Effects of the Presence of Suberin in the Cork of Cerasus jamasakura (Siebold ex Koidz.) H. Ohba on the High Toughness Behaviour

Abstract

Cork, the outermost tissue of bark, plays an important role in protecting trees from the surrounding environment and is used for various purposes, including flooring and insulation materials for buildings. This study focused on the amount and distribution of hydrophobic substances such as suberin and lignin in cork, as well as moisture conditions, to understand the mechanical properties of Cerasus jamasakura cork. Strips of cork were subjected to tensile tests after exposure to various moisture conditions (water-saturated, air-dried and oven-dried), and also after the desuberinisation and delignification of specimens. Cork with a high moisture content showed significant strain to the tensile load, whereas oven-dried specimens showed little toughness. The increased toughness of cork at higher moisture contents was due to the continued elongation in the plastic region, especially in the inner cork. The fibre length of the highly deformed cork differed significantly before and after the tensile test. Tensile tests of cork after desuberinisation and delignification indicated that the removal of suberin caused an earlier reduction in tensile properties than the removal of lignin. The presence of suberin in cork, distributed mainly in the inner cork, is believed to affect the tensile properties of cork.

1. Introduction

Wood, a natural organic polymer derived from the forest, can be a renewable resource through sustainable and appropriate forest management [1]. In addition, using wood, as opposed to other materials, in building construction provides a means for storing carbon, thus lowering the buildings’ carbon emissions. Timber production typically involves the generation of considerable amounts of offcuts, including bark, which accounts for 9–15% of the tree stem. Therefore, the use of bark has attracted attention from not only the timber industry but also researchers in recent years due to concerns over carbon dioxide emissions. However, little progress has been made with regard to industrial uses of bark [2] with the exception of the cork industry using bark from Quercus suber.

Cork, found in the outermost layer of the bark, is a unique material known for its low density, high elasticity and high impact resistance, and it has been used globally for a variety of applications, including bottle stoppers, musical materials, and flooring and insulation materials for buildings [3,4]. Bark has also been used in interesting ways in the field of Japanese traditional crafts. Figure 1 shows an example of the use of cork from Cerasus trees in Japan, in which the outer bark, containing cork, is used as a fastener for wooden lunchboxes, connecting thin boards; here, only the outer part of the cork, selectively peeled from the outer bark, is applied. The cork is stretched tangentially, which is followed by fixing to the overlapping portion of the curved wood board.

Recent studies focusing on the extensibility of cork in certain tree species, such as Cerasus, Prunus and Betula [5,6], have shown that cork elongation coincides with the length of cell tissues. Xu et al. [6] reported that longer cork cells resulted in a higher tensile strength and Young’s modulus but a lower strain at failure. Kobayashi et al. [7] suggested that the moisture conditions and the orientation of the crystalline cellulose in cork cell walls differed from those of xylem cell walls and affected the mechanical properties of cork in Cerasus sargentii var. sargentii. They also noted that the cork cell walls had a two-phase structure consisting of a cellulose microfibril region surrounded by lignin and a region containing only suberin. Kiyoto and Sugiyama [8] examined the factors contributing to the extensibility of cork cell walls based on microscopic observations of Betula platyphylla; their results showed that suberin deposition in the cell walls of cork contributed to the cork’s large deformation.

The anatomical structure and chemical component of bark is similar to, but not the same as, that of xylem tissue. Figure 2 shows a schematic diagram of the bark of Cerasus jamasakura that surrounds the xylem surface and the various layers. Cork forms the outermost layer of bark tissue, and it is pushed outward by the growth of newly formed cells. The main difference between the chemical components of bark and xylem is that bark has a high content of suberin, which consists of aliphatic and aromatic polymers [9]. More specifically, suberin is distributed in the outer cork cell walls, whereas the inner cork cell walls are lignified [7,8,10]. Suberin substantially influences the properties of cork [11]; however, few studies have provided a comprehensive evaluation of the mechanical properties, chemical composition, anatomical structure or moisture condition of cork. In addition, none have provided a fundamental answer to the question as to why the cork of some tree species, and not others, exhibits greater extensibility.

The purpose of this study was to understand the mechanical properties of cork, focusing on the amount and distribution of hydrophobic substances, such as suberin and lignin, in cork, as well as the moisture conditions. We examined the effects of both suberin and lignin on the tensile properties of cork, based on the application of selective and stepwise damage, in contrast to earlier studies that mainly considered the changes in the hydrophilic polysaccharides of cork.

2. Materials and Methods

2.1. Materials

The bark specimens used were taken from two branches of a wild standing tree of Cerasus jamasakura (Siebold ex Koidz.) H. Ohba in Mie Prefecture, Japan. The diameters of the two branches were in the range of 4–8 cm and 12–14 cm. C. jamasakura is a representative species of the tall cherry trees that grow wild throughout Japan; however, little is known about the mechanical properties of its cork. Figure 3 shows the process flow of the study. This study was broadly divided into two tests based on chemical composition analysis and mechanical testing of the tangential tension of cork specimens.

2.2. Chemical Composition Analysis of Cork

The process flow of chemical composition analysis of cork is shown in Figure 3. The part of cork stripped from the bark of tested specimen was mechanically crushed using a blender (WB-1, Osaka Chemical Co., Ltd., Osaka, Japan) to obtain a powder with a particle size of 250–355 µm. The ratio of each component was evaluated using the cork powder based on the weight loss during a series of removal processes [12]. First, a series of Soxhlet extractions were performed using each solution of dichloromethane, ethanol, and distilled water for 8, 10, and 24 h, respectively. After each extraction, solutions used were found to be clear and colourless. The cork powder that has undergone each extraction was air-dried in a draft chamber and then dried in an electric oven at 105 °C for 24 h to determine the weight at oven-dried condition after extractions. Next, the extracted powder was subjected to desuberinisation through a depolymerisation reaction (i.e., methanolysis) by refluxing with a 3% (v/v) methanolic solution of sodium methoxide for 3 h. The treated powder was collected via filtration and then refluxed with methanol for 15 min. The resulting solution was neutralised with an aqueous sulphuric acid solution.

The resulting solution and the residue powder were used to determine the suberin and lignin contents, respectively. The former was dried at room temperature to obtain the residue, which was dissolved in distilled water. Chloroform was then added, which was followed by drying with anhydrous sodium sulphate. After the chloroform evaporated, the residue was weighed to determine the suberin content. The latter was hydrolysed by immersing in a 72% (v/v) sulphuric acid aqueous solution for 4 h and then refluxed with 3% (v/v) dilute sulphuric acid for 4 h. After drying in an electric oven at 105 °C for 24 h, the residue powder was weighed to determine the lignin amount.

2.3. Preparation for Tensile Specimen

Figure 3 shows the process flow of the specimen preparation for the tensile tests. The thickness of the cork peeled from the outer bark was shaved with a knife to 1 mm. Then, half of the cork strips were further peeled into two pieces to prepare three types of specimens: whole cork, outer cork and inner cork. The strip specimens had dimensions of 7 mm × 1 mm × 30 mm (longitudinal × radial × tangential) and a thickness of 0.5 mm.

The prepared specimens were saturated in distilled water. Tensile tests were then performed under three moisture conditions after moisture conditioning: water-saturated, air-dried and oven-dried. Water-saturated specimens were used for the tensile test immediately after removal from the water. Air-dried specimens were conditioned in the laboratory at 20 °C until reaching a constant weight, and oven-dried specimens were dried in an electric oven at 105 °C for 24 h. The oven-dried specimens were stored in a sealed desiccator with phosphorus (V) oxide and cooled to room temperature. A constriction of 1 mm in width and 4 mm in length was applied to each specimen so that fracture would occur in the middle of the specimen during the test (Figure 3).

We also prepared samples from the xylem of C. jamasakura. The cell walls in xylem are arranged longitudinally. Thus, longitudinal specimens with dimensions of 7 mm × 1 mm × 30 mm (tangential × radial × longitudinal) were prepared and subjected to the same three moisture conditions as described for the cork specimens.

We also tested the tensile properties of cork after desuberinisation and delignification to examine the effects of suberin and lignin; the experimental process is shown in Figure 3. In this procedure, we used only the inner cork specimen, because most of the outer cork had lost its shape. The inner cork specimen was subjected to the same series of extractions as described above. Then, one quarter of the specimens were used for the tensile test. The remaining specimens were subjected to each chemical treatment: extraction, desuberinisation and delignification. The tensile test was performed on four types of treated specimens: untreated specimens; extraction-treated specimens; desuberinised specimens; and delignified specimens. Because we previously found a significant reduction in the mechanical properties after extraction [13], the tensile properties of the specimens were compared with the extraction-treated specimens. After the removal of each chemical component, all specimens were soaked in distilled water, and the tensile test was performed under water-saturated conditions.

The desuberinised and delignified specimens were prepared with different treatment times to understand the effect of the degree of chemical removal. The extraction-treated specimens were subjected to desuberinisation through a depolymerisation reaction (i.e., methanolysis) by refluxing with a 3% (v/v) methanolic solution of sodium methoxide for up to 6 h [12]. Specimens were removed from the solution after 1, 10, 30, 90 and 180 min of refluxing, resulting in a weight loss of 9.3%, 10.8%, 13.8%, 19.1% and 32.7%, respectively. The extraction-treated specimens were subjected to delignification via immersion in a 4% (v/v) sodium chlorite solution for up to 6 h [14]; specimens were removed from the solution after 0.5, 6, 24 and 48 h of immersion, resulting in a weight loss of 3.6%, 9.4%, 11.7% and 22.4%, respectively.

2.4. Tensile Test

Tensile tests of each type of cork specimen were performed under the following conditions in accordance with the test methods for wood [15], following the guidelines specified by ISO 13061-6:2014 [16]. A universal material testing machine (SV-201NA, Imada Seisakusho Co., Toyohashi, Japan) was used. Specimens were first placed on the load cell (TCLZ-2KNA, Tokyo Measuring Instruments Laboratory Co., Ltd., Tokyo, Japan) of the machine through a jig. The distance between both edges of the grips after installation on the machine was 14 mm. The temporal changes in load and displacement were recorded using a data logger (PCD-320 A, Kyowa Electronic Instruments Co., Ltd., Tokyo, Japan). A displacement rate of 0.1 mm/min was applied to most specimens; comparisons were made to understand the effects of loading speed: 0.1, 1, 50, and 100 mm/min. Data were collected at 10 Hz. The Young’s modulus, tensile strength, and fracture strain were determined based on the data [15]. In addition, the toughness was obtained to numerically express the high ductility of cork. The amount of mechanical work was evaluated based on integrated values of the stress–strain curve ranging from the starting point to fracture [17].

2.5. Measurement of Fibre Length

The fibre length of untreated inner cork was measured after the tensile test. After the tensile test, specimens were subdivided into the part stretched by the tensile load and the part gripped without the tensile load. The two parts were immersed in a mixed solution of sodium chlorite and acetic acid at 80 °C for 1 h to obtain single fibres, following the guidelines specified by IAWA [18]. Subsequently, the solution containing single fibres was diluted with distilled water in stages. A few drops of the solution mounted on a glass slide were covered with a cover glass. Sets were prepared until more than 100 single fibres were collected for each condition (oven-dried, air-dried and water-saturated). Digital images were then taken of each set using an optical microscope (BX41, Olympus Corp., Tokyo, Japan) equipped with a charge-coupled device camera (FX630, Olympus Corp.) to examine the effects of tensile loading on the fibre length. The fibres were measured using image processing software (ImageJ, version 1.52a, National Institutes of Health, Bethesda, MD, USA) of the obtained images.

3. Results and Discussion

3.1. Chemical Composition Ratio of Cork

Table 1. Chemical composition of cork from a branch of Cerasus jamasakura. The sum of values in this table is 67.5%; the remainder is considered to be polysaccharides.

Extractive Obtained by Each Solvent			Suberin	Lignin
Dichloromethane	Ethanol	Water
5.8	5.5	0.9	32.3	23.0

shows the proportions of the chemical components of cork of C. jamasakura. The hydrophobic components, lignin and suberin, accounted for more than half of the total. The lignin content was 20–30%, which was similar to the content in wood in a previous study [19]. The large suberin content and low polysaccharide content were consistent with the characteristics of cork [19].

3.2. Tensile Properties of Cork

Figure 4 shows representative results of the tensile tests. The shape of the stress–strain curves was impacted by the cork moisture condition. Whole cork specimens under water-saturated conditions showed significant strain, whereas the oven-dried samples showed little toughness without deformation after the proportional limit.

Table 2. Tensile test results of whole cork and xylem treated with different moisture conditions. The values in parentheses indicate the standard deviation.

Specimen	Moisture Condition	Moisture Content (%)	Density (g/cm3)	Young’s Modulus (GPa)	Strength (MPa)	Fracture Strain	Toughness (MJ/m3)
Whole cork	Oven-dried	0.0 (0.0)	0.98 (0.08)	3.0 (0.9)	36.9 (11.3)	0.03 (0.01)	0.5 (0.2)
	Air-dried	8.7 (1.6)	1.08 (0.12)	2.7 (0.9)	39.4 (15.0)	0.05 (0.03)	1.2 (0.9)
	Water-saturated	60.0 (10.5)	1.21 (0.17)	0.5 (0.2)	16.8 (3.2)	0.76 (0.46)	7.7 (5.6)
Xylem	Oven-dried	0.0 (0.0)	0.47 (0.05)	9.7 (2.1)	96.9 (24.3)	0.02 (0.00)	1.0 (0.3)
	Air-dried	11.2 (2.5)	0.54 (0.04)	11.0 (3.0)	111.1 (36.8)	0.02 (0.01)	1.7 (1.3)
	Water-saturated	166.8 (26.2)	1.25 (0.15)	3.9 (0.9)	67.8 (18.7)	0.03 (0.01)	1.2 (0.6)

summarises the tensile properties of whole cork and xylem under each moisture condition. Compared to xylem, whole cork had lower Young’s modulus, tensile strength and fracture strain values under all moisture conditions, despite its higher density. This may be due to the low content of polysaccharides such as cellulose that provide mechanical support (Table 1). Notably, the maximum toughness was found in water-saturated cork. The large strain of cork was attributed to this high toughness. Compared to cork, the tensile properties of xylem were less affected by the moisture condition.

The air-dried and water-saturated specimens revealed two apparent regions (Figure 4). That is, cork with a moisture content above a certain level maintained elongation in the plastic region even after a decrease in stress; this was particularly evident in the water-saturated specimens. We visually confirmed that the stress drop was due to the fracture of the outer cork while the inner cork continued to elongate.

Table 3. Comparison of the tensile properties of inner and outer cork in the water-saturated condition. The values in parentheses indicate the standard deviation.

Specimen	Moisture Content (%)	Density (g/cm3)	Young’s Modulus (GPa)	Strength (MPa)	Fracture Strain	Toughness (MJ/m3)
Inner cork	79.1 (14.9)	1.47 (0.19)	0.5 (0.2)	14.8 (3.7)	0.50 (0.19)	5.1 (2.8)
Outer cork	62.8 (11.4)	1.20 (0.15)	0.6 (0.2)	16.9 (4.7)	0.31 (0.17)	3.4 (1.9)

shows the tensile properties of the two regions within the cork (inner and outer). The inner cork exhibited higher fracture strain and toughness than the outer cork. Therefore, the high toughness was attributed to the inner cork, which became more pronounced at a higher moisture content. The fracture strain differences observed in the current study between the inner and outer cork agreed with previous findings [6].

Figure 5 shows the relationship between the moisture content and tensile properties of whole cork specimens. The Young’s modulus of C. jamasakura cork increased with decreasing moisture content (Figure 5a). This relationship agreed with the xylem content below the fibre saturation point [20]. The results also suggested that the fibre saturation point of cork was in the same range as the moisture content of xylem. The strain at maximum stress increased when the moisture content exceeded approximately 30% (Figure 5b). This suggests that moisture is essential for the extended elongation of cork.

Previous studies have similarly investigated the tensile properties of cork combined with the effects of moisture conditions for various tree species [5,6,7,8]; the findings are summarised in

Table 4. Comparison of the tensile properties of cork from different species measured in this and previous studies.

	This Study			Xu et al. (1997) [5]	Xu et al. (1998) [6]			Kobayashi et al. (2018) [7]		Kiyoto and Sugiyama (2022) [8]
Tree species	Cerasus jamazakura			Prunus serrula	Prunus serrula			Cerasus sargentii		Betula platyphylla
Collection site of cork specimen	Branch			—	—			—		—
Tree age (years)	32			—	—			—		—
Division in the radial direction	Undivided			—	outer	middle	inner	—		—
Moisture condition	Oven- dried	Air- dried	Water- saturated	—	—			Oven- dried	Air- dried	Oven- dried	Air- dried	Water-saturated
Specimen size (L × R × T mm)	7 × 1 × 30			5 × thin × 25	5 × thin × 25			5 × 0.2 × 30		5 × 0.2 × 30
Shape of specimen				—	—
Fibre length (μm)	191.8 (48.1)	207.5 (39.9)	224.8 (66.5)	—	187	116	—	—	—	—	—	—
Density (g/cm3)	0.98 (0.08)	1.08 (0.12)	1.21 (0.17)	1.24	1.11	1.24	1.31	—	1.12	—	—	—
Loading rate (mm/min)	0.1	0.1	0.1	12.5	12.5	12.5	12.5	10	10	10	10	10
Young’s modulus (GPa)	3.0 (0.9)	2.7 (0.9)	0.5 (0.2)	0.6	0.72	0.52	0.33	1.5	1.4	0.4	0.4	0.2
Strength (MPa)	36.9 (11.3)	39.4 (15.0)	16.8 (3.2)	98.4	56	37	26	43.7	42.7	18.3	20.1	15.9
Fracture strain	0.03 (0.01)	0.05 (0.03)	0.76 (0.46)	1.2	0.35	0.66	1.02	0.09	1.22	0.72	1.37	1.76
Toughness (MJ/m3)	0.5 (0.2)	1.2 (0.9)	7.7 (5.6)	62.3	—	—	—	3.4	44.1	11.0	21.7	19.5

. A universal law encompassing the entirety of cork’s tensile properties was not apparent; thus, the test conditions and species may affect the properties. To explore this possibility, we performed additional tensile tests under different loading speeds; the results are shown in Figure 6. Young’s modulus and the fracture strain of water-saturated cork could differ by a factor of ~2 with changes in loading speed (Figure 6b,d). However, air-dried cork exhibited minimal differences with the exception of the fracture strain at the two slower loading speeds (Figure 6a,c).

3.3. Differences in the Suberin Composition between the Inner and Outer Cork Layers

We further compared the tensile properties of cork peeled into inner and outer layers using Raman microspectroscopy. Cork sections (thickness: 3 µm) prepared using a sliding microtome were mounted on a confocal Raman microscope (LabRAM ARAMIS, Horiba Jobin Yvon, Longjumeau, France). The specimen was irradiated with a 532 nm excitation laser (Ventus VIS 532, Laser Quantum, Cheshire, UK). The output laser was brought into focus with an oil immersion objective lens (UPLSAPO 100×, 1.4 NA, Olympus Corp.), and the scattered Raman light from suberin was detected at several points in the radial direction. A previous study [21] noted that the changes in the intensity peaks at 1634 and 1597 cm⁻¹ may correspond to changes in the chemical composition of suberin. Thus, the intensity ratio of the Raman spectra at 1634 cm⁻¹ and 1597 cm⁻¹ was calculated for each irradiated spot in the cork specimen.

Figure 7 shows the Raman microspectroscopy results. The intensity ratio derived from changes in the chemical composition of suberin decreased from the inner to the outer cork layers, suggesting differences in the characteristics of suberin in these two regions. These differences likely contributed to the difference in toughness observed in the tensile tests under water-saturated conditions.

3.4. Changes in Fibre Length of Cork Due to Tensile Loading

Figure 8 shows a comparison of single fibre lengths of inner cork under the three moisture conditions with and without tensile loading. Only the water-saturated specimens exhibited a significant increase in the fibre length after loading (p < 0.01). Based on our previous study, suberin is found mainly in the cork fibres, whereas lignin is found in both the cork fibres and compound middle lamella that connects the fibres [22]. The substantial dimensional increase observed in the cork fibres with a high moisture content was likely due to the presence of suberin, which is found only in cork and not in xylem. Further verification, such as microscopic observation of the fracture site, is required to determine where the large strain, shown in Figure 4, occurs within the cork fibres.

3.5. Effects of Suberin and Lignin Removal on the Tensile Properties of Cork

Figure 9 shows the tensile test results of whole cork specimens after suberin and lignin removal; the plots show the tensile properties relative to those of the control after extraction. Changes in the tensile properties of cork due to suberin removal always preceded those due to lignin removal with the exception of the fracture strain with large variations. These tensile test results coincide with those of our previous study [22], showing that suberin in the cell walls plays the role of maintaining the arrangement of the cell wall structure. These findings indicate that suberin removal disrupts the cell wall structure, damaging the tensile properties earlier in the removal treatment. For both suberin and lignin, the tensile properties were nearly lost when approximately half of each component was removed. In a previous study, the shape of cork cells was generally preserved with delignification, whereas it was not maintained with desuberinisation [22]. In this study, the contribution of suberin to the tensile properties of cork exceeded that of lignin. This suggests that suberin has a major effect on the tensile properties of cork. Overall, C. jamasakura cork exhibited high toughness only with a high moisture content, indicating that the effect of suberin on tensile properties is greater than that of lignin.

4. Conclusions

This study investigated the tensile properties of C. jamasakura cork, focusing on the content and distribution of hydrophobic substances and moisture content. The following findings were drawn based on the results.

• Young’s modulus and tensile strength were lower for cork than xylem under all moisture conditions despite the higher density of cork. This may be due to the low content of polysaccharides such as cellulose that provide mechanical support.
• Only cork with a high moisture content showed significant strain to the tensile load; the oven-dried specimens showed low toughness. Xylem exhibited minimal differences in elongation under the different moisture conditions.
• Whole cork prepared under high moisture conditions continued to elongate in the plastic region after a drop in stress, because the inner cork exhibited particularly high toughness. In addition, a significant increase in fibre length after the tensile test was found only in cork prepared under high moisture conditions.
• Suberin removal from cork caused an earlier reduction in tensile properties than did lignin removal.

In the future, suberin might contribute to the use of cellulosic materials by enhancing their flexibility. Moreover, the manipulation of the mechanical properties of cork through controlling the suberin content could assist in creating a substitute for animal hide products.