CHARACTERIZATION OF THE STRUCTURE, PROPERTIES AND BIOCHEMICAL COMPOSITION OF NATURAL RUBBER OBTAINED FROM COAGULA MATURATED UNDER CONTROLLED CONDITION AND TREATED USING A TSR LIKE PROCESS􀀃

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Latex composition

The composition of latex derived from Hevea tree is a mixture of many different chemicals, both organic and inorganic. The major component of latex is rubber hydrocarbon poly(cis-1,4-isoprene) which forms 30 to 45% w/v latex. Besides rubber and water (55-60% w/v latex), fresh latex has been reported to contains 5-6% of non-isoprene components consisting of carbohydrates, proteins, lipids, amines, and other inorganic constituents. The quantity of these non-isoprene components varies by several factors such as age of the trees, weather, clones, soil and tapping system utilized (Eng and Tanaka, 1993 ; Nair, 2000). Early researchers reported that the tree sap of Hevea has three types of particles dispersed in the liquid phase. They are rubber hydrocarbon, Frey-Wyssling particles (Frey-Wyssling, 1929) and the globular particles named lutoids. Cook and Sekhar (1954) were the first to attempt separating the latex particles into fractions by means of ultracentrifugation. Their work was continued later by Moir (1959) under controlled conditions resulting in the identification of several distinct fractions after centrifugation as presented in Figure 3.

Lutoids

Next in abundance is lutoids accounting for 10 to 20% of the volume. Lutoids are a sub-cellular membrane-bound body ranging in size from 2 to 5 µm. This membrane encloses a fluid serum known as lutoids serum or B serum (as located in the bottom fraction of ultra-centrifuged latex) which was mentioned to destabilize rubber hydrocarbon (Nair, 2000). In addition, lutoids contain a wide range of hydrolytic enzymes and some of their enzymatic properties are analogous to the lysosomes of animal cells (Dupont et al., 1976). Approximately 20% of the dry matter in lutoids is made of water-soluble proteins, of which about 70% is hevein. This substance is anionic and shown to contain no less than 5% sulfur, all as cysteine (Webster and Baulkwil, 1989).

Frey-Wyssling particles

Frey-Wyssling particles are spherical and yellow or orange colored, larger in size and constitute 1% to 3% of latex volume. This particle’s diameter ranges from 0.5 to 2 µm. The membrane encloses a fluid serum known as lutoid serum or B serum which is a destabilizer of NR hydrocarbon (D’Auzac and Jacob, 1989).

C-serum

Both rubber particles and the membrane-bound organelles compose most of the bottom fraction to form a colloidal mixture in an aqueous suspension called cytosol. Cytosol represents 40 to 50% of latex volume and has the highest rubber synthesis activity and general metabolism. The latex cytosol contains minerals, organic acids, amino acids, nitrogenous bases, reducing agents and high molecular weight compounds such as proteins, lipids and nucleotides (Jacob et al., 1993).

Non-isoprene components

Around 10% of the dry matter of H. brasiliensis comprises non-isoprene components. They comprise different biochemical compounds such as proteins, carbohydrates, lipids, and inorganic constituents. The nature and quantity of non-isoprene components in Hevea latex can vary greatly depending on the clones, the exploitation system and the environmental conditions (Vaysse et al., 2012).

Proteins

Fresh Hevea latex contains about 1-2% proteins (w/w) of which about 20% is adsorbed on the surface of rubber particle; an equal quantity is found in bottom fraction (lutoids) and the remainder in the serum phase (Yeang et al., 2002). Adsorbed proteins and phospholipids impart a net negative charge to rubber particles, thereby contributing to the colloidal stability of latex. Moreover, the protein content was also reported to show clonal variations (Wititsuwannakul and Wititsuwannakul, 2001). While over 200 species of proteins can be found in NR latex, most of them are removed during processing. Only a small fraction remains in the products as residual extractable proteins. Though some proteins have been identified to be potential allergens in latex, information about whether all proteins could survive the stringent manufacturing process is presently incomplete (Yip and Cacioli, 2002).

Lipids

Lipids in Hevea latex occur mainly in the rubber particles and bottom fractions. Ho et al. (1975) indicated that lipids associated with the rubber particles and the latex fractions were important in the stability of rubber particles. Depending on the clone, lipids amount to 2.5–3.8% versus dry rubber in latex. The three main families of NR lipids were classified as neutral lipids, glycolipids and phospholipids (Hasma, 1984 ; Hasma and Subramaniam, 1986 ; Liengprayoon et al., 2013).

Carbohydrates and cyclitols

Quebrachitol (monomethyl-1-inositol), sucrose and glucose are the major low molecular weight organic solutes in latex. Quebrachitol, a cyclitol, is the most concentrated single component in the serum phase (75-95% of the total carbohydrates and cyclitols present in latex) (Ohya and Koyama, 2001). Its concentration is found to vary with clones and amounts for around 1% of whole latex. It is a major contributor to the osmotic pressure of the cytosol (D’Auzac and Jacob, 1989). Sucrose is the main sugar in latex as it is the initial molecule in isoprene synthesis and the main element in the laticiferous metabolism. Sucrose concentration in latex is one of the key measurements of latex diagnosis that used to express the physiological state of the exploited trees (Le Roux et al., 2000 ; Silpi et al., 2006). The concentration of sucrose in latex varies with clones and is influenced by exploitation techniques (Nair, 2000).
Non-isoprene components could be dissolved or suspended in the aqueous medium of the latex or absorbed on the surface on rubber particles including contained in other organelles such as lutiods or Frey-Wyssling particles. However, around 50% of these non-isoprene components are leached or degraded during dry rubber processing and their location could not be precisely indicated. In dry matter of latex, 10% is accounted for non-isoprene components and only 5% of those components remained with NR after primary processing (Vaysse et al., 2012).

Natural rubber mesostructure

Industrial utilization of dry NR in various products originated from its special structure and properties. Due to its natural origin as other biopolymers, NR has a complex structure. Mesostructure is a term used to describe dry NR macromolecular structure and gel. Macromolecular structure concerns the average-molar masses, molar mass distribution (MMD), branching, etc. For polymer characterization, average molar masses and MMD are important features because they could significantly impact on physical properties.
The most common averaged-molar masses used in establishing molar mass-property relationships are the number-averaged molar mass (Mn), the weight-average molar mass (Mw), and the z-average molar mass (Mz). The Mw is inevitably higher than the Mn and a measure of the spread of molar masses within a sample is provided by the ratio Mw/Mn which is defined as polydipersity index (Ip). Any polymer defined as monodisperse indicated that all the molecules are of the same sizes. NR from different clones was reported to have a wide range of Ip from 4 to 10 (Subramaniam, 1993). Currently, size exclusion chromatography (SEC) multi-angle light scattering (MALS) is a useful technique applied for polymer average molar masses and MMD determination. It importantly aids in the establishment of structure-property relationships for polymers. A new innovative and alternative technique is under study to assess the mesostructure of natural rubber such as asymmetrical flow field flow fractionation-multi-angle light scattering (A4F-MALS) (Dubascoux et al., 2012).

Molar masses and molar masse distribution

The molar massed and molar masse distribution in polymer systems play an important role in determining their bulk properties. Subramaniam (1972) was the first who studied MMD of fresh NR latex samples with SEC, expressed as MMD0. The MMD0 of NR is an important criterion to estimate certain NR properties that will be obtained after processing (Bonfils et al., 2000). Depending on the clone as well as ages of rubber trees, MMD0 expresses two types of distribution: bimodal or unimodal with a shoulder (sometimes also called ‘quasi-unimodal’) (Figure 4). In young trees, a situation where the lower molar mass peak is larger may be found which was claimed to be due to incomplete biosynthesis of rubber chain (Subramaniam, 1993). This explanation was in accordance with the work of Tangpakdee et al. (1996) who found the increase of number-average molar mass (Mn) with an increase of H. brasiliensis seedling (1, 3, 7, 36 and 84 months) by GPC and osmometry. In addition, though the shapes of distribution curves are different, the range of molar mass is approximately the same in rubber from all clones, normally in the region of 105-107 g.mol-1 (Eng and Tanaka, 1993). Furthermore, the type of harvesting and processing also influenced on MMD as mentioned in the work of Bonfils et al. (2000) which compared the MMD of TSR10 rubber prepared from an unimodal rubber clone (PB217) and bimodal rubber clone (PR107) by SEC. It was found that after processing the inherent MMD (MMD0) was no longer maintained. Figure 4 Examples of inherent molar mass distribution for several natural rubber clones (SEC in cyclohexane according to Bonfils and Char (2005)).

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Gel

Gel is defined as a network of polymers formed as a result of extensive branching or crosslinking, which is usually swollen by solvents but does not dissolve in non-destructive solvents (Lee, 1993 ; Eng et al., 1997). Two types of gel exist in NR: macrogel and microgel. Macrogel (macroaggregates) is the part of NR that is visible and insoluble in a conventional polyisoprene solvent and that can be eliminated by centrifugation. Microgel (microaggregates), contained in the soluble part, cannot be eliminated by centrifugation but can be retained by filtration (porosity < 1 Pm) (Bonfils and Char, 2005). Campbell and Fuller (1984) also used the term microgel to interpret the cloudiness of the solutions obtained when dissolving a sample of NR in dichloromethane. Regarding gel quantification method from ASTM, swelling index is used to define type of macrogel. Low and high swelling index refer to hard gel and loose gel, respectively. Hard gel usually indicates the presence of a material that does not break down readily by milling while the latter gel type is opposite (ASTM, 2000). Recently, it was proposed to quantify indirectly NR gel content in terms of “total gel” that was the sum of macrogel and the microgel calculated from the difference between the initial concentration on rubber put in solution and the injected concentration of the rubber solution measured by SEC-MALS (Bonfils and Char, 2005 ; Wisunthorn et al., 2012).
NR gel content was reported to vary with many conditions. Latex from virgin trees and from the tree which is not tapped for long period were found to contain very high gel content; the amount of macrogel can be as high as 70%. However, the macrogel content decreases on every tapping and reaches a value of about 5% on regular tapping (Sekhar, 1962). Storage of latex after tapping may also influence gel quantity. Freshly prepared NR has low gel content, of about 5 to 10%. During storage, the gel content increases and may reach 50% or even higher after a long storage (Subramaniam, 1987). Under dry storage, this phenomenon is called “storage hardening” (Rodriguez and De Paoli, 1985 ; Subramaniam, 1993 ; Gan, 1996). Gel content can also be increased by other factors such as clonal characteristics and rubber process (Dogadkin and Kuleznev, 1960 ; Ngolemasango et al., 2003).

Table of contents :

LITERATURE REVIEW􀀃
1. Present situation of natural rubber
2. Natural rubber latex and raw rubber production
2.1 Tapping and latex collection
2.2 Raw natural rubber production
3. Natural rubber structure and properties
3.1 Latex composition
3.2 Natural rubber mesostructure
3.3 Natural rubber properties
4. Natural rubber maturation
4.1 Modification of latex biochemical composition during maturation
4.2 Evolution of NR structure and properties during maturation
5. NR oxidation and antioxidants
5.1 NR oxidation
5.2 Antioxidants
5.3 Native antioxidant in natural rubber
5.4 Antioxidant activity assay
MATERIALS AND METHODS􀀃
1. Standards and chemicals
2. Latex sources
2.1 Latex source for uncontrolled maturation conditions experiment
2.2 Latex source for controlled maturation conditions experiment
3. Maturated sample preparation
3.1 Unsmoked rubber sheet (USS) preparation
3.2 NR sample prepared under uncontrolled maturation conditions (mini-sheets)
3.3 Natural rubber sample prepared under controlled maturation conditions
3.4 Maturation box device
4. Lipid extraction from dry rubber
5. Lipid composition analysis
5.1 Qualitative analysis of lipids by thin layer chromatography (TLC)
5.2 Quantitative analysis of lipids by high performance thin layer chromatography (HPTLC)
5.3 Total fatty acid and unsaponifiable compositions analysis by GC-FID/MS
5.4 􀈖-tocotrienol derivertives identification
6. Natural rubber properties measurement
6.1 Determination of dry rubber properties, Initial plasticity (P0) and PRI
6.2 Mesostructure analysis by size exclusion chromatography coupled with a multi-angle scattering detector, SEC-MALS
7. Antioxidant activity test of total lipid using 2,2-diphenyl-1-picrylhydrazyl, DPPH method .
7.1 􀈖-tocotrienol and NR lipid extracts samples antioxidant measurement
7.2 Method repeatability and reproducibility
CHAPTER 1: METHODOLOGY DEVELOPMENT􀀃
1. Introduction
2. Maturated mini-sheet preparation
2.1 Effect of volume reduction
2.2 Effect of latex dilution
2.3 Effect of the drying method
2.4 Maturation duration
3. Optimization of High Performance Thin Layer Chromatography (HPTLC) for NR lipid composition analysis
3.1 Mobile phase optimization
3.2 Standard calibration curve
3.3 Repeatability and reproducibility of the HPTLC method
3.4 Conclusion
4. Optimization of 2,2-di-phenyl-1-picrylhydrazyl (DPPH) antioxidant activity assay for NR lipids
4.1 Solvent modification
4.2 Comparison of a microplate spectrophotometer and cuvette spectrophotometer measurements .
4.3 Standard concentration range
4.4 Measurement repeatability and reproducibility
4.5 Conclusion
5. Structural identification of 􀈖-tocotrienol derivatives
5.1 􀈖-tocotrienol derivatives identification from 􀈖-tocotrienol standard
5.2 Tocotrienol derivatives identification from NR lipid extract
5.3 Conclusion
CHAPTER 2: CHARACTERIZATION OF THE STRUCTURE, PROPERTIES AND BIOCHEMICAL COMPOSITION OF NATURAL RUBBER OBTAINED FROM COAGULA MATURATED UNDER UNCONTROLLED CONDITION AND TREATED USING A USS OR RSS LIKE PROCESSING􀀃
1. Initial pH of latex, and pHs of coagula and serum during maturation
2. Fresh and dry matter of laminated mini-sheets
3. Evolution of bulk properties of NR from matured coagula under uncontrolled maturation conditions (USS/RSS like process)
4. Evolution of lipid extract composition of NR from matured coagula under uncontrolled maturation conditions (USS/RSS like process)
4.1 Lipid quantity
4.2 Qualitative analysis of lipid evolution during maturation
4.3 Quantitative analysis of lipid evolution during maturation
5. In vitro antioxidant activity test of NR lipid extract by 2,2-di-phenyl-1-picrylhydrazyl (DPPH) method
5.1 Antioxidant activity of lipid extracts from rubber obtained from maturated coagula
5.2 Antioxidant activity of purified 􀈖-tocotrienol derivatives
6. Conclusion
CHAPTER 3: CHARACTERIZATION OF THE STRUCTURE, PROPERTIES AND BIOCHEMICAL COMPOSITION OF NATURAL RUBBER OBTAINED FROM COAGULA MATURATED UNDER CONTROLLED CONDITION AND TREATED USING A TSR LIKE PROCESS􀀃
1. Initial pH of latex, and pHs of coagula and serum during maturation
1.1 Comparison with uncontrolled conditions
1.2 pH follow-up of mini-coagula samples under controlled conditions
2. Fresh and dry matter of mini-crepes
2.1 Comparison with uncontrolled conditions
2.1 Evolutions of fresh and dry matter of maturated coagula under controlled conditions
3. Evolution of bulk properties of NR from maturated coagula under controlled maturation conditions (TSR like process)
3.1 Comparison with uncontrolled conditions
3.2 Evolution of bulk properties of mini-crepes under controlled conditions
4. Mesostructure analysis
4.1 Total gel content
4.2 Weight-average molar mass (Mw) and number-average molar mass (Mn)
5. Evolution of lipid extract composition from matured coagula under controlled maturation conditions (TSR like process)
5.1 Lipid quantity
5.2 Qualitative analysis of lipid evolution during maturation
5.3 Quantitative analysis of lipid evolution during maturation
6. In vitro antioxidant activity test of NR lipid extract by 2,2-di-phenyl-1-picrylhydrazyl (DPPH) method
6.1 Comparison with uncontrolled experiment
6.2 Evolution of lipids extract antioxidant activity under controlled conditions
7. Conclusion
GENERAL CONCLUSION􀀃
REFERENCES􀀃

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