# Impact of size and biochemical composition of rubber particles on their interfacial behavior using two Hevea brasiliensis genotypes

Marion Baudoin, Siriluck Liengprayoon, Thongchai Khaeduang, Gilles

Paboeuf, Natedao Musigamart, Saowalak Jantarasunthorn, Tucksin

Lerksamran, Pisamai Chantuma, Christine Char, Maxime Geniez, et al.

► To cite this version:

Marion Baudoin, Siriluck Liengprayoon, Thongchai Khaeduang, Gilles Paboeuf, Natedao Musigamart, et al. Impact of size and biochemical composition of rubber particles on their interfacial behavior using two Hevea brasiliensis genotypes. *Surfaces and Interfaces*, 2025, 67, pp.106622. 10.1016/j.surfin.2025.106622. *hal-05105466*

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# Impact of size and biochemical composition of rubber particles on their interfacial behavior using two *Hevea brasiliensis* genotypes

Marion Baudoin<sup>a,b,c</sup>, Siriluck Liengprayoon<sup>d</sup>, Thongchai Khaeduang<sup>e,f</sup>, Gilles Paboeuf<sup>a</sup>, Natedao Musigamart<sup>d</sup>, Saowalak Jantarasunthorn<sup>d</sup>, Tucksin Lerksamran<sup>d</sup>, Pisamai Chantuma<sup>e,f</sup>, Christine Char<sup>b,c</sup>, Maxime Geniez<sup>b,c</sup>, Sylvie Beaufils<sup>a</sup>, Frédéric Bonfils<sup>b,c</sup>, Laurent Vaysse<sup>b,c</sup>, Véronique Vié<sup>a</sup>, Céline Bottier<sup>b,c,*</sup>

## ARTICLE INFO

### Keywords:

*Hevea brasiliensis* latex

Small rubber particles (SRP)

Large rubber particles (LRP)

Biochemical composition

Fluid films

Interfacial rheology

## ABSTRACT

**Hypothesis:** The interfacial behavior and properties of fluid films of both rubber particle (RP) fractions (i.e. small rubber particles (SRP) and large rubber particles (LRP)) from *Hevea brasiliensis* latex depends on the clone, and more specifically on the RP biochemical composition.

**Experiments:** SRP and LRP fractions from two clones of *Hevea brasiliensis* (RRIM600 and PB235) were characterized: biochemical composition (protein, lipid, *cis*-1,4 polyisoprene chain), RP film formation kinetics at the air-liquid interface, organization of films and interfacial rheological properties.

**Findings:** Both RP fractions from PB235 clone contain more lipids than the ones from RRIM600. Particle size influences all measured indicators in the same way for both clones: SRP adsorb faster, reach a higher surface pressure and display stronger viscoelastic response than LRP. SRP and LRP also show different organizations at various scales, as observed by Brewster angle and atomic force microscopies. For each RP fraction, the clone influences most indicators but with various tendencies. This study highlights the effect of the high lipid content of clone PB235, particularly neutral lipids, on film formation kinetics. The faster formation kinetics of films from clone PB235 is attributed to the saturation of the interface by lipids. SRP films from both clones have similar rheological responses with a strong viscous component, assigned to the complete destructuration of RP membrane and the spreading of the polymer chains. In contrast, LRP retain their individual particle topology thus limiting long-distance interactions at the interface.

## 1. Introduction

Latex, a white fluid exuded from *Hevea brasiliensis*, is a valuable source of natural rubber (NR) which is used in various industrial sectors such as automotive, medical, and consumer applications. Latex is the cytoplasm of the laticiferous cells [1] which is obtained by incision of the tree bark allowing the latex to flow under the effect of high turgor pressure in the laticifer vessels [2]. This complex and opaque liquid is a stable colloidal suspension composed of various micrometric particles such as lutoids and Frey-Wyssling particles, but mainly rubber particles

(RP) in suspension in a cytoplasmic serum called C-serum [3]. RP contain the hydrophobic *cis*-1,4-polyisoprene polymer chains whose stability in aqueous C-serum is ensured by a core/membrane structure, where the polyisoprene core is surrounded by a monolayer membrane composed of proteins and lipids [4,5]. RP display a bimodal size distribution, resulting into two RP categories: small rubber particles (SRP, diameter ~ 0.05–0.4 µm) and large rubber particles (LRP, diameter ~ 0.4–3.0 µm) [6]. SRP and LRP represent, respectively, 94 % and 6 % of the total RP population and respectively, 7 % and 93 % of the total RP volume [7].

<footnote><sup>a</sup> IPR – UMR UR1 CNRS 6251, ScanMAT – UAR 2025, Rennes University, F-35000 Rennes, France</footnote>

<footnote><sup>b</sup> CIRAD, UPR BioWooEB, F-34398 Montpellier, France</footnote>

<footnote><sup>c</sup> BioWooEB, Univ Montpellier, CIRAD, Montpellier, France</footnote>

<footnote><sup>d</sup> Kasetsart Agricultural and Agro-Industrial Product Improvement Institute (KAPI), Kasetsart University, 10900 Bangkok, Thailand</footnote>

<footnote><sup>e</sup> Chachoengsao Rubber Research Center (CRRC), Thailand</footnote>

<footnote><sup>f</sup> Rubber Authority of Thailand (RAOT), Thailand</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: celine.bottier@cirad.fr (C. Bottier).</footnote>
In recent years, SRP and LRP have sparked more and more interest, as described in a recent review [8]. In addition to their size, SRP and LRP differ in their biochemical composition, with notable variations in terms of protein and lipid contents as well as *cis*-1,4-polyisoprene chain lengths and distribution. LRP have a bimodal *cis*-1,4 polyisoprene chain length distribution with short and long chains, while SRP have a unimodal distribution composed overwhelmingly of long chains [9,10]. Both RP types exhibit lipid diversity in their composition including phospholipids, glycolipids, and neutral lipids, but proportions vary according to their size [11]. Disparities in protein composition are also observed between SRP and LRP, with a high protein diversity in their membrane [12]. Rubber Elongation Factor (REF1, 138 amino acids, 14.6 kDa), although present in both RP types, is mainly localized at the surface of LRP [13]. In SRP, the most abundant protein is the Small Rubber Particle Protein (SRPP1, 204 amino acids, 22.4 kDa) [14]. Some studies suggested that REF1 and SRPP1 are involved in the biosynthesis of *cis*-1, 4 polyisoprene chains [15–17]. Although various models of the RP proteolipid membrane have been proposed [4,5,18], several works have shown that proteins SRPP1 and REF1 are not implanted in the same way in the lipid layer surrounding the *cis*-1,4 polyisoprene core. REF1 has the capacity to implant itself deeply into the membrane, whereas SRPP1 remains anchored superficially to the membrane with bonds to the hydrophilic heads of the lipids [19–21].

The biochemical composition of *Hevea* latex is subjected to several factors of variation such as genetic diversity of clones, tapping systems, soil quality and seasonal variations [22–25]. Among the clones, RRIM600 has been extensively studied, in terms of film formation kinetics [26–28] and mechanical properties [29–33]. Because of their marked differences in latex composition, RRIM600 clone is often compared to PB235 one [11,34]. While PB235 produces a latex rich in lipids [35], the latex of RRIM600 is known for its high protein content [36,37]. Moreover, the *cis*-1,4 polyisoprene chains in RRIM600 latex have a bimodal molar mass distribution (MMD), unlike PB235, which is unimodal [38–40]. Finally, the genotype influences not only the biochemical composition of RP but also the mechanical properties of unvulcanized [41] and vulcanized [37] NR samples derived from latex coagulation and process. Therefore, understanding the role of isoprene and non-isoprene biochemical compositions on NR coagulation phenomena is important.

Characterizing the air-liquid interface in a Langmuir trough enabled us to characterize very thin films and to visualize small-scale phenomena [42–45]. Langmuir films are also used to mimic membrane systems [46,47] or synthetic lattice [48,49]. In 1942, Wren was the only one to use this technic to study ammoniated latex spread at the air-liquid interface [50]. Recently, we also used Langmuir trough implemented with different tools and showed that the size of ammonia-free RP from RRIM600 clone influences the interfacial film formation and its structuration [51]. The interface was used to destabilize and aggregate RP to induce the first steps of coagulation, without chemical modification [52, 53]. In the present study, we hypothesized that RP size is not the only factor of variability, but that the biochemical composition of RP could also play a significant role. To further explore this hypothesis, this work aims at investigating the relationship between the *Hevea brasiliensis* genotype, and more specifically the biochemical composition of SRP and LRP (proteins, lipids and polymer chains), and the film formation kinetics as well as structuration and rheological properties of interfacial RP films.

In this study, two contrasted *Hevea* genotypes in terms of biochemical composition, i.e. clones RRIM600 and PB235, have been chosen and the same protocol as previously described was applied to separate both RP fractions [11,51]. The biochemical composition of SRP and LRP fractions from both clones was analyzed in detail: protein (nitrogen) content and SDS-PAGE electrophoresis of protein extracts, total lipid content and thin layer chromatography of phospholipids, glycolipids and neutral lipids and finally molar mass distribution of *cis*-1,4-polyisoprene chains. The kinetics of film formation at the interface were

monitored over time by various methods including ellipsometry, tensiometry and Brewster angle microscopy (BAM). The interfacial films were also transferred to a solid substrate by Langmuir-Blodgett method and their organization was imaged by atomic force microscopy (AFM). Finally, interfacial rheology measurements allowed access to the rheological properties during film formation kinetics. This work complements our previous study by providing new insights through the study of SRP and LRP interfacial fluid films of two *Hevea brasiliensis* genotypes and the relationship between their biochemical composition and the rheological behavior of the films.

## 2. Materials and methods

### 2.1. Latex collection of latex and preparation of SRP and LRP fractions

Clonal certified *Hevea brasiliensis* trees of RRIM600 (30 trees) and PB235 (30 trees) clones were selected from a plantation of RAOT (Rubber Authority of Thailand) at the Chachoengsao Rubber Research Centre (CRRC), Chachoengsao, Thailand. The trees were tapped in early morning (5 a.m.). One hour later, latex that dropped in a clean plastic cup placed on ice (4 °C) was collected and stored in ice until further analysis in the laboratory (Bangkok). Three independent repetitions of latex were collected for each clone (10 trees per repetition). The two fractions of latex containing small rubber particles (SRP) and large rubber particles (LRP) were obtained by latex centrifugation, as previously described [51]. Briefly, latex was first centrifuged at 16,000 g for 45 min at 4 °C. After first centrifugation, the upper rubber fraction (LRP) was collected with a spatula while the C-serum containing SRP was sucked with a syringe and centrifuged at 45,000 g for 45 min at 4 °C. After second centrifugation, the upper rubber fraction (SRP) was collected with a spatula. After separation, fresh SRP and LRP fractions were kept for protein extraction and freeze-drying. The rest was dispersed in a buffer (Tris 100 mM, pH 10) at a concentration of 10 mg mL⁻¹ and the obtained suspensions were stored at 4 °C throughout the study.

### 2.2. Freeze-drying

SRP and LRP were diluted with distilled water at a ratio of 1:4 (w/w). Diluted RP fractions were stored at –20 °C overnight. The precooled RP fractions were subjected to freeze-drying for 48 h. The obtained freeze-dried SRP and LRP were used for the measurement of nitrogen, total lipid and macrogel contents, as well as for the determination of the molar mass distribution of *cis*-1,4 polyisoprenes chains (SEC-MALS).

### 2.3. Biochemical composition: lipids, proteins and cis-1,4 polyisoprene chains

#### 2.3.1. Lipid analysis

##### 2.3.1.1. Extraction of lipids.
Lipids from SRP and LRP freeze-dried samples were extracted using a method established for dry rubber by Liengprayoon et al. [54]. Frozen 2.5 g pieces of the lyophilized fractions were powdered under liquid nitrogen and transferred to 50 ml chloroform/methanol solution at a ratio of 2:1 (v/v). After stirring for 6 h at 200 rpm, the rubber was filtered, and the total lipids were concentrated using a rotary evaporator. The whole extract was then dissolved in 4 ml of the same solvent and the water-soluble components removed with 1 ml of 0.9 % NaCl solution. The lower lipid-containing layer was collected and evaporated to obtain the total lipid extract.

##### 2.3.1.2. Lipid class separation by solid-phase extraction (SPE).
The total lipid extract was fractionated into three lipid classes: neutral lipids (NL), glycolipids (GL) and phospholipids (PL), by solid phase extraction (SPE). Prior to use, an SPE cartridge was activated by sequential rinsing with
2.7 mL methanol and 2.7 mL chloroform. A lipid solution containing 30 mg lipid in 0.5 mL chloroform was then loaded into the activated SPE cartridge. NL, GL and PL were then eluted with 8.1 mL chloroform, 8.1 mL acetone/methanol (9:1; v/v) and 8.1 mL methanol, respectively. The flow rate was maintained at around 3 ml/min using a vacuum pump. Each eluted lipid class was collected, evaporated and weighed. The identity of each lipid class was confirmed by thin-layer chromatography (TLC), following the procedure described below.

### 2.3.1.3. Characterization of lipid extracts by thin layer chromatography (TLC).
Identification of the different lipid classes NL, GL and PL by thin-layer chromatography (TLC) was carried out according to the procedure described by Liengprayoon et al. [35]. We compared the NL profiles of SRP and LRP from RRIM600 and PB235 clones. A 5 µL volume of lipid solution, with a concentration of 10 mg/mL, was applied onto a silica gel 60 G TLC plate and placed in a TLC chamber. The mobile phase for NL separation consisted of n-hexane/diethyl ether/acetic acid (80:20:1; v/v/v). After development, the TLC plate was sprayed with a mixture of 40 % orthophosphoric acid and saturated copper acetate aqueous solution (1:1; v/v), followed by heating at 180 °C for 10 min in an oven. NL spots appeared brownish against a white background. For GL separation, the mobile phase was chloroform/methanol/water (95:20:2.5; v/v/v). After development, the TLC plate was sprayed with a mixture of sulphuric ornicol, then heated to 100 °C for 10-min 15 minutes in an oven. GL spots appear as orange-purple spots on a white background. For PL, the mobile phase was chloroform/methanol/ammonia 30 % (65:25:5; v/v/v). After migration, PL spots are directly visualized after spraying with molybdenum blue and appear as blue spots on a white background. The relative intensity of each spot of NL and GL TLC was analyzed by image analysis using Phoretix 1D software (version 628, TotalLab Ltd.).

### 2.3.2. Protein analysis

#### 2.3.2.1. Nitrogen content.
The nitrogen content of freeze-dried SRP and LRP fractions was determined with a CHN Determinator (LECO CHN628) on a 100 mg sub-sample.

#### 2.3.2.2. Extraction of proteins.
Fresh SRP and LRP fractions were mixed with an extraction buffer containing Tris (100 mM), EDTA (100 mM), glycerol (10 %), Triton X-100 (2 %), DTT (20 mM) and PMSF (2 mM) in a ratio of 1:2 (w/w). The mixture was stirred on a rotating apparatus at 4 °C for 40 min, then centrifuged at 20,000 g for 30 min at the same temperature. After centrifugation, the RP layer was displaced to access the protein-enriched intermediate phase. This solution underwent two further centrifugation cycles at 35,000 g for 30 min at 4 °C to remove residual rubber particles. Protein extracts were stored at -20 °C for further analysis.

#### 2.3.2.3. Separation of protein extracts by Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
SDS-PAGE analysis was performed in 3.75 % acrylamide/bis-acrylamide (Sigma-A9926, USA) of stacking gel and 14 % acrylamide/bis-acrylamide of resolving gel. 30 µl of protein extract were mixed with 30 µl of Laemmli buffer 2X. β-mercaptoethanol was added at a final concentration of 4 % and the obtained mixture was heated for 5 min at 95 °C. Glycerol was then added at a final concentration of 10 %. 45 µl of the final mixture was deposited per well corresponding to proteins extracted from 6.5 µg of fresh RP fraction. Protein migration occurred at room temperature by applying a constant voltage (200 Volts) for 45 min using a Bio-Rad apparatus filled with migration buffer (25 mM Tris, 192 mM glycine, 0.1 % SDS, pH 8.3). Staining of the gels was achieved by agitation during 2 hours in staining solution (40 % ethanol, 10 % acetic acid, 0.1 % Coomassie Blue R-250). Gels were then transferred to a 10 % acetic acid solution and agitated until complete destaining of the background (2 to 3 hours). Gels were scanned on a GS-900 calibrated densitometer (Bio-Rad) and analyzed using Phoretix 1D software (version 628, TotalLab Ltd.).

Lanes of similar width were drawn on the gels where protein bands were automatically detected using single-edge mode. The molecular weights of protein bands were assigned after a calibration procedure using a protein standard containing 10 proteins of known molecular weights: 250, 150, 100, 75, 50, 37, 25, 20, 15 and 10 kDa (Precision Plus Protein 1610,363, Bio-Rad). The intensity of each band (% of the total lane intensity) is obtained from the densitograms of the gels.

### 2.4. Macrogel content

Freeze-dried SRP and LRP samples (30 mg, mass $m_0$) were dissolved in tetrahydrofuran (THF, 30 mL, HPLC grade, Riedel-de-Hans France) stabilized with 250 mg/L of 2,6-di-tert-butyl-4-methylphenol (BHT). The solutions were kept at 30 °C in the dark in a temperature-controlled water bath. After 3 days, the soluble fraction and macrogel were separated by centrifugation at 30 000 g (Beckman Coulter Avanti J-E centrifuge, Paris, France) for 1 hour at 20 °C. The upper soluble fraction was recovered and the macrogel (bottom fraction) was dried (overnight at room temperature under a fume cupboard plus 4 hours at 40 °C in an oven under low pressure) then weighted (mass $m_1$). The macrogel rate was calculated according to this equation: macrogel (%) = ($m_1/m_0$) × 100.

### 2.5. Size exclusion chromatography coupled to a multi angle light scattering detector (SEC-MALS)

Freeze-dried SRP and LRP samples (30 ± 5 mg) were dissolved in pure tetrahydrofuran (THF, 30 mL, HPLC grade, Riedel-de-Hans, France) stabilized with 2,6-di-tert-butyl-4-methylphenol (BHT, 250 mg/L). After storing for 7 days in the dark at 30 °C, the solutions were gently stirred by hands, filtered (Acrodisc 1 µm, glass fiber, Pall, France) and injected into a SEC-MALS system. Three solutions were prepared for each sample, and each solution was injected once. SEC-MALS chromatography line consists of a refractometer (dRI) (Shimadzu RID2000, Montpellier, France, 30 °C), a Wyatt multiangle light scattering detector at 633 nm (Dawn Heleos II, Wyatt, Toulouse, France) and a Shimadzu HPLC line (DGU-20A3R degasser, LC20AD pump, CTO-20A column oven, SIL40 injector, Montpellier, France). The columns, maintained at 45 °C, are two PLgel, Mixed-A LS (Varian, 20 µm, 300 mm × 7.5 mm I. D., Montpellier, France) with a guard column. The mobile phase is THF stabilized with BHT at a flow rate of 0.65 ml/min. The injected volume is 100 µL. All diode detectors at all 18 angles in the MALS detector were normalized using a THF solution of low polydisperse polystyrene standard ($M_w$ = 34 kg/mol, Polymer Standards Service). Number-average molar mass ($M_n$) and weight-average molar mass ($M_w$) were calculated using Astra software version 6.1.7.16 (Wyatt technology). The order of polynomial fit used with the Berry method is two. All light scattering angles and dRI baselines were adjusted for the calculation. The differential refractive index increment (dn/dc) value used was 0.130 ml/g for NR in THF [55]. Astra software performed all the calculations from the chromatogram by integrating the whole peak corresponding to polyisoprene on the chromatogram.

### 2.6. Zeta potential measurement

A sample capillary cell (DT1070) coupled with a zeta potential analyzer (Malvern Nanosizer) was used to measure the zeta potential (ζ-potential) of RP dispersions diluted at 0.02 mg mL⁻¹ in Tris buffer (100 mM, pH 10). Zeta potential measurements were conducted at 25 °C for one minute, with each measurement being repeated three times for accuracy. The stability of the dispersions relies on how the charges are distributed on the surface of the suspended RP and the electrostatic interactions between them. The zeta potential quantifies the electrical potential at the surface arising from these interactions, allowing us to ascertain whether the dispersion remains stable or if there is a tendency using Phoretix 1D software (version 628, TotalLab Ltd.).
for aggregation.

## 2.7. Nanoparticle tracking analysis (NTA)

The size distribution of SRP and LRP dispersions was measured using nanoparticle tracking analysis (NTA) on a NanoSight LM10 instrument (Malvern Instruments, Malvern, UK). Before analysis, SRP and LRP dispersions were diluted in buffer solution (Tris 100 mM, pH 10) to a concentration of 0.02 mg mL⁻¹. Measurements were performed using a 638 nm laser beam at 25 °C, with three 30 s video captures per sample. Videos were recorded using a CCD camera, then analyzed using NanoSight NTA 3.1 software (Malvern, UK). The presented curves are the average of three measurements, expressed as the percentage of RP per sample for different diameters. This RP size analysis enabled us to assess the stability of the dispersions over time and to ensure that no coagulation nor creaming occurred throughout the experiments.

## 2.8. Ellipsometry and surface pressure measurements

Interfacial film formation of SRP and LRP was monitored using ellipsometric angle and surface pressure measurements over time, as previously described [51]. Briefly, ellipsometric angle (Δ, °) measurements are conducted with a homemade ellipsometer equipped with a polarized He-Ne laser (wavelength 632 nm). The angle is linked to the reflective index and thickness of the film, typically indicating the amount of substance at the interface. The laser has a surface area of 1 mm² and probes a depth of 1 µm. The measurement accuracy for the delta angle is 0.2 ° Surface pressure (π, mN m⁻¹) is measured using the Wilhelmy method, utilizing a filter paper plate connected to a microelectronic feedback system (Nima Technology, UK). The surface pressure measurement accuracy is 0.5 mN m⁻¹.

Measurements of ellipsometric angles and surface pressures were conducted simultaneously over time at room temperature (19–21 °C) using a 8 mL circular trough. The cleanliness of the interface was ensured by measuring ultrapure water and buffer for 5 min or under compression. Initial values of both parameters were considered zero unless there are changes observed in the ellipsometric angle or surface pressure. RP dispersions (0.6 mg mL⁻¹) were added directly to the trough after dilution from the stock solution (10 mg mL⁻¹). To counteract evaporation during long experiments (> 20 hours), an automatic syringe pump regularly injected water to maintain a constant volume in the trough. The curves presented in the study are representative of at least two experiments.

## 2.9. Brewster angle microscopy (BAM)

Brewster angle microscopy (BAM) allows visualization at a micrometric scale of the air-liquid interface using a Nanofilm EP3 microscope (Accurion, DE), as previously described [51]. A 532 nm laser beam, reflected by the interface at the Brewster angle $\theta_{Brewster}$, was captured by a 10 × objective and CCD detector to produce images. The Brewster angle is determined by the Fresnel equation with values typically around 53.1° At this angle, p-polarized light is perfectly transmitted through the interface without reflection, resulting in minimal light intensity on pure water. However, when RP adsorb at the interface, creating a refractive index difference from water, grayscale variations are observed, depending on the density of matter at the interface. Samples, diluted to 0.6 mg mL⁻¹, were directly placed in the trough, with water continuously injected using an automated syringe pump to compensate for evaporation. Experiments were conducted over a 20 h period at room temperature (19–21 °C).

## 2.10. Atomic force microscopy (AFM)

Atomic force microscopy (AFM) was used to observe the organization of interfacial SRP and LRP films at a nanometric scale, as previously

described [51]. The interfacial film was transferred to a mica plate using the Langmuir-Blodgett method and imaged by AFM (Multimode Nanoscope 8, Bruker, France) in PeakForce Quantitative Nanomechanical Mapping (QNM in air) mode. This mode provides a detailed height representation of the sample surface at the nanoscale without any degradation, capturing images of height variation and deformation. All images were taken using a standard silicon cantilever (SNL-10, Bruker, France) with a stiffness of 0.06 N m⁻¹, a scan rate set at 1 Hz, and an image resolution of 512 × 512 pixels. Analysis of the AFM images in terms of height variation and deformation was carried out using Gwyddion software (version 2.55).

## 2.11. Interfacial rheology

The rheological properties of interfacial RP films were studied by surface shear rheology measurements carried out using a homemade interfacial rheometer, as previously described [56–59]. An aluminum float containing a mirror was placed at the interface, in the center of the 10 mm diameter Teflon tank. The magnet placed in the float has a permanent magnetic field of 2.10⁻⁵ T and was positioned parallel to the terrestrial field by means of a solenoid. Two Helmholtz coils were used to apply torque to the magnet placed in a float. The amplitude and displacement of the float were recovered by reflecting a laser through a mirror on the magnet to a CCD sensor. Both parameters depend on the rheological properties of the interfacial layer. The Kelvin-Voight solid model was used to calculate the storage modulus (μ=G's, mN m⁻¹), the loss modulus (G''s, mN m⁻¹) and the surface viscosity (η, N s m⁻¹) [47, 60].

Calibration was performed on pure water with a frequency sweep in the range 0.01–100 Hz. Moduli values G's and G''s were then calculated. Diluted samples at a concentration of 0.6 mg mL⁻¹ were directly introduced into the trough, where water was continuously supplied via an automated syringe pump to counteract evaporation. The experiments were conducted at room temperature (19–21 °C) over a 20 hours duration, employing a shear sweep ranging from 1–50 Hz in 0.5 Hz steps, every 30 min. Each measurement was an average of 3 measurements.

# 3. Results and discussions

## 3.1. Hevea genotype influences the biochemical composition (lipids, proteins, cis-1,4 polyisoprene chains) of both SRP and LRP

Ammonia-free fresh lattices from *H. brasiliensis* clones RRIM600 and PB235 were centrifuged to isolate both SRP and LRP fractions which were stabilized in the form of highly-diluted dispersions (0.02 mg mL⁻¹) in a basic buffer (Tris 100 mM, pH 10). These RP dispersions were characterized by measuring their size distribution and zeta potential values (Fig. 1). For both clones, the size distributions of SRP and LRP are centered on very contrasting diameters, reflecting a good separation of the two RP fractions. Moreover, for both clones, the SRP peaks have a particle concentration about 10 times higher than the LRP ones. For RRIM600 clone, the size distribution of SRP presents two peaks of similar intensities centered on 83 and 132 nm whereas the one of LRP shows one broad peak at 560 nm. These data on RRIM600 clone (origin: Chachoengsao province) are in good agreement with our previous study on this clone (origin: Chanthaburi province) where values of 141/187 nm and 575/637 nm were measured for SRP and LRP, respectively [51]. For PB235 clone, SRP show a unimodal size distribution with a thin and intense peak centered on 190 nm, while the size distribution of LRP shows a broad peak centered on 562 nm. The size of the two RP fractions are in agreement with values reported in the literature, varying from 50 to 400 nm and 400 to 3000 nm for SRP and LRP, respectively [6,61].

For clone RRIM600, zeta potential values of –57 mV and –70 mV were measured for SRP and LRP, respectively. The zeta potential absolute values measured for PB235 clones were slightly higher with –62
<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Size distribution profiles of SRP (blue color) and LRP (red color) for the clones RRIM600 (full lines) and PB235 (dotted lines) obtained by NTA. The values of zeta potential (ζ-potential) are added for each sample.</figcaption></figure>

mV and -71 mV for SRP and LRP, respectively. For both clones, the zeta potential of LRP has a higher absolute value than the one of SRP. This trend and range of values are in good agreement with measurements carried out in our previous study [51]. With absolute values above ±30 mV, the stability and good repulsion of RP from each other in solution is validated [62]. Strong RP repulsion is ensured by the presence of a charged membrane surrounding the hydrophobic polyisoprene chain cores [15,63,64].

The presence of negatively charged proteins at the surface of RP contributes to their stability in solution. In this work, the nitrogen content of whole latex is higher for RRIM600 (0.71 ± 0.09 % w/w dry matter) than PB235 (0.56 ± 0.04 % w/w dry matter), in agreement with literature [22,37,65]. Table 1 gathers the nitrogen contents of SRP and LRP fractions from clones RRIM600 and PB235, which are in the same range as previous studies [11,66]. Although the whole latex of RRIM600 is richer in proteins than PB235, this clonal difference is no longer observed for SRP and LRP whose nitrogen contents are respectively comprised within range of 0.53–0.57 and 0.22–0.23 % w/w dry matter. For both clones, SRP are about 2.4 times richer in proteins than LRP: 0.57 vs 0.23 % w/w dry matter for RRIM600 and 0.53 vs 0.22 % w/w dry matter for PB235. This is explained by the specific membrane surface area between the two RP sizes, SRP having a larger specific membrane surface area than LRP.

The SDS-PAGE gels of proteins extracted from SRP and LRP fractions of RRIM600 and PB235 clones, and the corresponding densitograms, are shown in Fig. 2A and Fig. 2B, respectively. In total, 24 bands were identified in each sample (Fig. S1) whose molecular weight and intensity are listed in Supporting Information (Table S2). For all SDS-PAGE lanes, band number 22 located at 14 kDa is the most intense band and corresponds to REF1 protein [13]. For SRP fractions, band number 18 located at 23 kDa is the second most intense band and corresponds to SRPP1 protein [67]. These major isoforms of REF and SRPP proteins are known to be located at the surface of RP [15]. For LRP, the

ratios of the intensity of band 22 (REF1) over the one of band 18 (SRPP1) are 4.1 and 3.0 for RRIM600 and PB235, respectively. This ratio is roughly two times lower for SRP: 2.2 and 1.3 for RRIM600 and PB235, respectively. These observations agree with the presence of REF1 in both RP fractions while SRPP1 is more concentrated on SRP than on LRP [14]. Finally, the bands of high molecular weight (> 35 kDa) present in all SDS-PAGE lanes mainly arise from proteins present in C-serum [11] as RP were not washed after separation.

The contents of total extracted lipids from SRP and LRP of both clones are reported in Table 2. As observed for proteins, SRP are richer in lipids than LRP, due to their larger specific membrane surface. This observation is valid for both clones. As previously reported [11], the RP fractions from PB235 are richer in lipids compared to those from RRIM600. Specifically, SRP from PB235 and RRIM600 contain 4.85 % and 3.96 % w/w dry matter of total lipids, respectively, while LRP contain 3.48 % and 2.02 % w/w dry matter of total lipids. Total lipid extracts were separated into different lipid classes: neutral lipids (NL), glycolipids (GL) and phospholipids (PL) (Table 1). For RRIM600, SRP has similar proportions (~ 33 %) of PL, GL and NL. The majority of LRP lipids is made of NL which represent 65.6 % of total lipids whereas PL and GL represent 16.1 % and 18.3 % of total lipids, respectively. In the case of PB235, NL is also found as the major lipid in LRP representing 70.2 % of total lipids, followed by GL (17.9 %) and PL (11.9 %). In SRP, NL account for 40.7 % of total lipids, followed by GL (33.4 %) and PL (25.9 %). These values are in accordance with those reported in the literature [11,68]. Based on the measured total lipid content (% w/w dry matter) and the proportions of PL, GL, and NL in total lipids (Table 2), the respective contents of PL, GL, and NL (% w/w dry matter) were calculated. For SRP from RRIM600 and PB235 respectively, the contents (% w/w dry matter) are as follows: 1.32 and 1.26 for PL, 1.31 and 1.62 for GL, and 1.22 and 1.97 for NL. Similarly, for LRP from RRIM600 and PB235, the contents are respectively: 0.33 and 0.41 for PL, 0.37 and 0.62 for GL, and 1.33 and 2.44 for NL.

The total lipid extracts were characterized by TLC to qualify the lipid composition for each NL, PL and GL class (Fig. 3), using standards to identify the different spots. Nine spots were observed in the TLC of NL (Fig. 3A) corresponding to free sterols (FS), free fatty acids (FFA), γ-tocotrienol (γ-toco), α-tocotrienol (α-toco), trifuranoyl glycerols (TGF), triacyl glycerols (TG), tocotrienol ester (TE), sterol esters (SE) and the most polar lipids of NL. A major difference between both genotypes is the presence for both RP fractions of PB235 of a strong spot of TGF which is almost absent for both RP fractions of RRIM600, as previously observed for whole latex [35,69]. The relative intensity of NL spots differs between the four samples as indicated by image analysis in Supporting Information (Fig. S3). For RRIM600 clone, the spot of highest relative intensity is FS for SRP and TE for LRP, while for PB235 clone, TGF is the spot of highest relative intensity for both SRP and LRP. The TLC of GL (Fig. 3B) shows four major spots attributed to digalactosyldiacylglycerol (DGDG), steryl glucoside (SG), monogalactosyldiacylglycerol (MGDG) and esterified steryl glucoside (ESG). As observed for NL, the relative intensity of GL spots differs between the four samples as indicated by image analysis in Supporting Information (Fig. S4). For RRIM600 clone, the spot of highest relative intensity is DGDG for SRP and SG for LRP, while for PB235 clone, the contrary is observed as the spot of highest relative intensity is SG for SRP and DGDG

<table><caption>**Table 1**

Averaged nitrogen, total lipid, phospholipid, glycolipid and neutral lipid contents from SRP and LRP of clones RRIM600 and PB235. Data are the means of 3 independent repetitions and are presented with their standard deviation.</caption><thead><tr><th>Hevea brasiliensis Clone</th><th>Fraction</th><th>Nitrogen<br/>[% w/w dry matter]</th><th>Total lipids<br/>[% w/w dry matter]</th><th>Phospholipids<br/>[% w/w of total lipids]</th><th>Glycolipids<br/>[% w/w of total lipids]</th><th>Neutral lipids<br/>[% w/w of total lipids]</th></tr></thead><tbody><tr><td rowspan="2">RRIM600</td><td>SRP</td><td>0.57 ± 0.04</td><td>3.96 ± 0.41</td><td>33.3 ± 7.3</td><td>33.0 ± 2.5</td><td>33.7 ± 5.0</td></tr><tr><td>LRP</td><td>0.23 ± 0.01</td><td>2.02 ± 0.67</td><td>16.1 ± 0.6</td><td>18.3 ± 2.4</td><td>65.6 ± 1.8</td></tr><tr><td rowspan="2">PB235</td><td>SRP</td><td>0.53 ± 0.04</td><td>4.85 ± 0.49</td><td>25.9 ± 8.2</td><td>33.4 ± 4.3</td><td>40.7 ± 5.6</td></tr><tr><td>LRP</td><td>0.22 ± 0.01</td><td>3.48 ± 0.43</td><td>11.9 ± 1.1</td><td>17.9 ± 3.3</td><td>70.2 ± 3.4</td></tr></tbody></table>
<figure><img src="image_5.png" /><figcaption>Fig. 2. A) SDS-PAGE gels of protein extracts from LRP and SRP of RRIM600 and PB235 clones, B) SDS-PAGE densitograms of the gels shown in A.</figcaption></figure>

<caption>**Table 2**</caption>

Averaged molecular mass in number (M<sub>n</sub>), in weight (M<sub>w</sub>) and the polydispersity index (PI) from SRP and LRP of clones RRIM600 and PB235. Data are the mean of 3 independent repetitions and are presented with their standard deviation.

<table><thead><tr><th>Hevea brasiliensis<br>Clone</th><th>Fraction</th><th>Macrogel<br>(%)</th><th>M<sub>n</sub> (kg/mol)</th><th>M<sub>w</sub> (kg/mol)</th></tr></thead><tbody><tr><td rowspan="2">RRIM600</td><td>SRP</td><td>21.7 ± 0.8</td><td>1138 ± 21</td><td>1688 ± 14</td></tr><tr><td>LRP</td><td>11.0 ± 0.7</td><td>387 ± 57</td><td>1289 ± 73</td></tr><tr><td rowspan="2">PB235</td><td>SRP</td><td>14.9 ± 0.7</td><td>1064 ± 27</td><td>1589 ± 24</td></tr><tr><td>LRP</td><td>8.1 ± 2.1</td><td>887 ± 42</td><td>1489 ± 30</td></tr></tbody></table>

for LRP. Finally, the TLC of PL (Fig. 3C) shows a single spot corresponding to phosphatidylcholine (PC). For all samples, no spot corresponding to lysophosphatidylcholine (LPC) was observed indicating that no partial hydrolysis of PC occurred.

Macrogel contents were measured on both freeze-dried RP fractions from clones RRIM600 and PB235 and are reported in Table 2. The macrogel is the part of the sample that is still visible and insoluble after several days in a conventional solvent for polyisoprene. It is a network formed by polyisoprene chains interacting with certain non-isoprene compounds [70,71]. For both fractions (SRP and LRP), the macrogel is higher for RRIM600 than for PB235 (SRP: 21.7 % > 14.9 %, LRP: 11.0 %

<figure><img src="image_6.png" /><figcaption>Fig. 3. TLC of total lipids extracts (deposit of 5 µL volume of lipid solution, with a concentration of 10 mg ml<sup>-1</sup>) from SRP and LRP from both clones RRIM600 and PB235 developed in: A) n-hexane/diethyl ether/acetic acid (80:20:1; v/v/v) to reveal NL, B) chloroform/methanol/water (95:20:2.5; v/v/v) to reveal GL and C) chloroform/methanol/ammonia 30 % (65:25:5; v/v/v) to reveal PL. The positions and the abbreviated names of the standards (detailed in the text below) are indicated on the left part of each TLC.</figcaption></figure>
> 9.2 %). For both clones, SRP contain more macrogel than LRP (SRP/LRP = 2.0 for RRIM600 and 1.6 for PB235), as previously shown for latex films from RRIM600 clone [66,72]. The higher protein contents of SRP as compared to LRP could result in stronger protein/polyisoprene interactions in agreement with higher macrogel measured in SRP [66, 73].

Both freeze-dried RP fractions from clones RRIM600 and PB235 were analyzed by size exclusion chromatography coupled to a multiangular light scattering detector (SEC-MALS). The SEC-MALS elution profiles are presented in Supporting Information (Fig. S5). Freeze-dried LRP from RRIM600 exhibited a bimodal elution profile. The first peak (short elution time) corresponds to a population of long polyisoprene chains and the second one to a population of short polyisoprene chains. Unlike RRIM600, freeze-dried LRP from PB235 exhibited a quasi-unimodal elution profile. The main difference between the LRP of the two clones lies in the short-chain population. SRP of both clones contain mainly long polyisoprene chains (unimodal elution profile). These results agree with the literature reporting unimodal and bimodal elution profiles for latex from PB235 and RRIM600 clones, respectively [22,38,72,74]. As a consequence of these elution profiles, number-average ($M_n$) and weight-average ($M_w$) molar masses are lower for LRP from RRIM600 clone as compared to the three other samples (LRP from PB235 and SRP from both clones). These $M_n$ and $M_w$ are in the same trend as previously observed by Thepchalerm et al. [72].

The above biochemical description (proteins, lipids, polyisoprene chains) of SRP and LRP fractions from clones RRIM600 and PB235 is consistent with findings reported in the literature. This step was essential for gaining a comprehensive knowledge of our samples and for supporting the interpretation of the measurements at the interfaces described below.

## 3.2. Lipids from clone PB235 saturate the interface resulting in different kinetic formation and organization of RP films as compared to clone RRIM600

Film formation kinetics at the air-liquid interface of SRP and LRP from both clones were investigated over a 20 hours period using various complementary techniques including ellipsometry, tensiometry and BAM. The results of surface pressure $\pi$ and ellipsometric angle $\Delta$ measurements over time are shown in Fig. 4A and 4B, respectively. For both clones and both RP fractions, an increase in surface pressure is observed over time until a stabilization plateau is reached from ~ 10 hours (Fig. 4A). This surface pressure increase reflects RP adsorption at the air-liquid interface. Whatever the clone, SRP adsorb faster than LRP which is explained by a faster diffusion due to smaller RP size. SRP also reach a

higher maximum surface pressure ($\pi_{max}$ = 23.5 mN m$^{-1}$) than LRP ($\pi_{max}$ = 19.0 mN m$^{-1}$) reflecting stronger lateral interactions for SRP than LRP due to higher lipid content in SRP that spread at the interface. This observation agrees with our previous study on film formation kinetics at the air-liquid interface of RP from clone RRIM600 [51].

Although the surface pressure increase over time of RP from clone PB235 shows the same pattern as the ones of RRIM600, the maximum surface pressure is reached faster for PB235 than RRIM600. It is believed that the RP size is not the cause of this difference as the size of LRP is similar for both clones while the ones of SRP are very close (Fig. 1). So, we assume that the differences in RP biochemical composition induced by the genotype might influence the destabilization and destructuration rate of RP at the interface, increasing the number of lateral interactions in the film more rapidly for clone PB235 than for clone RRIM600. As the protein contents are in the same range for both clones, the higher proportion of total lipids for both RP fractions of clone PB235 may be the cause of the differences observed in surface pressure kinetics (Table 1).

In addition to surface pressure, the delta angle was measured during adsorption kinetics by ellipsometry and its temporal evolution is shown in Fig. 4B From 0 to 4 hours of kinetics, the delta angle increases for both RP fractions and both clones. This increase reflects the arrival of matter at the interface and hence RP adsorption. This is consistent with the rapid increase in surface pressure over the first 4 hours of kinetics. After 4 hours, SRP and LRP do not follow the same trend over time, as observed in our previous study [51]. The delta angle of SRP stabilizes for both clones, forming a plateau around $\Delta$ = 8°, reflecting a constant amount of material at the interface. In contrast, the LRP films of both clones regularly densify during kinetics as shown by the continuous increase of delta angle. Clones RRIM600 and PB235 reach $\Delta_{max}$ values of 27.3° and 18.6°, respectively. This difference indicates that, at the end of the kinetics, LRP from clone RRIM600 form a denser layer with more matter at the interface than clone PB235. This might be caused by a more saturated interface for clone PB235, which would limit the adsorption of additional RP at the interface. The more rapid saturation of the interface would be consistent with the faster increase in surface pressure in the LRP of clone PB235.

From the BAM images shown in Fig. 5, two different interfacial organizations after 20 hours of kinetics are highlighted for SRP and LRP. For clone RRIM600, the organizations at 20 hours agree with those already observed [51], i.e. the presence of white spots for SRP (Fig. 5A) and aggregate networks for LRP (Fig. 5C). This is consistent with the lower delta angle of SRP, as compared to LRP which have a denser interface in terms of amount of matter at 20 hours. For SRP, the white spots present at 20 hours (Fig. 5A) are not visible anymore at 30 hours (Fig. 5B). We assume that after a certain time, the white spots spread out

<figure><img src="image_7.png" /><figcaption>**Fig. 4.** Temporal evolution of surface pressure (A) and ellipsometric angle (B) during the 20 hours adsorption kinetic at the air-liquid interface of SRP (blue color) and LRP (red color) from clones RRIM600 (full symbols) and PB235 (empty symbols).</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>**Fig. 5.** BAM images (395 × 494 µm²) recorded at different times of adsorption kinetics at the air-liquid interface of SRP (A-B) and LRP (C) from RRIM600 clone, SRP (D-E) and LRP (F) from PB235 clone.</figcaption></figure>

and lose thickness to form a homogeneous film but with no change in the amount of matter at the interface. The interface of SRP from PB235 clone at 20 hours (Fig. 5E) shows a homogeneous gray level across the entire image, indicating a film with regular height at this scale. This homogeneous surface is similar to that observed for SRP from RRIM600 at 30 hours of kinetics (Fig. 5B) but different from the one observed at 6 hours showing small white spots at the interface (Fig. 5D).

At BAM scale, the SRP of both clones reach a similar organization, but at different kinetic times (i.e. 20 hours for PB235 and 30 hours for RRIM600), in agreement with the surface pressure kinetics. Apart from this difference in interfacial film formation speed, the SRP film organization is not affected by the genotype at this scale. LRP from both clones show aggregate networks at the air-liquid interface with similar structures at 20 hours kinetics (Fig. 5C and Fig. 5F for RRIM600 and PB235, respectively). However, the interface of LRP shows fewer aggregates for PB235 than for RRIM600, in agreement with ellipsometry. The hypothesis of the role of RP size difference between the two clones having been ruled out previously, the interpretation of these differences in interfacial film formation kinetics and final state (in terms of aggregates) turns to the lipid composition of the particles, which is influenced by the clone (Table 1).

After transferring the RP film on a solid support using the Langmuir-Blodgett method, the samples were visualized by atomic force microscopy (AFM) at different surface pressures that are gathered in Supporting Information (Fig. S6) and in Fig. 6. For more clarity, Fig. 6 only shows the AFM images at the end of the kinetics (20 hours).

The SRP images of both clones (Fig. 6A and 6B) show a similar organization with high zones of bright color (highlighted by white

arrows), whose height from the bottom varies from 85 to 103 nm. These high zones have a crater shape surrounded by zones lower than the crater but higher than the background of the image. These hollows are believed to result from aging of the film at the interface between the beginning of the surface pressure plateau (~ 10 hours) and the end of the kinetics (20 hours). As a limitation of AFM image analysis is the attribution of topographical variation to molecular nature, it is helpful to monitor the evolution of the film at different pressures and/or times. From the comparison of images recorded at 5 hours (π =10 mN m⁻¹), 10 hours (π =19 mN m⁻¹) and 20 h (π =19 mN m⁻¹) (Fig. S6A), the crater shape seems to be related to the initial position of the observed aggregates. In these different bright zones, the RP no longer appear either in isolated particles nor as aggregates of spherical particles. These observations agree with our previous study on SRP of RRIM600 [51], where we observed an organization with loss of individual RP topology, assumed to be linked to destructuring and spreading of the membrane and cis-1,4 polyisoprene chain core over the interface during kinetics. It is important to note that the interior of the craters (background) is at the same level, sometimes slightly higher, than the rest of the image, indicating that the core of the SRP aggregates is spread out over a first layer of amphiphilic molecules, probably RP membrane lipids. Nevertheless, some differences appear between both clones concerning the background. For RRIM600, it presents a homogeneous surface with depression of 5 nm (Fig. S6A) while for PB235 it presents a roughness of few nm. The different lipid contents and, more specifically, higher NL contents in PB235 (major specie TGF), of the two clones may play a key role in the kinetics of SRP deployment and spreading at the interface.

AFM images of LRP samples from clone RRIM600 and PB235 at 20
<figure><img src="image_9.png" /><figcaption>**Fig. 6.** AFM images (20 × 20 µm²) in height mode at 20 hours kinetics of SRP with Δz = 155 nm (A, B) and LRP with Δz = 2.1 µm (C, D) films from clones RRIM600 and PB235. Images 1, 3 (Δz = 750 nm) and 2, 4 (Δz = 10 nm) are zooms (5 × 5 µm²) of aggregates and backgrounds in height mode. Images 1' and 3' correspond to images in deformation mode of images 1 and 3, respectively.</figcaption></figure>

hours are shown in Fig. 6C and Fig. 6D, respectively. The organization of the two samples shows similarities in the shape and height of the structures visible at the interface. These structures reveal the aggregation of individual LRP, even if, for PB235, the contrast between individual particles is less clear. This interfacial aggregation of LRP is in accordance with the observations made for RRIM600 [51]. Although of similar shape, the structures of RRIM600 form a network on the mica surface, as observed at the liquid interface on BAM images (Fig. 5C). For the clone PB235, these structures are independent of each other's or less connected, in line with BAM images (Fig. 5F). This difference in densification or quantity of matter at the interface, should be related with higher ellipsometric angle values observed for RRIM600 than for PB235 but also between LRP and SRP whatever the clones. In addition, the aggregates of two clones show differences in their surface state in topographic and deformation images (Fig. 6C, images 1, 1' and Fig. 6D, images 3, 3'). For RRIM600, the surface presents individual particles in close packing, which are linked by brighter zones (Fig. 6C, image 1). These higher zones exhibit lower deformability than the LRP (Fig. 6C, image 1'), suggesting different chemical composition or organization. In contrast, PB235 has a more complex surface, with several low variations in height, and unlike the RRIM600 clone, there is no delineation between LRP (Fig. 6D, image 3). Similarly, there are several deformability values (Fig. 6D, image 3') that can also be assimilated to several chemical compositions or organizations. The difference in lipid composition between the two clones may be at the origin of these differences. Indeed, clone PB235 contains a higher total lipid content than RRIM600 and among them, NL, and most specifically TGF, are predominant. The precise location of NL within RP fractions is not reported in the literature, but we can assume their presence, at least partially, in the hydrophobic RP core where they could interact with the hydrophobic part of the RP protein/lipid membrane resulting in membrane rigidification thus maintaining the membrane around the *cis*-1,4

polyisoprene core. This assumption is supported by a recent study showing that, lipid droplets (storage organelles accumulating hydrophobic molecules whose structure is similar to RP [75]) in *A. thaliana* leaves, contain enzymes associated with furan-containing fatty acid biosynthesis [76], the main fatty acid found in NL from PB235 clone [69], and especially in LRP fraction (Fig. 3A). The AFM images of the backgrounds observed for LRP from clones RRIM600 and PB235 are shown in Fig. 6C (image 2) and Fig. 6D (image 4), respectively. The space between the particle aggregates is covered by a molecular film, as attested by a high surface pressure (around 20 mN m⁻¹), resulting from the spreading of the first particles. For RRIM600, the film is continuous with a low average roughness of 0.78 nm and with a few bright domains or spots. Regarding the literature, the collapse surface pressure is lower for native NL from latex than for GL and PL, reflecting lower compressibility and increased interfacial stiffness [21]. It is thus believed that the background is composed of a PL/GL monolayer added with aggregates of NL, and possibly sprinkled with membrane proteins [77]. In the same way, the background of LRP from PB235 shows a roughness greater than for RRIM600, with the presence of spherical white objects, possibly linked to a larger amount of total lipids (in particular NL) in the LRP of PB235 as compared to RRIM600. These background observations reinforce the hypothesis of interface saturation by lipids during kinetics in the case of PB235, preventing new particles from adsorbing to the interface and thus limiting the increase in delta angle over time. This rapid saturation of the interface by lipids from the PB235 clone decreasing the interfacial accessibility would explain the difference in surface pressure kinetics over time with clone RRIM600.

### 3.3. SRP film has a high viscoelastic response, whereas LRP film does not form a cohesive layer

The temporal evolutions of rheological parameters calculated from
the Kelvin Voight solid model (G's: storage modulus and G''s: loss modulus) during the film formation of SRP and LRP of both clones under shear stress are shown in Fig. 7A and Fig. 7C, respectively. In addition, knowing the surface pressure evolution over time, both G's and G''s moduli are presented as a function of the surface pressure in Fig. 7B and Fig. 7D for SRP and LRP, respectively.

As shown in Fig. 7A and Fig. 7C, all RP films display a viscoelastic response as indicated by the regular increase of G's and G''s moduli along time [64]. At all times, all samples exhibit G''s > G's, reflecting very fluid RP films where viscous interactions dominate over the elastic ones. For SRP, both G's and G''s are null until ~2.5 hours and then start to regularly increase until they reach a plateau at ~16 hours (Fig. 7A). For LRP, both G's and G''s remain null until ~5 hours and reach their plateau earlier, at ~10 hours (Fig. 7C). At 20 hours, SRP display higher values of G's and G''s than the ones of LRP (Table 3). This difference in viscoelastic response shows a stronger layer cohesion in SRP, in agreement with the spread of polymer chains over the interface evidenced by other methods. In contrast to SRP, the polymeric cores of LRP remain in their original particulate form which limits interactions between chains.

Although the SRP of both clones have close values of G's, η's and G''s after 20 hours (Table 3), the kinetics over time differ (Fig. 7A). Indeed, the storage modulus response, G's, increases more rapidly and reaches a slightly higher value for SRP from RRIM600 (G's = 1.55 ± 0.08 mN m⁻¹) as compared to PB235 (G's = 1.14 ± 0.14 mN m⁻¹). This greater storage modulus response to shear could originate from a thicker background in RRIM600 induced by the spreading of cis-1,4 polyisoprene chains over the interface. The chains would act as a spring in the layer, providing RRIM600 a higher storage modulus than PB235, where the chains,

<table><caption>**Table 3**

Surface rheological properties of SRP and LRP for RRIM600 and PB235 clones at 20 hours of kinetics.</caption><thead><tr><th rowspan="2">Indicator</th><th colspan="2">RRIM600<br/>t = 20 hours</th><th colspan="2">PB235<br/>t = 20 hours</th></tr><tr><th>SRP</th><th>LRP</th><th>SRP</th><th>LRP</th></tr></thead><tbody><tr><td>μ = G's [mN m⁻¹]</td><td>1.55 ± 0.08</td><td>0.28 ± 0.03</td><td>1.14 ± 0.14</td><td>&lt;0.03</td></tr><tr><td>ηs [m s m⁻¹]</td><td>3.25 ± 0.14<br/>× 10⁻⁵</td><td>1.77 ± 0.14<br/>× 10⁻⁵</td><td>3.63 ± 0.15<br/>× 10⁻⁵</td><td>2.60 ± 3.06<br/>× 10⁻⁶</td></tr><tr><td>G''s [mN m⁻¹]</td><td>2.69 ± 0.30</td><td>0.92 ± 0.08</td><td>2.96 ± 0.18</td><td>0.20 ± 0.08</td></tr></tbody></table>

although spread out, would not occupy the entire interface. In the case of PB235, chain spreading would be limited due to interface saturation by lipids. Looking at viscosity ηs and loss modulus G'', the opposite effect is observed: SRP from clone PB235 (ηs = 3.46 10⁻⁵ m s m⁻¹, G''s = 2.96 ± 0.18 mN m⁻¹) have a higher response than clone RRIM600 (ηs = 3.33 10⁻⁵ m s m⁻¹, G''s = 2.69 ± 0.30 mN m⁻¹). The high loss modulus reflects the high viscosity and liquid behavior of the interfacial layer. This viscous response may be induced by friction between the polymer chains during shear loading of the layer. As shown in Fig. 7B, for surface pressures π < 20 mN m⁻¹, there is no rheological response of SRP interfacial layers of both clones. This threshold in surface pressure corresponds to the point at which the interface is sufficiently dense and energy-stable to provide a shear solicitation response. This threshold is also the point at which the stabilization in surface pressure begins (Fig. 4A) and therefore the onset of particle aggregate spreading, as

<figure><img src="image_10.png" /><figcaption>**Fig. 7.** Evolutions of G's (full symbols) and G''s (empty symbols) during the 20 hours adsorption kinetic at the air-liquid interface of RP from clones RRIM600 (blue color) and PB235 (red color) under shear stress as a function of time for SRP (A) and LRP (C) and as a function of surface pressure for SRP (B) and LRP (D).</figcaption></figure>
observed in a previous study [51]. This corroborates the hypothesis that the rheological response is caused by the spreading of *cis*-1,4 polyisoprene chains of SRP on the air-liquid interface.

The interfacial rheology response over time of LRP from both clones is shown in Fig. 7C. As compared to SRP, the clonal effect is more pronounced for LRP. While a weak but positive response in storage and loss moduli are observed for RRIM600 (G's = 0.28 ± 0.03 mN m⁻¹, G''s = 0.92 ± 0.08 mN m⁻¹), PB235 has almost no response to shear solicitation (G's < 0.03 mN m⁻¹, G''s = 0.20 ± 0.08 mN m⁻¹). LRP from RRIM600 display a higher ellipsometric angle than PB235 at 20 hours (Fig. 4B), corresponding to a larger amount of matter the air-liquid interface. Indeed, the BAM and AFM images show that the LRP interfacial film is constituted by aggregates formed by individual particles and linked together, resulting in a network across the entire interface. In contrast, for LRP of clone PB235, the particle aggregates are not linked directly to each other resulting in a weaker film cohesion and a rheological response close to zero, as observed in Fig. 7C. Fig. 7D shows the viscoelastic response of the LRP interfacial layer as a function of surface pressure. For both clones, and as observed for SRP, a threshold surface pressure appears at which the rheological response becomes greater than zero, particularly for clone RRIM600 (π = 18 mN m⁻¹ and π = 15 mN m⁻¹, for RRIM600 and PB235 respectively). The difference in surface pressure thresholds agrees with the faster film formation kinetics of PB235. LRP have a weaker rheological response than SRP probably because their polyisoprene chain core has not unfolded and spread out on the surface. Unlike SRP, there is no polymeric network formation at the interface during kinetics resulting in the absence of a rheological response.

## 4. Conclusion

RP size was previously shown to affect the interfacial behavior of RP fluid films from RRIM600 clone [51]. This study was carried out to clarify whether the RP biochemical composition could also affect the RP fluid film formation and properties. Two biochemically contrasted *Hevea* genotypes, i.e. RRIM600 and PB235 clones, were chosen and the biochemical composition (lipid, protein, *cis*-1,4 polyisoprene) of SRP and LRP was analyzed. While the protein contents of SRP and LRP are almost similar for both clones, both RP fractions of PB235 are richer in lipids, and particularly in NL, as compared to RRIM600. As previously observed for RRIM600 [51], the RP size for PB235 influences the formation kinetics (surface pressure, ellipsometry) and organization (BAM, AFM) of interfacial RP films. This work also highlights that RP size affects the rheological properties of RP films for both clones. Indeed, SRP display stronger viscoelastic response (G's and G''s) than LRP, which can be attributed to their complete membrane destructuring at the interface and consequently the spreading of *cis*-1,4-polyisoprene chains.

Although the general kinetics of film formation of both RP fractions are similar for both clones, differences in biochemical composition between clones, particularly in lipid content, influence other indicators. RP from PB235 show faster adsorption at the interface, likely due to lipid saturation, which also explains the formation of a less dense interfacial layer, especially in the case of LRP from PB235. For LRP from the RRIM600 clone, membrane extrusion and partial destructuring at the interface are more pronounced as compared to PB235. This difference in membrane reorganization may be due to a stiffer RP membrane in PB235, potentially caused by the higher NL content than RRIM600. The rheological response is also affected by the clone, with LRP from RRIM600 displaying a stronger viscoelastic response as compared to LRP from PB235. The partial destructuring of the membrane and the formation of a denser interfacial film for RRIM600 promote long-range interactions. In the case of SRP, the clone has no significant impact on the rheological response at this scale.

This detailed description of the behavior of SRP and LRP from two biochemically contrasted clones in their hydrated state contribute to better apprehend their coagulation mechanism. It could help to cope

with the sustainability challenges of the concentrated latex industry linked to the heavy use of acid required to coagulate SRP [78–80].

## Abbreviations

AFM, Atomic Force Microscopy; BAM, Brewster Angle Microscopy; GL, Glycolipid; LRP, Large Rubber Particles; MMD, Molar Mass Distribution; NL, Neutral lipid; NTA, Nanoparticles Tacking Analysis; PI, Polydispersity index; PL, Phospholipid; REF, Rubber Elongation Factor; RP, Rubber Particles; SRP, Small Rubber Particles; SRPP, Small Rubber Particle Protein; TLC, Thin Layer Chromatography.

## CRediT authorship contribution statement

**Marion Baudoin:** Writing – original draft, Methodology, Investigation, Formal analysis, Data curation.
**Siriluck Liengprayoon:** Methodology, Formal analysis.
**Thongchai Khaeduang:** Project administration.
**Gilles Paboeuf:** Software, Methodology.
**Natedao Musigamart:** Project administration.
**Saowalak Jantarasunthorn:** Methodology, Formal analysis.
**Tucksin Lerksamran:** Methodology.
**Pisamai Chantuma:** Project administration.
**Christine Char:** Methodology.
**Maxime Geniez:** Methodology.
**Sylvie Beaufils:** Software, Methodology.
**Frédéric Bonfils:** Methodology, Investigation, Formal analysis.
**Laurent Vaysse:** Methodology, Investigation, Formal analysis.
**Véronique Vié:** Writing – review & editing, Writing – original draft, Supervision, Project administration, Funding acquisition, Formal analysis, Data curation, Conceptualization.
**Céline Bottier:** Writing – review & editing, Writing – original draft, Supervision, Project administration, Funding acquisition, Formal analysis, Data curation, Conceptualization.

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgments

We would like to thank Région Bretagne and CIRAD for funding the PhD grant of M. Baudoin, as well as Campus France, France (Ministères des Affaires Etrangères et de l'Enseignement Supérieur et de la Recherche) and OHEC, Thailand (Office of the Higher Education Commission, Thailand) for supporting the missions between France and Thailand (PHC SIAM, project N 49493QB). We are very grateful to F. Gauffre for the NTA measurements, to B. Lefeuvre for the zeta potential measurements and to Chachoengsao Rubber Research Center (CRRC) for kindly providing latex. Finally, the 2CBioMIF platform (ScanMAT-UMS 2001, Rennes, France) is acknowledged for allowing the biophysical characterization of the samples presented in this article. This work was undertaken as part of the Hevea Research Platform in Partnership (HRPP).

## Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.surfin.2025.106622.

## Data availability

Data will be made available on request.

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