Investigating the Mechanistic and Structural Role of Lipid Hydrolysis in

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Article Cite This: Langmuir 2018, 34, 12730−12738

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Investigating the Mechanistic and Structural Role of Lipid Hydrolysis in the Stabilization of Ammonia-Preserved Hevea Rubber Latex Sirirat Kumarn,† Nut Churinthorn,‡ Adun Nimpaiboon,§ Manus Sriring,‡ Chee-Cheong Ho,∥ Atsushi Takahara,⊥ and Jitladda Sakdapipanich*,†,‡ †

Institute of Molecular Biosciences, Mahidol University, 25/25 Phuttamonthon 4 Road, Salaya, Nakhon Pathom 73170, Thailand Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Mahidol University, Phayathai, Bangkok 10400, Thailand § Rubber Technology Research Centre (RTEC), Faculty of Science, Mahidol University, Salaya, Nakhon Pathom 73170, Thailand ∥ Universiti Tunku Abdul Rahman, Sungai Long Campus, Chera 43000, Kajang, Selangor Malaysia ⊥ Graduate School of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan

Langmuir 2018.34:12730-12738. Downloaded from pubs.acs.org by UNIV OF SUNDERLAND on 11/02/18. For personal use only.

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S Supporting Information *

ABSTRACT: The stabilization mechanism of natural rubber (NR) latex from Hevea brasiliensis was studied to investigate the components involved in base-catalyzed ester hydrolysis, namely, hydrolyzable lipids, ammonia, and the products responsible for the desired phenomenon observed in ammonia-preserved NR latex. Latex stability is generally thought to come from a rubber particle (RP) dispersion in the serum, which is encouraged by negatively charged species distributed on the RP surface. The mechanical stability time (MST) and zeta potential were measured to monitor field latices preserved in high (FNR-HA) and low ammonia (FNRLA) contents as well as that with the ester-containing components removed (saponified NR) at different storage times. Amounts of carboxylates of free fatty acids (FFAs), which were released by the transformation and also hypothesized to be responsible for the like-charge repulsion of RPs, were measured as the higher fatty acid (HFA) number and corroborated by confocal laser scanning microscopy (CLSM) both qualitatively and quantitatively. The lipids and their FFA products interact differently with Nile red, which is a lipid-selective and polarity-sensitive fluorophore, and consequently re-emit characteristically. The results were confirmed by conventional ester content determination utilizing different solvent extraction systems to reveal that the lipids hydrolyzed to provide negatively charged fatty acid species were mainly the polar lipids (glycolipids and phospholipids) at the RP membrane but not those directly linked to the rubber molecule and, to a certain extent, those suspended in the serum. From new findings disclosed herein together with those already reported, a new model for the Hevea rubber particle in the latex form is proposed.

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INTRODUCTION Latex produced by plants is usually considered to be involved in a biochemical mechanism of defense against damage from, for example, herbivores.1−4 Hevea brasiliensis is the most common source of natural rubber (NR) latex used in the rubber industry to produce both latex-based and dry-rubberbased products. The NR latex is a colloid of rubber particles (RP) dispersed in an aqueous medium of nonrubber components, such as proteins, lipids, carbohydrates, and inorganic salts, commonly known as serum. Within a few hours after tapping, the NR latex undergoes putrefaction and coagulation due to volatile fatty acids (VFAs) produced by microorganisms.5 Both processes involve the rubber particles coming close together and aggregating, known as destabilization, which is caused by charge neutralization. Ammonia is often used as a preservative to treat both field natural rubber © 2018 American Chemical Society

(FNR) latex and latex concentrates during transportation and for longer-term storage. In this case, ammonia acts as a bactericide to prevent undesired enzymatic reactions of the microorganisms to produce VFAs.6,7 Additional outcomes of this ammonia preservation are known to include latex stabilization, which keeps the dispersion of the rubber particles to a maximum.7 Such stabilization is thought to come from the repulsion of negatively charged particles, which are derived from the ammonia-catalyzed hydrolysis of phospholipids present in the latex, adhered to the outer surface of the rubber particles.8,9 However, the actual source of the hydrolyzable lipids involved in the hydrolysis and therefore Received: July 10, 2018 Revised: October 4, 2018 Published: October 18, 2018 12730

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time required before distributing commercial latex or latex concentrates from latex manufacturers to latex-based product manufacturing plants. In the presence of ammonia, water ionizes to create ammonium cations and hydroxide anions, where the latter acts as the nucleophile in the base-catalyzed ester hydrolysis of hydrolyzable lipids present in the latex (Scheme 1). Lipids found in the Hevea latex are approximately 50% neutral lipids, such as triglycerides and free fatty acids (FFAs),22 25% glycolipids (1), mainly digalactosyl diacylglycerols (DGDG, e.g., 1a),23 and 25% phospholipids (4), mainly phosphatidylcholines (PC, e.g., 4a),22,24 especially in clone RRIM 600. Considering the polar−nonpolar interface, the best way to maintain maximum negative charges at the RP surface is for these charged species to be amphiphilic in nature so that they can be adsorbed onto the membrane with their hydrophobic tails anchoring toward the rubber core and their hydrophilic heads in contact with the serum. Carboxylates of fatty acids with the tails long enough to secure themselves to the membrane are therefore the most suitable and are also found to be responsible for such a stabilization mechanism.8,9 Consequently, the polar lipids, namely, glycolipids and phospholipids, are more susceptible to hydrolysis at the interface than the neutral ones, rendering them hydrolyzable. In the initial study, the latex stability was assessed using the mechanical stability time (MST), which is the time taken for a latex to start coagulating under a constant shear force, and the zeta potential, which determines the surface charges of a colloidal system at a given pH and can be used to indicate the degree of electrostatic repulsion between charged particles. This would account for the mechanical and electrostatic aspects of latex stabilization. The number of negatively charged species, specifically, carboxylates of fatty acids (3) in ammoniapreserved NR latex, could also be determined, for example, as the higher fatty acid (HFA) number,8 to correlate with the two stability parameters of latex samples preserved with standard high-ammonia content (0.6% v/v) and low-ammonia content (0.1% v/v with a secondary preservative to match the bactericidal effect) preservation systems. For comparison, another latex was also prepared by saponification to remove any ester-containing particles at the RP surface to confirm their importance in the stabilization mechanism. Additionally, lipidselective fluorescence labeling was employed to visually analyze the different components at the RP surface by confocal laser scanning microscopy (CLSM) as different lipids interact with a fluorophore of choice selectively.25−27 Fluorescence emissions at characteristic wavelengths were analyzed and compared with the ester contents of the corresponding lipids sequentially extracted using different solvent systems determined by quantitative Fourier transform infrared spectroscopy (FTIR) analysis. Finally, the hydrolyzed molecular species responsible for latex stability were identified on the basis of their mass-tocharge ratios (m/z) using gas chromatography−mass spectrometry (GC−MS).

responsible for the latex stability, whether they are those bound to the rubber molecules, adsorbed on the outer surface of the rubber particles, or freely suspended in the serum, has yet to be proven and elucidated. Each rubber particle is a spherical rubber core encased in a well-defined membrane10,11 of a protein−lipid monolayer of approximately 20 nm in thickness.12 The rubber molecule is a polymer of cis-1,4-isoprene units. It is commonly accepted that one end of the rubber molecule (the α-end) is capped with a mono- (OP) or diphosphate (OPP) group,13,14 present since chain elongation in the biosynthesis of the NR latex,15 and the other end (the ω-end) is an isoprene-derived group next to two trans-1,4-isoprene units connecting the main polyisoprene chain16,17 (Figure 1). Although a recent NMR study suggested

Figure 1. Structures of the natural rubber molecule.

the presence of a group of four possible structures between the main cis-polyisoprene chain and the OP/OPP group at the αend (Figure 1, in brown),17 it is likely an isoprene-based unit, possibly just another cis-1,4-isoprene unit.15 To date, no definite NMR structural evidence has been reported for the ωgroup (in yellow) of Hevea NR specifically.16 The dimethylallyl structure has been suggested for this, based on the ω-terminal of NR from other species with much shorter rubber main chains.18,19 However, there have been a number of reports indicating that the α-end is associated with phospholipids13,14 and the ω-end is associated with proteins.15,20,21 In the latex form, where the serum is water-based, it is energetically more stable for the rubber molecules to have their nonpolar chains of polyisoprene packed together to form the rubber core and their polar ends pointing outward, forming an interface with the serum. The minimized polar−nonpolar interaction makes the rubber particle spherical and causes it to be covered with a layer of phospholipids and proteins, which are coordinated with the respective ends. Latex stabilization is essential for storing NR latex suitable for latex-based product manufacturing. One possible mechanism of maximizing the effect is by encouraging the basecatalyzed ester hydrolysis of relevant lipids using ammonia as a base as well as maintaining the resulting negatively charged species on the RP surface. It is therefore very important to understand the chemical reaction taking place at this rubber− serum interface, together with the actual source and location of the hydrolyzable lipids and their resulting carboxylates responsible for the desired RP−RP repulsion. This will help to facilitate potential applications, such as mimicking the natural stabilization mechanism and shortening the storage

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EXPERIMENTAL SECTION

Materials. Reagents were used as supplied. Solvents were purchased as reagent (AR) grade and used without further purification. The water used throughout was pure (type 2) or ultrapure (type 1), as indicated where applicable, purified by a Milli-Q purification system. Freshly tapped NR latex, obtained from H. brasiliensis (clone RRIM 600), was provided by the Thai Rubber Latex Corporation (Thailand) Public Co., Ltd. and subjected to 12731

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Langmuir Scheme 1. Base-Catalyzed Ester Hydrolysis of Glycolipids and Phospholipids Using Ammonia

under high-speed agitation (14 000 rpm) at 35 °C to record the time taken to observe the first sign of flocculation in seconds (s). Determination of the Zeta Potential. A latex sample (100 μL) was added to deionized water (type 1, 20 mL). The resulting mixture was adjusted to pH 10 using a 0.1% w/v potassium hydroxide (KOH) solution. Prior to determining the zeta potential using a zeta potential analyzer (Malvern Autosizer 4700), the sample solution was injected into a sample cell (capillary flow) to flush the cell. The zeta potential was then measured at 25 °C where the dielectric constant was 79, the cell field was 28.9 V/cm, and the current was 1.5 mA. Determination of the Higher Fatty Acid (HFA) Number.8 A latex sample (approximately 10 mL) was deposited on four pieces of filter paper (Whatman grade 542, ø 125 mm), which had been desiccated over concentrated sulfuric acid (conc. H2SO4). The papers were dried using a fan blower at room temperature for 24 h and kept over conc. H2SO4 for at least 16 h. They were then weighed together before being subjected to Soxhlet extraction with acetone in the dark for 24 h. After removing the solvent under reduced pressure, the residue was dissolved in a 0.1 M KOH solution (22 mL) and then acidified using a 0.1 M hydrochloric acid (HCl) solution until pH 2 was reached. The resulting mixture was extracted with diethyl ether (3 × 50 mL). The combined organic phase was washed with a 3% w/v sodium chloride (NaCl) solution at least seven times until the pH was

immediate ammonia preservation: (i) 0.6% v/v ammonia or (ii) 0.1% v/v ammonia with 0.025% w/v tetramethylthiuram disulfide (TMTD) and 0.025% w/v zinc oxide (ZnO), using a commercially available 30% ammonia solution, to afford FNR-HA and FNR-LA latices, respectively. Determination of Total Solids Content (TSC). A latex sample of interest (approximately 3 g) was weighed in a Petri dish before drying in a hot-air oven at 70 °C for 12 h. The dried rubber was weighed again to calculate the TSC using TSC = (mrub/mlat) × 100, where mrub was the mass of the dried rubber and mlat was the mass of the latex sample. Each value of TSC was obtained as an average of three samples. Preparation of Saponified Natural Rubber (SPNR) Latex. The FNR-HA latex (i.e., 0.6% v/v ammonia) was treated with a 1% w/v solution of Triton X-100 in a 1% w/v sodium hydroxide (NaOH) solution at 70 °C for 3 h. The resulting mixture was centrifuged at 20 292g for 30 min twice. The cream fraction collected was diluted with water (type 2) to make 30% total solids content (TSC) and further treated with ammonia to reach 0.6% v/v. Determination of the Mechanical Stability Time (MST). A latex sample adjusted to 30% TSC (approximately 80 g) was subjected to mechanical stability testing (Unitronics Vision120) 12732

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Langmuir close to that of the NaCl solution. After solvent removal under reduced pressure, the residual HFA was dissolved in warm ethanol (approximately 70 °C, 30 mL) and filtered under vacuum (Whatman grade 541). The solution was titrated with a standard 0.01 M KOH solution in the presence of phenolphthalein as an indicator. Water (type 2, 30 mL) was acidified to pH 2 and titrated with the standard KOH solution as a blank. The volume of the KOH solution required to neutralize the HFA solution was subtracted with that used for the blank and then converted to the HFA number using HFA number = 5.61 M V/m, where M is the molar concentration of the KOH solution, V was the volume (mL) of the KOH solution used, and m was the mass (g) of latex total solids. Fluorescence Labeling and Analysis of NR Particles Using Confocal Laser Scanning Microscopy (CLSM). A CLSM sample was prepared by subjecting a latex sample (1 mL) to centrifugation at 20 292g to remove the serum. The cream fraction obtained was redispersed in deionized water (type 1, 1 mL) and incubated with a 1 mM solution of Nile red in a 1:1 mixture of DMSO/glycerol (1 mL) for 1 h. The excess dye was removed from the sample using a dialysis membrane with a molecular-weight cutoff of 50 kDa for 6 h. The resulting RP sample was deposited on a glass slide and covered with a coverslip. Fluorescent images of the RPs were captured using a confocal laser scanning microscope (Olympus FV-1000) equipped with four laser systems (Multi AR, HeNe-G, HeNe-R, and LD405/ 440 laser diode) and a transmitted light detector with an oilimmersion objective lens (60×) and processed using an integrated image analysis program (Olympus Fluoview). The optimal sampling z thickness was fixed at 550 nm/slice. Different combinations of filter sets, which were TRITC and Alexa Fluor 633, were used to measure the fluorescence emission intensities of Nile red-bound lipids on the NR particles (determined from the images and subtracted with that of the background). The fluorescence intensity obtained was divided by the cross-sectional area of the respective particle: ΔFIparticle = (FINR − FIbackground)/area of a particle (μm2). Each value reported was an average of all rubber particles in the selected field of view area. Determination of Ester Content. A dried rubber sample was dissolved in chloroform to make a 1% w/v mixture, which was then cast onto a potassium bromide (KBr) disk and dried under a stream of nitrogen to form a thin film. The film was analyzed quantitatively using FTIR (JASCO FT/IR 4100) by measuring peak heights at 1739 (CO) and 1664 (CC) cm−1 to convert the intensity ratio (A1739/ A1664) to an ester concentration using a calibration curve. The calibration curve was constructed by plotting the intensity ratios observed against the ester concentrations of a known mixture of methyl stearate and synthetic cis-1,4-polyisoprene (Kuraprene IR10) at six concentrations of methyl stearate to obtain a linear equation: y = 0.0092x + 0.0046 (R2 = 0.9876), where y was A1739/A1664 and x was the ester concentration (mmol/kg rubber). Sample Preparation for Determining Polar Lipid Content. A latex of interest was subjected to centrifugation at 20 292g to separate the rubber part, which was then spread on a glass plate and dried in a hot-air oven at 70 °C for 12 h. The dried rubber was cut into small pieces (approximately 30 mg per piece) and Soxhlet-extracted with acetone for 24 h to remove any neutral lipids. The remaining rubber was dried and then dissolved in chloroform at a constant concentration suitable for determining the ester content using FTIR as previously described. Sample Preparation for Determining the Remaining Polar Lipid Content. The rubber sample remaining after the previously described acetone extraction was immersed in a 2:1 mixture of chloroform/methanol at room temperature for 24 h to remove polar lipids at the RP surface.9,28 The sample was then dried, and the remaining polar lipid content was determined using quantitative FTIR analysis described above for determining the ester content. Free Fatty Acid Characterization by GC−MS. The acetone extract previously obtained was subjected to gas chromatography− mass spectrometry (Agilent GC6890N, with ECD-TCD/MSD 5973N) equipped with an HP-INNOWax capillary column (30 m × 0.25 mm i.d., 0.25 μm film thickness). Helium gas was used as a mobile phase with a constant pressure of 10.7 psi. The split ratio was

set to 1:2. The temperatures of the column and the injector were set to 230 and 250 °C, respectively. The oven temperature was raised at 3 °C/min until 230 °C was reached, and it was held constant at this temperature for 18 min. The mass spectrometer was set to the electron impact ionization mode at 70 eV. The temperatures of the transfer line and the ion source were set to 280 °C. The FFA species were identified on the basis of their chain lengths (number of carbon atoms present in a molecule) against a library of known FFAs. In quantitative GC−MS analysis, each sample was analyzed at least twice to obtain the average area under the peak. For ionization accuracy of self-comparison, a commercial sample of each fatty acid was used as a standard to construct a calibration curve by plotting the areas under the peak against four known injected concentrations.29 The gradient of the linear equation obtained was used to convert the areas under the peak of interest into concentrations (mg/mL) and, subsequently, actual amounts (mg/g dry rubber).

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RESULTS AND DISCUSSION For base-catalyzed ester hydrolysis to take place, two main components, namely, ammonia (base) and hydrolyzable lipids (esters), are required. The importance of ester hydrolysis to latex stabilization was investigated by determining the mechanical stability times (MST) of high-ammonia (FNRHA), low-ammonia (FNR-LA), and saponified (SPNR) latex samples after 1, 7, 14, 21, 28, and 35 days of storage (Figure 2).

Figure 2. Mechanical stability time (MST) as a function of storage time for FNR-HA, FNR-LA, and SPNR latices.

The products of the transformation are ammonium salts of respective fatty acids (3) whose molecular structures are negatively charged (carboxylates) with ammonium counterions. Their presence and amounts in the latex samples were confirmed and measured as HFA numbers and zeta potentials at different storage times (Figures 3 and 4). The results from MSTs, HFA numbers, and zeta potentials were found to be in agreement, where the mechanical stability, amount of hydrolyzed lipids, and surface potential all increased in magnitude with storage time in the latex sample with both hydrolyzable lipids and high ammonia present (FNR-HA). The negative values observed for the zeta potential, which became even more negative over storage time, indicated the anionic property of the species produced upon hydrolysis (Figure 4). When the amount of ammonia added was low (FNR-LA), both the mechanical stability (monitored by MSTs) and surface potential (observed using zeta potentials) were unchanged. Although its HFA number increased slightly 12733

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used to characterize or analyze NR particles.30 However, the technique offers visual information on the microscopic structure of a sample surface and allows optical sectioning to study its interior and exterior surfaces. Nile red was chosen as a fluorophore because it is polarity-sensitive (nonfluorescent in water), making it selective to lipids, and therefore it stains lipids characteristically due to the degree of hydrophobicity of the lipids.25,27,31 In other words, Nile red interacts with polar membrane lipids, such as phospholipids, to re-emit at the maximum of 633 nm (red) upon excitation at the maximum of approximately 550 nm, while it interacts with neutral lipids to re-emit at approximately 550−580 nm (green-yellow) upon excitation at the maximum of approximately 510 nm.27 Ammonia-preserved latices stored for 7 and 21 days were labeled with Nile red for CLSM analysis. The preparation technique involved the removal of the serum of each latex sample for efficient staining, so the images obtained were of corresponding RP samples. The fluorescence emission of Nile red for neutral lipids and polar membrane lipids was then recorded at 550 and 633 nm, respectively (Figure 5).

Figure 3. HFA number as a function of storage time for FNR-HA, FNR-LA, and SPNR latices.

Figure 4. Zeta potential as a function of storage time for FNR-HA, FNR-LA, and SPNR latices. Figure 5. Fluorescence images of unmounted Nile red-stained NR particles from ammonia-preserved (FNR-HA) latices stored for 7 (left) and 21 days (right) observed at emission wavelengths of 550 (top) and 633 nm (bottom).

during the first 7 days, the values became constant thereafter, which was likely due to the limited availability of ammonia in the system (Figure 3). In SPNR latex, where ester-containing species at the RP surface and in the serum had been removed but which still had a high ammonia content, no changes were observed in MST, the HFA number, and the zeta potential. The high magnitude of the zeta potentials observed in the SPNR sample could be a result of the nonionic surfactant used in sample preparation. This was because the residual surfactant present at the RP−serum interface pushed the shear plane away from the actual particle surface and the potential of the hydroxide ions used to adjust the pH was measured instead. However, the constant values observed suggested that no hydrolysis had taken place. These results therefore confirmed the occurrence, as well as the importance in latex stabilization, of the base-catalyzed ester hydrolysis of lipids in ammoniapreserved NR latex. Lipid components in NR latex are usually determined by measuring the ester contents, and these can sometimes be obscured depending on the methods of extraction used. Other than NR biosynthesis investigations and synthetic material analyses, confocal laser scanning microscopy (CLSM) is rarely

It can be seen that both neutral and polar lipids were present on the RP surfaces when analyzed with the aid of optical sectioning at 550 and 633 nm, respectively, for the corresponding latices kept for 7 and 21 days. The quantitative results revealed that the averaged fluorescence intensity of emission at 550 nm of the sample prepared from the FNR-HA latex increased linearly with storage time while that from the FNR-LA latex remained constant at its initial value (Figure 6). This suggested that the increase observed only when the latex was in a high ammonia composition corresponded to the FFAs released as a result of hydrolysis. The fluorescence data not only confirmed our initial findings that the ester hydrolysis really took place but also showed how the products from the reaction arranged themselves at the RP surface upon formation, enabling latex stabilization. The fluorescence emission data recorded at 633 nm, on the other hand, showed no changes in the polar lipid content on the RP surface, whether the latex was kept under high (FNR12734

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Figure 6. Averaged fluorescence intensity of emission at 550 nm as a function of storage time for RP samples prepared from FNR-HA and FNR-LA latices.

Figure 8. Total polar lipid content as a function of storage time for rubber samples prepared from FNR-HA and FNR-LA latices after acetone extraction, by quantitative FTIR.

HA) or low ammonia (FNR-LA) conditions (Figure 7). This is surprisingly different from our expectation for the polar lipids

Figure 9. Remaining polar lipid content as a function of storage time for rubber samples prepared from FNR-HA and FNR-LA latices after chloroform/methanol extraction, by quantitative FTIR. Figure 7. Averaged fluorescence intensity of emission at 633 nm as a function of storage time for RP samples prepared from FNR-HA and FNR-LA latices.

(FNR-HA) decreased more markedly than those stored under low ammonia conditions (FNR-LA). The value of the former, however, tended to reach a minimum at around 6.5 mmol per kg of rubber. In Figure 9, a constant polar lipid content of approximately 4 mmol per kg of rubber was observed for either sample at different storage times. This suggested that there were polar lipids that should otherwise be chemically hydrolyzable but remained intact due to their unavailability or less-accessible location at the RP membrane. These lipids, together with the hydrolyzable ones, were also likely to be responsible for the constant fluorescence emission intensities observed at 633 nm in the CLSM studies (Figure 7). The unavailable polar lipids could be those bound to the α-ends of the rubber chains forming the RP membrane. In the case of phospholipids, the interaction between the unavailable lipid and the α-end could therefore be a covalent linkage between their phosphate groups, as supported by the 1H NMR information from our previous enzymatic studies,13 rendering these linked lipids unavailable for ester hydrolysis. The lipid compositions in Hevea latex (3.36−3.67% of dry rubber) and in sheet rubber (2.31−3.27% of dry rubber)

to be consumed through ester hydrolysis over time. A more conventional investigation was therefore carried out to determine the changes in the amounts of the different lipids based on their ester contents using different solvent extraction systems. Acetone is normally used to extract any lipid except polar lipids,8 while a 2:1 mixture of chloroform/methanol will extract all forms of lipid9,28 from solid rubber. Each latex sample of interest was centrifuged before casting the cream into film. To distinguish these lipids, acetone was used first to remove any neutral lipids, and the remaining rubber was then analyzed by FTIR to quantify its ester content, which corresponded to the total polar lipids (Figure 8). Any polar lipids at the RP surface should then be removed by chloroform/methanol extraction. The ester content of the remaining material therefore corresponded to those nonhydrolyzable lipids (Figure 9). From Figure 8, it could be suggested that the polar lipids in the rubber sample prepared from high ammonia conditions 12735

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structure of the Hevea rubber particle in the latex form can be proposed (Figure 10a). To minimize the undesired interactions between hydrophobic rubber chains and the aqueous medium, the NR rubber chain is elongated inward during biosynthesis, pointing the ω-end (which is attached to the protein responsible for the polyisoprene biosynthesis)15 outward. The α-terminus of the rubber chain, which is capped with a hydrophilic mono- or diphosphate group also present since biosynthesis, is linked to a phospholipid13,14 and placed to face the serum. The three-dimensional arrangement which minimizes the rubber−water interfacial surface area is spherical. The proteins and phospholipids linked to the respective rubber chain ends, along with the unbound polar lipids and the initial FFAs, form a membrane to prevent the rubber core from being exposed to the serum. Such linkages at the rubber chain ends are so strong, as they are formed during biosynthesis, that they are responsible for the superior mechanical properties of NR compared to those of its synthetic counterparts.32−35 Hydrolyzable lipids at the membrane surface and those suspended in the serum get hydrolyzed in the presence of ammonia to liberate their fatty acids in the form of ammonium carboxylates, known as salts (Figure 10b). These negatively charged species are adhered to the RP surface by anchoring their hydrophobic tails inward and exposing their charged heads to the serum, making the RPs well dispersed, which is otherwise known as latex stability.

reported by Liengprayoon et al. suggested that the lipid content in NR was mainly associated with the rubber part (i.e., 70−90% of total lipids) rather than the serum.22 Although our sample preparation methods did not differentiate polar lipids suspended in the serum from those at the RP surface completely, it could still be concluded that those involved in base-catalyzed ester hydrolysis were the hydrolyzable ones mainly in the RP membrane, together with those in the serum existing as micelles to minimize the polar−nonpolar interaction. In addition, ammonium salts of FFAs (3), once formed, apparently remained adsorbed on the RP membrane and were responsible for latex stability (Figure 5 at 550 nm). Finally, the fatty acid chains of 3 were identified using GC− MS against a library of known compounds (Table 1). The Table 1. Free Fatty Acid Compositions from FNR-HA and FNR-LA Latices after 1 and 35 Days of Storagea

entry 1 2 3 4 5 6

FFA type b

lauric acid (C12) myristic acid (C14)c palmitic acid (C16)d stearic acid (C18)e oleic acid (C18:1)f linoleic acid (C18:2)g

amount from FNR-HA (mg/g dry rubber)

amount from FNR-LA (mg/g dry rubber)

1 day

35 days

1 day

35 days

0.27 0.33 0.28 0.29 0.24 0.62

0.33 0.41 0.36 0.40 0.30 1.27

0.28 0.34 0.30 0.31 0.26 0.68

0.30 0.35 0.32 0.36 0.29 0.84

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CONCLUSIONS The MST, zeta potential, and HFA number results were all well correlated to confirm that base-catalyzed ester hydrolysis actually took place, converting polar lipids (esters) on the RP surface to corresponding FFA products in the presence of ammonia (base). Further studies employing Nile red, a lipidselective and polarity-sensitive fluorophore, in confocal laser scanning microscopy, revealed visual evidence of the polar lipids at the RP surface forming a membranelike layer and also how their FFA products were distributed. Together with FTIR analysis to compare the ester contents of the samples with the different lipid components sequentially removed, they were able to distinguish the polar lipids that were hydrolyzed to be those at the RP membrane but not directly linked to the α-end of rubber molecules. The FFA species produced were negatively charged under basic conditions and therefore were

a

Footnotes b−g: determined by GC−MS using calibration curves. by = 1.1176 × 109x − 1.7568 × 108, R2 = 0.9903. cy = 1.3795 × 109x − 2.3110 × 108, R2 = 0.9986. dy = 7.5999 × 108x − 1.0335 × 108, R2 = 0.9995. ey = 4.2269 × 108x − 5.2967 × 107, R2 = 0.9522. fy = 4.5156 × 108x − 5.5336 × 107, R2 = 0.9502. gy = 1.4703 × 108x − 2.4071 × 107, R2 = 0.9902.

results obtained, especially where linoleic acid (C18:2) was found in highest abundance (entry 6), were in good agreement with those reported in lipid compositions by Liengprayoon22 and again confirmed that the formation of these species was associated with latex stabilization. However, it is of note that ammonium laurate and oleate are used extensively in the latex industry as stabilizers. Armed with the information obtained herein together with that previously reported,12,21 a new model for the micro-

Figure 10. Proposed Hevea rubber particle model in the latex form: (a) FNR and (b) FNR-HA after storage. 12736

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FNR-LA, 0.1% v/v ammonia with 0.025% w/v tetramethylthiuram disulfide (TMTD) and 0.025% w/v zinc oxide (ZnO) field natural rubber; SPNR, saponified natural rubber; FFA, free fatty acid; CLSM, confocal laser scanning microscopy; VFAs, volatile fatty acids; FTIR, Fourier transform infrared spectroscopy; GC−MS, gas chromatography−mass spectrometry; DRC, dry rubber content; TSC, total solids content.

likely to be the cause of latex stability as they were distributed on the RP surface, orienting their charged ends to face the polar serum at the interface. This keeps the RPs well dispersed through like-charge repulsion and therefore stabilizes the latex. The FFA species were identified using GC−MS as mainly linoleic acid (C18:2, an 18-carbon chain length with 2 double bonds) which is in accordance with the lipid compositions found in Hevea latex in general. To explain the observation, a new model for the microstructure of the NR particle has thus been proposed to include the unbound polar lipids (glycolipids and phospholipids), which can be hydrolyzed in the presence of ammonia at the particle membrane, and FFAs. After storage under high ammonia conditions, the negatively charged fatty acid species generated as a result of hydrolysis of the unbound polar lipids at the particle surface keep the particle dispersion uniform, bringing about latex stabilization.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.8b02321. Confocal laser scanning microscopic (CLSM) images observed at emission wavelengths of 550 and 633 nm for the unmounted Nile red-stained RP samples prepared from ammonia-preserved latex (FNR-HA) stored for 1, 7, 21, and 35 days (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Phone: +66-2-889-3116. Fax: +66-2-889-3116. E-mail: [email protected]. ORCID

Sirirat Kumarn: 0000-0002-1959-1782 Adun Nimpaiboon: 0000-0002-4663-8240 Chee-Cheong Ho: 0000-0001-7683-4237 Atsushi Takahara: 0000-0002-0584-1525 Jitladda Sakdapipanich: 0000-0001-5812-4186 Funding

We acknowledge the Thailand Research Fund (TRG5780141), Mahidol University (A15/2557, MU-PD-2017-7 and MU-PD2018-8), and the Center of Excellence for Innovation in Chemistry (PERCH-CIC), Commission on Higher Education, Ministry of Education, for funding. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully acknowledge the Thailand Research Fund (TRG5780141), Mahidol University (A15/2557, MUPD-2017-7 and MU-PD-2018-8), and the Center of Excellence for Innovation in Chemistry (PERCH-CIC), Commission on Higher Education, Ministry of Education for financial support. Sincere appreciation is extended to the Thai Rubber Latex Corporation (Thailand) Public Co., Ltd. for providing the NR latex.

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ABBREVIATIONS NR, natural rubber; RP, rubber particle; MST, mechanical stability time; HFA, higher fatty acid; FNR, field natural rubber; FNR-HA, 0.6% v/v ammonia field natural rubber; 12737

DOI: 10.1021/acs.langmuir.8b02321 Langmuir 2018, 34, 12730−12738

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DOI: 10.1021/acs.langmuir.8b02321 Langmuir 2018, 34, 12730−12738