HRPP 1st Annual Seminar, 27 – 28 December 2009 (THAILAND)

Lipid Composition of Natural Rubber Sheet

and Relationship With Its Structure and Properties

S. Liengprayoon 1, 2, F. Bonfils 3, J.Sainte-Beuve 3, K. Sriroth 2, E. Dubreucq 1, L. Vaysse 4

1 -Montpellier SupAgro, UMR IATE, Montpellier, France,

2 -Kasetsart University, Faculty of Agro-Industry, Bangkok, Thailand,
3 -CIRAD, UMR IATE, Montpellier, France, 4 -CIRAD, UMR IATE, Bangkok, Thailand
 INTRODUCTION

Fresh latex
Poly-(cis)-1,4-isoprene
(30-45% / latex volume)
 
  
Non-isoprene component 







: Lipids  : Proteins 
: Carbohydrates  : Others 

Rubber Processing

cleaning



  Loss of hydrophilic components

(Proteins, carbohydrates)
   

Dry rubber
  
 
 

Lipids are the main non-isoprene components in dry rubber

 INTRODUCTION

Roles of lipids on physical and chemical properties of rubber

Neutral lipid and phospholipid in lutoid membrane were correlated
positively with the colloidal stability of latex
(Jacob et al.,1975; Ho et al.1996)
Differences
Phospholipids as initiator to form branch points (Sakdapipanich, 2007)
Saturated FA act
samples as nucleating
(grades, clones…) agent in crystallization of NR
(Kawahara et al., 1996) Hardly comparable
conditions (raw, vulcanized…)
Unsaturated FA act as plasticizer (Kakubo et al., 1997)
Some lipid
fattyanalysis methods
acids (such as oleic and linoleic acids) exhibited
synergistic pro-oxidation activity (Arnold and Evans, 1991)
Antioxidant activity (i.e tocotrienol, phenolic compound) (Hasma, 1984)

Research question

Are lipids related to the lack of quality consistency of natural rubber ?

 METHODOLOGY

Ages Clone Planted year

RRIM600 (old) 1988
RRIM600 (young) 1995

Lipid compositions
: Fresh latex
: Dry rubber => Unsmoked sheet Natural rubber structure
[Rodphukdeekul et al., 2008] Molar mass distribution (MMD)
Gel content

Natural rubber properties

Physical properties
Clones Breakdown behavior
Vulcanization characteristics
Clone Planted year Season

RRIM600 1988 April 2004 – December 2006

RRIM600 1995 Rainfall
GT1 1991 • Dry (< 100 mm.)
PB235 1994 • Intermediate (100 - 300 mm.)
BPM24 1997 • Rainy (> 300 mm.)
 METHODOLOGY

1. Lipid content

 Lipid extraction [Liengprayoon et. al, 2008]

2. Lipid classes

 Silica gel column chromatography (LC)

Neutral lipids / Glycolipids / Phospholipids

3. Lipid composition

 Thin layer chromatography (TLC)

Global lipid profile

 R6G spectrophotometry
Free fatty acids

 Gas Chromatography (GC) / GC-mass spectrometry (GC-MS)

Total fatty acid composition Unsaponifiables

 High Performance Liquid Chromatography-MS (HPLC-MS)

Glycolipid families Phospholipid families
 METHODOLOGY

 NR Physical Properties
: Determination of initial Wallace plasticity (P0)/ ISO 2007:1995

: Determination of plasticity retention index (PRI)/ ISO 2930:1995

: Determination of Mooney viscosity (ML)/ ISO 289-1:1994

 Breakdown index (Lim and Ong ,1985)

: Thermal degradation (140 oC starting temperature, 50 rpm)

: Mechanical degradation (50 oC starting temperature, 100 rpm)

 Vulcanization Characteristics
: Minimum torque (Tmin) : Maximum torque (Tmax)
: Scorch time (ts2) : Optimum cure time (tC90)

 Correlations
: JMP IN version 5.1.2 (SAS Institute Inc., USA)
 RESULTS (lipids composition)

Total lipid content of fresh latex

4
17.8% Phospholipids

%Total lipid extract (w/w dry rubber)

25.8% 22.4%
Glycolipids

3
25.0% Neutral lipids
26.0%
3.36ab 3.67a
21.3%
3.36a
23.5%
2 25.3%
2.47c 2.80bc
36.0%
33.0%
61.0%
1
49.0% 54.2%
41.0% 39.0%

0
RRIM600 (old) RRIM600 GT1 PB235 BPM24
(young)

• Low lipid content (RRIM600 (old) and GT1: 2.5 – 2.8% w/w dry rubber)

• High lipid content (RRIM600 (young), BPM24 and PB235: 3.4 – 3.7% w/w dry rubber)

• Neutral lipids => major lipid class ~ 40% of total lipid weight
 RESULTS (lipids composition)

Total lipid content of unsmoked sheet (USS)

4
Phospholipids

%Total lipid extract (w/w dry rubber)

Glycolipids
3 Neutral lipids

2
2.31c 3.28a
2.67b

1
1.99e 2.15d

0
RRIM600 (old) RRIM600 GT1 PB235 BPM24
(young)

: Less lipids in rubber sheet than fresh latex (2.0 – 3.3% w/w dry rubber)

: Less clonal difference in lipid classes

: Neutral lipids : 82 – 86% of total lipids weight
: Glycolipids : 10 – 13 % of total lipids weight
Hydrolysis
: Phospholipids : 4 – 5 % of total lipids weight
 RESULTS (lipids composition)

Free fatty acids of fresh latex and USS lipids

Thin layer chromatography (TLC)

More free fatty acids in USS lipids

Spectrophotometry (Rhodamine 6G)

Free fatty acids

• Fresh latex
1.5 – 2.2 % (w/w total lipids)

1 2 3 4 1 2 3 4
• Unsmoked sheet (USS)
Unsmoked sheet fresh latex
18 – 22 % (w/w total lipids) Lipid extracts

1: RRIM600 (young), 2: GT1, 3: PB235 and 4: BPM24

Lipase activity assay (mobile phase: n-hexane/diethyl ether/acetic acid (80:20:1, v/v/v))

Observed lipase activity in fresh latex

Free fatty acid could be released by enzyme-catalyzed hydrolysis

 RESULTS (lipids composition)

Total fatty acids composition of fresh latex and USS lipids

Table 1 Fatty acid composition (%w/w total fatty acids) of lipid extract from fresh latex
clones C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 FFA C20:0
RRIM600 (old) 0.31b 7.73a 0.58b 14.07a 13.52ab 53.21a 2.34b 7.44c 0.81b
RRIM600 (young) 0.29b 7.36a 0.42b 13.51a 10.41b 47.38b 3.01a 17.00c 0.86b
GT1 0.41a 7.51a 0.43b 13.42a 15.76a 46.99b 1.64c 12.84c 1.00ab
PB235 0.25b 3.33c 1.34a 5.38b 10.41b 11.47dd
11.47 1.04d 73.94aa 0.51c
BPM24 0.42a 5.91b 0.44b 13.16a 12.17ab 37.31c 2.07bc 27.75b 1.08a
Mean of 6 samplings; 3 repetitions was performed for each sampling except RRIM600 (old) and GT1 (mean of 3 repetitions from 1 sampling)

Linoleic acid (C18:2): 32 - 53% (w/w total fatty acids) of RRIM600, GT1 and BPM24

Furan
Table 2 Fatty acid fatty acids (FFA):
composition (%w/w74total
% (w/w total
fatty fatty acids)
acids) ofextract
of lipid PB235 from USS
clones C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 FFA C20:0
RRIM600 (old) 0.29bc 6.53a 0.51ab 19.56a 13.00ab 40.92ab 1.95b 15.78c 1.45ab
RRIM600 (young) 0.27c 6.97a 0.41b 14.19b 11.49b 44.41a 2.58a 18.09c 1.27b
GT1 0.39a 6.42a 0.44b 15.22b 14.72a 37.34bc 1.45c 22.89c 1.58ab
PB235 0.16d 2.37c 0.72a 4.37c 2.44c 7.88d 0.58d 81.17a 0.51c
BPM24 0.34ab 5.72b 0.40b 14.72b 11.81ab 32.97c 1.69bc 30.31b 1.61a
Mean of 15 samplings; 3 repetitions was performed for each sampling

Similar total fatty acid composition between latex and USS lipids
 RESULTS (lipids composition)

Unsaponifiable composition of fresh latex and USS lipids

(x1,000,000)

(b) USS unsaponifiable Peak position Previously reported to

2.50
1. octadecanol be fucosterol
2.25
2. Eicosanol
2.00 bbunsaponifiable of USS
unsaponifiable of USS lipid
total3. -tocotrienol
lipid extract
extracts
Different retention times
1.75 4. -tocotrienol between unknown and
1.50
5. Stigmasterol fucosterol standard
6. -sitosterol
1.25 1 2 3 45 6 7
7. unknown
1.00 Identification
0.75 aaunsaponifiable
(a) Latex unsaponifiable
unsaponifiable of latex
of latex lipid
totalextracts
lipid extract
0.50

0.25 1 2 3 45 6 7
0.00

-0.25

5.0 7.5 10.0 12.5 15.0 17.5 20.0

Figure 1 Total ion count GC-MS chromatogram of silylated unsaponifiable fractions from total lipids
of H. brasiliensis PB235 latex (a) and rubber sheet (b)
 RESULTS (lipids composition)
(x1,000,000) %

100.0 129 (a) Fucosterol

2.50 (a) Fucosterol standard Fucosterol
Peak87, RT 21.43 min
Peak
75.0 386
2.25 Fucosterol
296
(21.43 min)
50.0 119
2.00
145 281
25.0
1.75 257
173 211 371
199 227 343 394 484
469
0.0 327
1.50 100 150 200 250 300 350 400 450 500
%
1.25 129 (b) ∆-5 avenasterol
5-Avenasterol
100.0
Peak
Peak 78, RT 21.68 min
(b) Oat oil unsaponifiable
1.00 75.0 386
-sitosterol 296
∆-5 avenasterol 119
0.75 (21.42 min)
(21.68 min) 50.0
281
145
0.50 25.0 211
257
(c) Latex unsaponifiable 371
173 227
199
343 469 484
0.25
-sitosterol 5-avenasterol
Unknown peak 0.0
100 150 200 250 300 350 400 450 500
(21.42 min) (21.68min)
(21.68 min) %
0.00
100.0 129 (c) Unknown from PB235
21.0 21.5 22.0 22.5
5-Avenasterol
Unknown
Peak
Peak78, RT 21.68 min
Figure 2 Total ion count GC-MS chromatogram 75.0 386
296
of unsaponifiable fractions
119
50.0 281
145
Retention time 25.0 211 257
371
173 199 227 484
0.0
Mass Spectrum 100 150 200 250 300 350 400 450 500

Figure 3 Mass spectrum of fucosterol and 5-avenasterol

 RESULTS (lipids composition)

Table 3 Composition of unsaponifiable from fresh latex (in %w/w of total unsaponifiable)

Octa Eico -toco -toco Stigma -sito ∆-5 avena

Clones
decanol sanol trienol trienol sterol sterol sterol
RRIM600 (old) 3.60b 0.90b 22.20a 4.10a 7.10a 45.88a 16.20ab
RRIM600 (young) 6.63b 1.75b 18.95a 4.30a 8.10a 45.80a 20.68ab
GT1 7.60ab 2.10ab 22.50a 5.70a 6.20a 37.90a 17.90ab
PB235 10.87a 2.17a 17.17a 2.83a 7.15a 34.37a 29.45a
BPM24 9.68ab 2.88a 19.63a 6.55a 6.63a 44.22a 13.06b
Mean of 6 samplings; 3 repetitions was performed for each sampling

-sitosterol was the main sterol (34 - 46% of total unsaponifiable)

Table 4 Composition of unsaponifiable from USS (in %w/w of total unsaponifiable)
Octa Eico -toco -toco Stigma -sito ∆-5 avena
Clones
decanol sanol trienol trienol sterol sterol sterol
RRIM600 (old) 5.19c 0.79c 10.32b 4.68b 8.10a 58.65a 11.79c
RRIM600 (young) 5.52c 1.30bc 13.48ab 5.19b 7.51ab 50.59b 17.12b
GT1 6.74bc 1.53bc 12.17ab 7.20a 7.50ab 57.47bc 11.44c
PB235 8.38ab 1.96b 13.44ab 4.57b 7.27bc 39.93c 24.55a
BPM24 10.36a 3.16a 14.33a 8.05a 6.71c 41.74c 11.44c
Mean of 15 samplings; 3 repetitions was performed for each sampling

Similar unsaponifiable composition between latex and USS lipids

 RESULTS (lipids composition)

Polar lipids of fresh latex analyzed by HPLC-MS

Chromatographic separation
(c) MGDG
2.82
100
%
0
(b) ESG
2.64
100
%
0
7.84
Molecular weight calculation
(a) TIC
5.04
100
3.25
%
e 0 Time
5.00 10.00
(2) H. brasiliensis latex
Molecular species identification
(c) P B 235
1 00 1019
10 19
PB235
m/z 1019 : [FFA/FFA + Na+]
1020
Integration of appropriate % 96 7
968 102 1 1035
939 965
m/z range extracted from 0
937
941 991 995
997 1051

(d ) R R IM 60 0
chromatographic peak 967
96 7
1 00
RRIM600 m/z 967 : [C18:0/C18:2 + Na+]
965 968
: [C18:1/C18:1 + Na+]
% : [C16:0/FFA + Na+]
963 970
939
941 10 19
937 97 1 993 995 10 35
0 m /z
Quantification 850 87 5 9 00 925 95 0 975 1 0 00 10 2 5 1 0 50 1 0 75 1 10 0
 RESULTS (lipids composition)

60
DGDG SG ESG MGDG Digalactosyl diglycerides (DGDG) : 43- 50%
51
50 47 Steryl Glucoside (SG) : 30- 34%
43

Esterified-SG (ESG) : 7-19%

40
% relative

34 34
Monogalctosyl-DG (MGDG) : 8%
30
30

20 19

12
10 8 8 7 8

0
RRIM600 PB235 BPM24

Figure 4 Glycolipid composition of latices from RRIM600, PB235 and BPM24 clones

 DGDG was the main glycolipid family in natural rubber latex

 Different in ESG content => High ESG (19%) in PB235 / the other clones (7-12%)
 RESULTS (lipids composition)

80
PC LPC PE PI LPI PA
70
62 Phosphatidylcholine (PC): 55-62%
60 55 55
Lyso-PC (LPC): 19-28%

50
% relative

Phosphatidic acid (PA): 7-11%

40 Phosphatidylethanolamine (PE): 4-5%

28 28
30 Phosphatidylinositol (PI): 1-3%
19
20 Lyso – PI (LPI): 2-5%
11 10
10 7
4 4 5 5
3 2 2
1 1
0
RRIM600 PB235 BPM24

Figure 5 Phospholipid composition of latices from RRIM600, PB235 and BPM24 clones

 PC was the main phospholipids found in natural rubber latex

 PA and Lysophospholipids (LPC, LPI) => artifacts from phospholipases

 RESULTS (NR Properties)

Table 5 Initial plasticity (P0), Mooney viscosity (ML(1+4) 100) and plasticity retention index (PRI)
of USS from different H. brasiliensis clones.

Clones P0 ML(1+4) 100 Clones PRI

PB235 47.8a 78.8a RRIM600 (old) 104.4a
RRIM600 (old) 39.1b 68.9b RRIM600 (young) 102.1ab
GT1 35.7c 63.3c GT1 101.2ab
RRIM600 (young) 35.3c 61.9c BPM24 100.9b
BPM24 33.6d 58.9d PB235 91.9c
Mean of 15 samplings;
Molar3mass
repetitions were performed for each sampling
distribution

7
PB235 Remarks : PRI > 100 => Crosslinking
6
GT1
Involved with short chain
5 RRIM600 (old)

polyisoprene
Intensity (%)

RRIM600 (young) (Ngolemasango et al., 2003)

4
BPM24

2
Less short polyisoprene chains for PB235
1
=> Low crosslinking ability
0
1.E+03 1.E+04 1.E+05 1.E+06 1.E+0
molar masses (g/mol)
 RESULTS (Breakdown behavior of NR)

Table 6 Breakdown index of USS

Breakdown index (BI)

clones
Thermal conditions* Mechanical conditions**
RRIM600 (young) 2.12a 1.24a
GT1 1.89ab 1.18ab
RRIM600 (old) 1.58b 1.14ab
BPM24 1.86b 1.06b
PB235 0.60c 0.54c
Mean of 15 samplings; 3 repetitions were performed for each sampling
* Thermal conditions; 140oC starting temperature, 50 rpm rotor speed
** Mechanical conditions; 50oC starting temperature, 100 rpm rotor speed

• The rubber with lowest ML value could be masticated easier in both conditions

: RRIM600 (young): the most degraded rubber

: PB235: the least degraded rubber

 RESULTS (Vulcanization characteristics of NR)

Table 7 Vulcanization parameters from different H. brasiliensis clones

clones ts2 (min) tc(90) (min) TL (in.lb) TH (in.lb)

RRIM600 (old) 6.63b 11.65b 12.14b 67.94a
RRIM600 (young) 6.15bc 10.95bc 11.28c 66.12b
GT1 6.19bc 11.24bc 11.16cd 65.35bc
PB235 7.48a 13.27a 12.82a 64.20d
BPM24 5.90c 10.65c 10.70d 65.04c
Mean of 15 samplings; 3 repetitions were performed for each sampling

 The shortest scorch time (ts2) was found in BPM24 / longest ts2 from PB235

 Delay of scorch time consequently resulted in longer optimum cure time (tc(90))

 Minimum torque (TL) was well correlated with ML of unvulcanized rubber

 PB235 / RRIM600 (old) = high ML / high TL

 Maximum torque (TL) did not correspond with the unvulcanized rubber properties
 RESULTS (Correlations)

Correlations between lipid composition & NR structure and properties

15
Axis 2
(13.7%)

 RRIM600 (young)
10

 PB235 BPM24 

Figure 6 Principle component

analysis (PCA) of mean values
Axis 1 of lipid composition and natural
0
(26.4%) rubber structure and properties
GT1 
clones: season:
: RRIM600 (old)  dry season
-5
: RRIM600 (young)  intermediate
: GT1  rainy season

RRIM600 (old) : PB235
-10 : BPM24
-15 -10 -5 0 5 10

Clonal variation RRIM600 (old and young), GT1 and BPM24 PB235
 RESULTS (Correlations)

1. Plasticizing effect

• lipid extracts • Initial Plasticity (P0)

• total fatty acids (furan fatty acid) • Mooney viscosity (ML (1+4)100)
• 5-avenasterol • Cumulated mechanical energy (BIthermal)

• Initial Plasticity (P0)

• neutral lipids
• Mooney viscosity (ML (1+4)100)

55 55
(a) RRIM600, GT1 and BPM24 (b) PB235
50 50
Mean(Po_RRIT)

Mean(Po_RRIT)
45 45

Mean (Po)
Mean (Po)

40 40

35 35

30 30 No correlation from PB235

25 25
2.0 2.5 3.0 3.5 2.0 2.5 3.0 3.5
Mean(%Lipid
Mean
Mean(%(%
lipids extract
lipidsw/w dryvs
w/wdry rubber)
rubber)
rubber) Mean
Mean(%(%lipids
lipids w/w
Mean(%Lipid w/w dry
extract rubber)
dryvs
rubber)
rubber)

Correlation between lipids and P0

 RESULTS (Correlations)

2. Auto-oxidation Enhancer 3. Antioxidant

115
110 (a) (a)
110

100 105

Mean(PRI_RRIT)
Mean(PRI_RRIT)
Mean (PRI)

Mean (PRI)
100
90
95
80 90

70 85
RRIM600, GT1 and BPM24 80 RRIM600, GT1 and BPM24
60
0 .1 .2 .3 .4 .5 .6 .7 .8 .9 .00 .01 .02 .03 .04 .05 .06 .07 .08 .09 .10 .11 .12 .13
Mean (%unsaturated
Mean(%fatty acids
unsat vsw/w
NR)dry rubber) Mean(%alpha toco vs NR)
Mean (%-tocotrienol w/w dry rubber)
Correlation between unsaturated fatty acids and PRI Correlation between -tocotrienol and PRI

• free fatty acids

• Stigmasterol
• unsaturated fatty acids PRI
• -tocotrienols
(Linoleic acid)
 RESULTS (Correlations)

4. Natural activators
12 12
(a)
RRIM600, GT1 and BPM24 (b)
PB235
10 10
(min))

(min))
(ts2 (min))
(min)
(min)
(ts2(min))

8 8
Mean(TS2

Mean(TS2
Mean (ts2
(ts2

6
Mean

Mean
Mean

4 4

2 2
.0 .2 .4 .6 .8 1.0 1.2 1.4 1.6 .0 .2 .4 .6 .8 1.0 1.2 1.4 1.6
Mean (%free fatty acidsvsw/w
Mean(FFA NR)dry rubber) Mean (%free fatty acids
Mean(FFA vs w/w
NR)dry rubber)
Correlation between free fatty acids and scorch time (ts2)

• Lipid extracts
• Neutral lipids
• Free fatty acids
Scorch time (ts2)
• Fatty alcohols
• α-tocotrienol
• 5-avenasterol
 CONCLUSIONS
Lipids compositions

Higher lipids quantity in latex than in USS

• Latex contained 2.5 - 3.7% of lipids and sheet contained 2 – 3.3% of lipids
(PB235 => RRIM600 (young) => BPM24 => GT1 => RRIM600 (old))

Different lipid class proportion between latex and USS

• Mainly by enzyme-catalyzed hydrolysis  High neutral lipids in USS

 High free fatty acids in USS

Similar lipids profile between latex and corresponding USS

• Fatty acid composition  Linoleic acid (C18:2) for RRIM600, GT1 and BPM24

 Furan fatty acid (FFA) for PB235

 Higher FFA in PB235 glycolipid molecular species

than in phospholipids

• Unsaponifiable composition  Mainly -sitosterol in both latex and USS

 Confirmation of 5-avenasterol presence

 CONCLUSIONS
NR structure and properties

Raw rubber

• PB235 showed the extreme characteristics  Highest P0, ML and lowest PRI
 Involvement of short chain polyisoprene
During mastication

• Rubber with low P0 (ML) value could be masticated easier (higher BI value)

During vulcanization

• Scorch time, optimum cure time and minimum torque were related to physical properties
of unvulcanized rubber except maximum torque

Correlations between lipid composition and NR structure and properties

Clonal variation was stronger than other studied variables

The observed correlations showed both advantages and disadvantages of lipids

1. Plasticizing effect 3. Antioxidant

2. Auto-oxidation enhancer 4. Natural activator during vulcanization

 PERSPECTIVES

Lipids and Natural rubber quality

 None of correlation was observed from PB235 => less variation in data

Work on wider range of natural rubber properties ex. degraded natural

rubber sample during maturation

 Mechanism of lipids on natural rubber properties observed from correlations

Ex. anti-oxidant, natural activators

Natural rubber quality

 Natural rubber quality is strongly influenced by clone compared to other

agronomical parameters

Important criteria for rubber plantation or manufacturer

Does monoclonal rubber cause a problem of quality inconsistency?

 HRPP 1st Annual Seminar, 27 – 28 December 2009 (THAILAND)

Thank You For Your Attention