Disentangling the origin of chemical differences using GHOST
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
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
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
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
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.
Notes de l'éditeur
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.