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Grape cell-wall polysaccharides : influence on the interaction between salivary proteins and tannins
Elsa Brandão, Mafalda Santos Silva, Ignacio Garcia-Estevez, A. Fernandes, Pascale Williams, Nuno Mateus, Thierry Doco, Victor de Freitas, Susana
Soares
To cite this version:
Elsa Brandão, Mafalda Santos Silva, Ignacio Garcia-Estevez, A. Fernandes, Pascale Williams, et al..
Grape cell-wall polysaccharides : influence on the interaction between salivary proteins and tannins.
10. Symposium In Vino Analytica Scientia, Jul 2017, Salamanque, Spain. 2017, IVAS 2017 Vino Analytica Scientia Symposium. �hal-01984275�
References
[1] Soares, S. et al. Critical Reviews in Food Science and Nutrition 2017, 57, 937-948 [2] Brandão, E. et al. Journal of Agricultural and Food Chemistry 2014, 62, 9562-956 [3] Soares, S. et al. Journal of Agricultural and Food Chemistry 2012, 60, 3966-3972.
[4] Vidal, S. et al. Carbohydrate Polymers 2003, 54, 439-447
aREQUIMTE/LAQV, Departamento de Química e Bioquímica, Faculdade de Ciências da Universidade do Porto, Porto, Portugal
bINRA Joint Research Unit 1083, Sciences for Enology, Montpellier, France
E. Brandão
a, M. Silva
a, I. García-Estévez
a, A. Fernandes
a, P. Williams
b, N. Mateus
a, T. Doco
b, V. de Freitas
aand S. Soares
a,*
(*) susana.soares@fc.up.pt
Polyphenols are secondary metabolites from plants widely present in vegetables, fruits and derived products (e.g. red wine and green tea) being responsible for their major organoleptic properties (color and taste attributes such as bitterness and astringency) [1]. Polyphenols’
ability to precipitate salivary proteins (SP) is at the origin of the well- known astringency sensation which is characterized by the dryness, tightening, shrinking and puckering sensations perceived in the oral cavity. Different SP families have been reported to interact with tannins, namely proline-rich proteins (PRPs), statherin, P-B peptide and cystatins [1,2].
However, SP-tannin interactions appear to be inhibited or reduced by the presence of some polysaccharides [3]. The main polysaccharides from wine can be originated from grape cell walls including arabinogalactan-proteins (AGPs) and rhamnogalacturonan type II (RGII), or from yeast cell walls, such as mannoproteins [4]. In this work it was intended to study, at molecular level, how SP-tannin interaction could be affected by the presence of some of wine polysaccharides. So, we focused on: a) isolate different families of SP, b) isolate tannins with different molecular structures (procyanidin B2, commonly present in red wine and punicalagin, an abundant ellagitannin present in pomegranate), c) isolate and characterize different grape skin fractions of polysaccharides, and d) study the interaction between SP, tannins and polysaccharides by High-Performance Liquid Chromatography (HPLC).
CONCLUSIONS
Grape cell-wall polysaccharides: Influence on the interaction between salivary proteins and tannins
Grape skin
Boiling water, 5 min Homogeneize with
UltraTurrax
Pellet
(Alcohol Insoluble Material) Fraction A
Supernatant
80% EtOH 10 min
EtOH
10 000 rpm, 30 min, 10°C
10 000 rpm, 30 min, 10°C
Pellet
Fraction B Supernatant
Ammonium oxalate 50 mM pH= 5.0
Ammonium oxalate 1 h, 40°C
Supernatant Fraction C
ISOLATION OF GRAPE CELL WALL POLYSACCHARIDES
H PL C RE SU LTS
CHARACTERIZATION OF FRACTION C OF POLYSACCHARIDES
PRPs_PNG
bPRPs gPRPs aPRPs
0 25 50 75 100
125 S+PNG+C new 2.0mg/mL
S+PNG+C new 2.4 mg/mL
Area (%) of cromatographic peaks of salivary proteins
Outras_PNG
Statherin P-B
Cystatins 0
25 50 75 100
125 controlo
Saliva+PNG 277 uM S+PNG+PS 0.4 mg/mL S+PNG+PS 0.7 mg/mL S+PNG+PS 1.2 mg/mL S+PNG+PS 1.5 mg/mL S+PNG+PS 2.0 mg/mL S+PNG+PS 2.4 mg/mL
Area (%) of cromatographic peaks of salivary proteins
Statherin P-B
Cystatins 0
25 50 75 100 125
Saliva control
Saliva+PNG 0.3 mM
Saliva+PNG 0.3 mM+Fraction C 0.4 mg/mL Saliva+PNG 0.3 mM+Fraction C 0.7 mg/mL Saliva+PNG 0.3 mM+Fraction C 1.2 mg/mL Saliva+PNG 0.3 mM+Fraction C 1.5 mg/mL Saliva+PNG 0.3 mM+Fraction C 2.0 mg/mL Saliva+PNG 0.3 mM+Fraction C 2.4 mg/mL Area (%) of chromatographic peaks of salivary proteins
PRPs_B2
bPRPs gPRPs aPRPs
0 25 50 75 100
125 Legend
Legend
Area (%) of cromatographic peaks of salivary proteins
Statherin P-B
Cystatins 0
25 50 75 100 125
Saliva control
Saliva+PNG 0.3 mM Saliva+PNG 0.3 mM+PS 0.4 mg/mL
Saliva+PNG 0.3 mM+PS 0.7 mg/mL
Saliva+PNG 0.3 mM+PS 1.2 mg/mL
Saliva+PNG 0.3 mM+PS 1.5 mg/mL
Saliva+PNG 0.3 mM+PS 2.0 mg/mL
Saliva+PNG 0.3 mM+PS 2.4 mg/mL
Area (%) of cromatographic peaks of salivary proteins
Statherin P-B
Cystatins 0
25 50 75 100 125
Saliva control Saliva+B2 1.2 mM
Saliva+B2 1.2 mM+Fraction C 0.4 mg/mL Saliva+B2 1.2 mM+Fraction C 0.7 mg/mL Saliva+B2 1.2 mM+Fraction C 1.2 mg/mL Saliva+B2 1.2 mM+Fraction C 1.5 mg/mL Saliva+B2 1.2 mM+Fraction C 2.0 mg/mL Saliva+B2 1.2 mM+Fraction C 2.4 mg/mL
Area (%) of chromatographic peaks of salivary proteins
INTRODUCTION
Tannins
Procyanidin dimer B2 (1.2 mM)
Addition to tannins (30 min)
Collection of human saliva
• Healthy and non-smoking volunteers
• Saliva treatment with 10% of trifluoroacetic acid
Addition of human saliva 2
HPLC analysis
INFLUENCE OF FRACTION C OF POLYSACCHARIDES ON SALIVARY PROTEIN-TANNIN INTERACTION
v Different fractions of cell-wall polysaccharides can be obtained from grape skin using different solvents for extraction;
v Fraction C of polysaccharides is mainly rich in galactose, arabinose and galacturonic acid residues;
v In presence of both tannins and in the absence of Fraction C of polysaccharides, only the chromatographic peaks of acidic PRPs, statherin and P-B peptide presented a decrease over 50% of the initial value;
v Fraction C of polysaccharides seemed to be effective to cause a recovery of the chromatographic peaks of aPRPs, statherin and P-B peptide;
v In the case of cystatins interaction with procyanidin B2, the addition of Fraction C of polysaccharides caused a decrease of the chromatographic peak of this protein;
v Fraction C of polysaccharides could be used to modulate the interaction SP-polyphenols of wine and other foodstuffs.
Acknowledgments
The authors would like to thanks Fundação para a Ciência e Tecnologia for financial support by three fellowships (SFRH/BPD/88866/2012, SFRH/BPD/112465/2015 and SFRH/BD/105295/2014) and by the project PTDC/AGR- TEC/6547/2014. The authors would also like to thanks the LAQV (UID/QUI/50006/2013- POCI/01/0145/FEDER/007265) for financial support from FCT/MEC through national funds and co-financed by FEDER, under the Partnership Agreement PT2020. This study has been also supported by Project IBERPHENOL – Red, Research cooperative within the scope of polyphenols and its industrial applications
3
Hydrolysis Reduction Acetylation
Neutral sugars determination
Neutral and acidic sugar composition
Solvolysis (Methanol anhydrous) per-O-trimethylsilylation
Acidic sugars determination
GC-MS analysis
Pellet
(Following extractions)
Chromatographic profile of human saliva detected at 214 nm
1
Glycosyl/Methyl Residue % Moles
Arabinose 22.8
Rhamnose 2.9
Galactose 12.2
Glucose 6.5
Mannose 2.1
Xylose 12.3
Galacturonic acid 37.8
Glucuronic acid 2.6
Table 1. Glycosyl and methyl residues composition (% Moles) of Polysaccharides of Fraction C
Freeze-dried Fraction C
Fraction C
Punicalagin (0.3 mM)
