Dr. Merve Kaya- Structure of Citrus Pectine
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Dr. Merve Kaya- Structure of Citrus Pectine
- 1. supervisor: Dr. Marie-Christine Ralet INRA Nantes, France jury members: Prof. Marc Hendrickx KU Leuven, Belgium Prof. Jørn Dalgaard Mikkelsen Technical University of Copenhagen, Denmark Prof. Peter A. Williams Glyndwr University Wrexham, United Kingdom Prof. Phillippe Delavault University of Nantes, France July 9, 2015 Nantes, France
- 2. 1 Outline Brief overview of pectin Objectives Materials & Methods Strategy Results I. Characterisation of citrus pectin samples extracted under different conditions II. Characterisation of isolated pectic domains Summary Perspectives
- 3. Plant cells are surounded by rigid cell walls • cellulose • hemicelluloses • pectin • mainly pectin • mainly lignin (polyphenolic) polysaccharides make up the major part of the primary cell walls Albersheim et al., 2010 2 PERSPECTIVESINTRODUCTION OBJECTIVES STRATEGYMATERIALS & METHODS RESULTS SUMMARY
- 4. Why studying plant cell walls? 3 food ingredient pharmacy product industrial chemical wood technology textile dietary benefits animal feed PERSPECTIVESINTRODUCTION OBJECTIVES STRATEGYMATERIALS & METHODS RESULTS SUMMARY
- 5. Pectin is a gelling, emulsifying, and thickening agent • high sugar jam • low sugar jam • confectionery jellies • yoghurt fruit preparations • acidified dairy beverages 4 PERSPECTIVESINTRODUCTION OBJECTIVES STRATEGYMATERIALS & METHODS RESULTS SUMMARY
- 6. CP Kelco, WallTraC training 2015 Commercial pectin is mainly derived from citrus peel 5 • commercial availability • pectin quality • pectin yield high production of citrus in the USA, Mexico, Brazil, China, and Spain PERSPECTIVESINTRODUCTION OBJECTIVES STRATEGYMATERIALS & METHODS RESULTS SUMMARY
- 7. Orange production represent 60% of the world citrus production 6 2010-world citrus production by fruit type Turner & Burri, 2013 orange juice industry • mostly preferred ones ≠ pectin structure and functionality grapefruit and pomelos lemons and limes oranges tangerines, mandarins, clementines other PERSPECTIVESINTRODUCTION OBJECTIVES STRATEGYMATERIALS & METHODS RESULTS SUMMARY
- 8. Pectin extraction 7 chopping and washing after drying transportation • alcohol precipitation • acidified hot water (nitric acid) extraction filtration purification PERSPECTIVESINTRODUCTION OBJECTIVES STRATEGYMATERIALS & METHODS RESULTS SUMMARY
- 9. 8 xylogalacturonan (XGA) arabinogalactan II arabinans homogalacturonan (HG) rhamnogalacturonan II (RGII) Pectin comprises structurally distinct pectic domains α–D-GalpA α-L-Rhap α-L-Araf β–D-Galp α-D-Xylp β–D-DHAp β-L-Araf α-D-KDOp β-D-Apif β-L-Rhap β–L-Galp β-D-GlcpA α–L-Galp β-D-GalpA β-D-Fucp α-L-Arap β-L-AcefA Ropartz, 2015 methyl esterification acetyl esterification rhamnogalacturonan I (RGI) two main domains arabinogalactan I PERSPECTIVESINTRODUCTION OBJECTIVES STRATEGYMATERIALS & METHODS RESULTS SUMMARY
- 10. 9 homogalacturonan HG is the predominant domain • exclusively composed of α–(1, 4)-linked D-GalpA • unbranched polymer • chain length is approximately 100 GalA residues Voragen et al., 1995; Thibault et al., 1993; Hellin et al., 2005 α–D-GalpA PERSPECTIVESINTRODUCTION OBJECTIVES STRATEGYMATERIALS & METHODS RESULTS SUMMARY
- 11. 10 homogalacturonan HG is the predominant domain • exclusively composed of α–(1, 4)-linked D-GalpA • unbranched polymer • chain length is approximately 100 GalA residues • GalA residues are partly methyl/ acetyl esterified Degree of methyl esterification (DM) • DM > 50 %, High methyl-esterified pectin (HM) • DM < 50 %, Low methyl-esterified pectin (LM) Voragen et al., 1995; Thibault et al., 1993; Hellin et al., 2005 α–D-GalpA methyl esterification acetyl esterification PERSPECTIVESINTRODUCTION OBJECTIVES STRATEGYMATERIALS & METHODS RESULTS SUMMARY
- 12. 11 Gelling mechanism Voragen et al., 1995; Powell et al., 1982; Oakenful & Scott, 1984; Rolin, 2002 Calcium • LM pectin gel according to ‘’egg box’’ model • 7-20 non-esterified GalA residues are required • form gel at pH 3-6, sugar is not necessary • HM pectin gel due to hydrogen bonds and hydrophobic interaction between methylated groups • form gel at pH 2-3.8 and 60% sugar homogalacturonan α–D-GalpA methyl esterification PERSPECTIVESINTRODUCTION OBJECTIVES STRATEGYMATERIALS & METHODS RESULTS SUMMARY
- 13. • alternating GalA and Rha residues RGI backbone Lau et al., 1985 12 α–D-GalpA α-L-Rhap acetyl esterification on GalA residues acetyl esterification PERSPECTIVESINTRODUCTION OBJECTIVES STRATEGYMATERIALS & METHODS RESULTS SUMMARY
- 14. RGI side chains Lau et al., 1985; Albersheim et al., 1996; Ridley et al., 2001 13 arabinogalactan II arabinogalactan I arabinans • mainly Gal and Ara residues are attached to Rha • 20-80% of Rha branched with neutral sugar side chains α–D-GalpA α-L-Rhap α-L-Araf β–D-Galp • (1, 5)- α-L-Araf backbone • branched by α-L-Araf units • (1, 4)-β–D-Galp backbone • short side chains of (1, 5)- α-L-Araf • (1, 3)-β–D-Galp backbone • side chains of (1, 6)-β–D-Galp • Ara residues can be attached single β–D-Galp PERSPECTIVESINTRODUCTION OBJECTIVES STRATEGYMATERIALS & METHODS RESULTS SUMMARY
- 15. Smooth and Hairy Regions Rhamnogalacturonan-I Backbone Homogalacturonan Neutral sugar side chains Rhamnogalacturonan I backbone Model 1 Model 2 (de Vries, 1981; Schols and Voragen, 1996) (Vincken et al., 2003). 14 How these pectic domains are connected to each other? PERSPECTIVESINTRODUCTION OBJECTIVES STRATEGYMATERIALS & METHODS RESULTS SUMMARY
- 16. • GalA & neutral sugar content • molecular weight and intrinsic viscosity gelling strength stabilising power Structure- function relationship 15 • HG length • HG/ RGI proportion PERSPECTIVESINTRODUCTION OBJECTIVES STRATEGYMATERIALS & METHODS RESULTS SUMMARY
- 17. 16 Ara-containing side chains important for gel strength of Ca2+-pectin gels enzymatically modified pectin (Ara- containing side chain degrading enzymes) • significant reduction in Ara content WSP: water soluble carrot pectin DBr: debranched DEP: de-esterified pectin NS: Ara+Gal+Rha Ngouemazong et al., 2012 • ‘’weak’’ gel behaviour (induced entanglement of the polymer) PERSPECTIVESINTRODUCTION OBJECTIVES STRATEGYMATERIALS & METHODS RESULTS SUMMARY
- 18. 17 • to apprehend possible structural and macromolecular variation in extracted-pectin samples related to citrus source • to determine the effect of extraction conditions (pH and extraction agent) on pectin and pectic sub domain characteristics PERSPECTIVESINTRODUCTION OBJECTIVES STRATEGYMATERIALS & METHODS RESULTS SUMMARY
- 19. plant source acid type nitric acid oxalic acid pH mild harsh MN: mild nitric acid MO: mild oxalic acid HN: harsh nitric acid HO: harsh oxalic acid extraction conditions 18 PERSPECTIVESINTRODUCTION OBJECTIVES STRATEGYMATERIALS & METHODS RESULTS SUMMARY
- 20. plant source acid type nitric acid oxalic acid pH mild harsh MN: mild nitric acid MO: mild oxalic acid HN: harsh nitric acid HO: harsh oxalic acid extraction conditions 19 PERSPECTIVESINTRODUCTION OBJECTIVES STRATEGYMATERIALS & METHODS RESULTS SUMMARY
- 21. 20 Oxalic acid is a chelating agent • oxalic acid: isolation of cation-based (mostly Ca2+) cross-linking PERSPECTIVESINTRODUCTION OBJECTIVES STRATEGYMATERIALS & METHODS RESULTS SUMMARY
- 22. 21 2. isolated pectic domains acidic means hot alkali rhamnogalacturonan I-RGI homogalacturonan-HG hot alkali 1. characterisation of extracted-pectin samples PERSPECTIVESINTRODUCTION OBJECTIVES STRATEGYMATERIALS & METHODS RESULTS SUMMARY
- 23. 22 1. extracted pectin samples • yield • sugar composition • molecular weight & intrinsic viscosity PERSPECTIVESINTRODUCTION OBJECTIVES STRATEGYMATERIALS & METHODS RESULTS SUMMARY
- 24. Extraction yields harsh nitric acid harsh oxalic acid mild oxalic acid mild nitric acid mg extract/ g dry peel 23 pH of extraction orange lemon lime grapefruit PERSPECTIVESINTRODUCTION OBJECTIVES STRATEGYMATERIALS & METHODS RESULTS SUMMARY
- 25. Extraction yields the lowest yield with orange mg extract/ g dry peel 24 pH of extraction orange lemon lime grapefruit PERSPECTIVESINTRODUCTION OBJECTIVES STRATEGYMATERIALS & METHODS RESULTS SUMMARY
- 26. 25 1. extracted pectin samples • yield • sugar composition • molecular weight & intrinsic viscosity PERSPECTIVESINTRODUCTION OBJECTIVES STRATEGYMATERIALS & METHODS RESULTS SUMMARY
- 27. Arabinose + Galactose Rhamnose RGI “decoration” 26 arabinogalactan II arabinogalactan I arabinans Rha: branching point Ara and Gal: major sugars PERSPECTIVESINTRODUCTION OBJECTIVES STRATEGYMATERIALS & METHODS RESULTS SUMMARY
- 28. • nitric acid: trim side chains (especially Ara-containing ones) harsh nitric acid harsh oxalic acid mild oxalic acidmild nitric acid RGI “decoration” 27 Ara + Gal Rha pH of extraction orange lemon lime grapefruit PERSPECTIVESINTRODUCTION OBJECTIVES STRATEGYMATERIALS & METHODS RESULTS SUMMARY
- 29. 28 harsh nitric acid harsh oxalic acid mild oxalic acidmild nitric acid RGI “decoration” pH of extraction orange lemon lime grapefruit • nitric acid: trim side chains (especially Ara-containing ones) • oxalic acid: conserved abundant side chains Ara + Gal Rha PERSPECTIVESINTRODUCTION OBJECTIVES STRATEGYMATERIALS & METHODS RESULTS SUMMARY
- 30. 29 RGI “decoration” pH of extraction • grapefruit has low amount of neutral sugar • İt indicates shorter/fewer side chains orange lemon lime grapefruit Ara + Gal Rha PERSPECTIVESINTRODUCTION OBJECTIVES STRATEGYMATERIALS & METHODS RESULTS SUMMARY
- 31. Galacturonic acid Rhamnose HG/ RGI ratio 30 GalA Rha PERSPECTIVESINTRODUCTION OBJECTIVES STRATEGYMATERIALS & METHODS RESULTS SUMMARY
- 32. pH of extraction HG/ RGI ratio nitric acid isolated RGI rich pectin oxalic acid isolated HG rich pectin harsh nitric acid mild nitric acid harsh oxalic acid mild oxalic acid GalA/ Rha 31 orange lemon lime grapefruit PERSPECTIVESINTRODUCTION OBJECTIVES STRATEGYMATERIALS & METHODS RESULTS SUMMARY
- 33. pH of extraction HG/ RGI ratio GalA/ Rha 32 orange and grapefruit were rich in RGI orange lemon lime grapefruit PERSPECTIVESINTRODUCTION OBJECTIVES STRATEGYMATERIALS & METHODS RESULTS SUMMARY
- 34. 33 1. extracted pectin samples • yield • sugar composition • molecular weight & intrinsic viscosity PERSPECTIVESINTRODUCTION OBJECTIVES STRATEGYMATERIALS & METHODS RESULTS SUMMARY
- 35. 34 What is IV [η] (Intrinsic Viscosity)? • HG is a rigid polymer • RGI is more flexible • IV is lower the molecule is compact, occupying a relatively small volume PERSPECTIVESINTRODUCTION OBJECTIVES STRATEGYMATERIALS & METHODS RESULTS SUMMARY
- 36. • nitric acid: hydrolysis of neutral sugar side chains, breakdown of RGI backbone, and possible breakdown of HG domains harsh nitric acid mild nitric acid mild oxalic acidharsh oxalic acid (dL/ g) 35 Intrinsic viscosity pH of extraction orange lemon lime grapefruit PERSPECTIVESINTRODUCTION OBJECTIVES STRATEGYMATERIALS & METHODS RESULTS SUMMARY
- 37. pH of extraction Intrinsic viscosity • nitric acid: hydrolysis of neutral sugar side chains, breakdown of RGI backbone, and possible breakdown of HG domains 36 harsh nitric acid mild nitric acid mild oxalic acidharsh oxalic acid (dL/ g) orange lemon lime grapefruit • oxalic acid: dissolution of better- conserved pectin structure PERSPECTIVESINTRODUCTION OBJECTIVES STRATEGYMATERIALS & METHODS RESULTS SUMMARY
- 38. orange and grapefruit: lower IV Rha-rich samples are more flexible orange lemon lime grapefruit 37 pH of extraction (dL/ g) Intrinsic viscosity Axelos & Thibault, 1991; Ralet et al., 2008 PERSPECTIVESINTRODUCTION OBJECTIVES STRATEGYMATERIALS & METHODS RESULTS SUMMARY
- 39. • oxalic acid extracted pectins rich in HG & RGI stretches with conserved side chains 38 Homogalacturonan Neutral sugar side chains Rhamnogalacturonan I backbone PERSPECTIVESINTRODUCTION OBJECTIVES STRATEGYMATERIALS & METHODS RESULTS SUMMARY
- 40. • nitric acid extracted pectins exhibited lower Mw & IV & RGI stretches with few and/or short side chains 39 Homogalacturonan Neutral sugar side chains Rhamnogalacturonan I backbone PERSPECTIVESINTRODUCTION OBJECTIVES STRATEGYMATERIALS & METHODS RESULTS SUMMARY
- 41. 40 2. isolated pectic domains acidic means homogalacturonan-HG 1. characterisation of extracted-pectin samples yield sugar composition macromolecular characteristics PERSPECTIVESINTRODUCTION OBJECTIVES STRATEGYMATERIALS & METHODS RESULTS SUMMARY
- 42. 41 HG/ RGI ratio PERSPECTIVESINTRODUCTION OBJECTIVES STRATEGYMATERIALS & METHODS RESULTS SUMMARY REDACTED
- 43. HG/ RGI ratios of pectin samples 42 PERSPECTIVESINTRODUCTION OBJECTIVES STRATEGYMATERIALS & METHODS RESULTS SUMMARY REDACTED
- 44. Degree of polymerization of HG domains 43 PERSPECTIVESINTRODUCTION OBJECTIVES STRATEGYMATERIALS & METHODS RESULTS SUMMARY REDACTED
- 45. 44 2. isolated pectic domains acidic means hot alkali rhamnogalacturonan I-RGI homogalacturonan-HG hot alkali1. characterisation of extracted-pectin samples yield sugar composition macromolecular characteristics + purification by chromatography PERSPECTIVESINTRODUCTION OBJECTIVES STRATEGYMATERIALS & METHODS RESULTS SUMMARY
- 46. Sugar analyses of purified RGI regions of lemon 45 REDACTED PERSPECTIVESINTRODUCTION OBJECTIVES STRATEGYMATERIALS & METHODS RESULTS SUMMARY
- 47. HGs : • REDACTED 47 • REDACTED Key findings PERSPECTIVESINTRODUCTION OBJECTIVES STRATEGYMATERIALS & METHODS RESULTS SUMMARY
- 48. • GalA & neutral sugar content • molecular weight and intrinsic viscosity • HG length • HG/ RGI proportion important for pectin applicability gelling strength stabilising power Impact of pectin structural variances on rheological performances? 51 PERSPECTIVESINTRODUCTION OBJECTIVES STRATEGYMATERIALS & METHODS RESULTS SUMMARY
- 49. Acknowledgment WallTraC fellows & PIs Friends… INRA colleagues CP Kelco colleagues Family.. Dr. Marie Christine Ralet PVPP Team Special thanks: • Marie-Jeanne Crépeau • Jacqueline Viqouroux • Susanne Sorensen • Antonio Sousa
- 50. Acknowledgment This presentation reflects the author’s views only. The European Community is not liable for any use that may be made of the information contained herein. More information about the WallTraC project at www.walltrac-itn.eu. The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7 2007-2013) under Grant Agreement n°263916.
Hinweis der Redaktion
- Plant cells are surrounded by rigid cell walls. There are 3 different types of compartments: primary cell walls, secondary cell walls and middle lamella. Primary cell wall is present in young growing cells. Polysaccharides make up the major part of the primary cell walls. It contains cellulose, hemicelluloses, and pectin. The walls of two neighbouring cells are firmly connected to each other by a middle lamella, which contains pectic substances. After cessation of cell expansion, some cells such as those in xylems, schlerenchyma, and periderm develop a secondary cell wall. It is composed of mainly lignin. Lignin is a polymer, consisting of aromatic subunits.
- First of all, plant cell wall are beneficial for human health due to high fiber content. Digestibility of animal feed depends upon plant cell wall structure. Plant cell walls are important organic sources for industrial contexts such as textile, wood technology, industrial chemicals. TRANSITION HERE…The polymers isolated from the cell walls are widely used in diverse application as food ingredient and pharmacy products., that provides a method for reccycling agricultural wastes.
- One of the best known application of pectin are high jam. İts used for low sugar jam and confectionery jellies. İn yoghurt drinks as a stabilizer. And to some extent it is used in pharmacy products.
- More than 80% of commercial pectin is derived from citrus peel due to commercial availability of raw materiall, high quality, citrus pectin has satisfying functional properties in food, and high yield. There is a high citrus production in the USA, Mexico, Brazil, China, and Spain. Alternative sources for commercial pectin are sugar beet and apple.
- Amongst citrus, orange represent 60% of the world citrus production due to high demand in orange juice production. Its followed by grapefruit, lemon, and lime. Citrus sources have different pectin structure and functionality. Lemon and lime are mostly preferred for pectin production due to relatively higher yield and quality.
- The strating material is fresh citrus peel. The chopped peel is washed and then dried. Washing and drying are common pretreatments to de-activate enzymes in fruits. This way, pectin is kept stable for transportation. Pectin is extracted in acidified hot water. Different pectin are extracted due to their different solubility in extraction solvents. Nitric acid is mostly used in industry. After filtration of resulting solution from the insoluble solids, pectin is purified by precipitating in alcohol and then the precipitated pectin is dried.
- Pectin comprises structurally distinct pectic domains. It is tought that these building units are covalently linked to each other throughout the cell wall and middle lamella. There are some minor building blocks of pectin. rhamnogalacturonan II, has a backbone of at least 6-8 GalA residues and decorated with five different side chains. It is a complex MOLECULE, including 12 different monosaccharides and 20 different linkages. It was found in all higher plants analysed to date. xylogalacturonan. GalA backbone is highly substituted by xylose residues. Xylose substitution vary according to plant source. HG and RGI are the major domains of pectin, that I am going to focus on today.
- Commercial pectin is mainy classified according to their degree of methyl esterification. The degree of methyl esterification is defined as the number of moles of methanol per 100 mol of GalA. When 50% of or more of their GalA is methyl esterified is called HM pectin.
- LM pectin gel according to egg box model bu using ionic bridges between the HG chains. 7-20 GalA residues are required for association with Ca.
- RGI backbone is composed of alternating GalA and Rha residues. RGI backbone can me acetyl esterified.
- Rha residues are substituted by sde chains, incuding mainly Gal and Rha residues. The proportion of Rha branched with neutral sugars varies from 20-80% depending on plant source, extraction conditions, and developmental stage. These substitutions may be single Gal units or polymeric side chains of arabinans or arabinogalactans. Arabinans consist of (1, 5)-linked α-L-Araf backbone, and possibly further substituted with short side chains of Ara residues. Arabinogalactans are composed of galactan chains, carrying Ara and Gal residues. There are two types of arabinogalactans, type I and type II. AG type I consists of a linear backbone of (1, 4)-linked β–D-Galp residues, which might be substituted short side chains of (1, 5)-linked α-L-Araf residues. AG type II has a linear backbone of (1,3)-linked β–D-Galp backbone is substituted by numerous side chains, consisting of (1,6)-linked β–D-Galp, Ara residues can be attached to the secondary galactan chains.
- So far two models have been proposed for pectin structure. The first one is established in 1980s. Pectin can be represented as smooth and hairy region. The smooth region is mainly composed of HG, while hairy region comprises RGI, arabinans, AGI, AGII, and XGA. Pectin is composed of alternationg HG and RGI domain. In the second one, the backbone encompasses RGI only, HG is connected as side chains.
- Pectin has a wide range of application relating to GalA residues and neutrals ugar content, molecular weight and intrinsic viscosity. The length of HG and HG/ RGI ratio in the molecule may influence the pectin properties. Therefore, thorugh investigation of these characteristics, it could provide further information towards pectin applicability.
- Pectin samples were obtained from dry citrus peels using four different extraction methods and named according to the type of acid and the pH: Mild nitric acid, Mild oxalic acid, Harsh nitric acid, and harsh oxalic acid.
- Pectin samples were obtained from dry citrus peels using four different extraction methods and named according to the type of acid and the pH: Mild nitric acid, Mild oxalic acid, Harsh nitric acid, and harsh oxalic acid.
- Oxalic acid is a chelating agent, deconstructing pectin networks by entrapping ions (mainly Ca2+) from egg-box junction zones
- The main objective of the study is to apprehend possible structural and macromolecular variation related to citrus source and to determine the effect of extraction conditions on pectin and pectic sub domain characteristics.
- It was found that the highest pectin yield was obtained with lime pectin. Grapefruit, lemon, and orange followed it.
- The main objective of the study is to apprehend possible structural and macromolecular variation related to citrus source and to determine the effect of extraction conditions on pectin and pectic sub domain characteristics.
- In order to provide an overview of monosaccharide variation with respect to plant source and extraction conditions GalA/Rha and (Ara+Gal)/Rha ratios were calculated. Since Ara and Gal are the major sugars in side chains and Rha is the branching point, the molar ratio between the former and the latter, can provide a global insight into RGI ‘decoration’
- Whatever the extraction condition, grapefruit pectin samples exhibited particularly low (Ara+Gal)/Rha ratios compared to pectin samples extracted from the other citrus peel samples that all behaved similarly
- The ratio of GalA to Rha was calculated as a hypothetical representation of HG/RGI ratio within the pectin samples
- With respect to extraction conditions, the highest GalA/ Rha ratios were observed for oxalic acid extracts
- Low GalA/Rha ratios of pectin in orange and grapefruit were associated with high Rha amount in their structure. It suggests that these samples had abundant GalA-Rha repeats.
- The main objective of the study is to apprehend possible structural and macromolecular variation related to citrus source and to determine the effect of extraction conditions on pectin and pectic sub domain characteristics.
- the units of intrinsic viscosity are deciliters per gram
- irrespective of extraction conditions, orange and grapefruit pectin samples exhibited lower intrinsic viscosity values than lemon and lime pectin samples, showing that Rha-rich samples are more flexible.
- The main objective of the study is to apprehend possible structural and macromolecular variation related to citrus source and to determine the effect of extraction conditions on pectin and pectic sub domain characteristics.