms of the amount of phosphate released from ATP. Glycolytic pH drop was followed with a glass electrode. The grape extracts inhibited the activity of GTF B or C between 60-80% at concentrations as low as 31.25 micrograms/ml. The acid production by S. mutans cells was also disrupted by the grape extracts, which was due in part by inhibition of F-ATPase activity. In general, Pinot Noir extracts were more effective than those from other grape varieties. The grape and pomace extracts displayed remarkable biological activity against some of the
virulence traits of S. mutans involved in glucan formation and acidogenicity. However, the effects were variable depending on the source and variety of grapes likely due to differences on their phenolic content.
Impacts
Red wine grapes and pomace are promising sources of active polyphenolic extracts for oral disease prevention. Further research will determine the specific fractions or compounds responsible for the biological activity and their putative mechanisms of action. In addition, the outcome of our studies will determine the feasibility of using these extracts for commercially viable applications in oral health care and food industries.
Publications
- No publications reported this period
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