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Analysis of Free Sugar and Starch in Oil Palm Trunks ( Elaeis Guineensis Jacq. )
from Various Cultivars as a Feedstock for Bioethanol Production

Article  in  International Journal of Green Energy · February 2015

DOI: 10.1080/15435075.2014.910786

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Analysis of Free Sugar and Starch in Oil Palm Trunks

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(Elaeis Guineensis Jacq.) from Various Cultivars as a
Feedstock for Bioethanol Production
a b a b
Zubaidah Aimi Abdul Hamid , Takamitsu Arai , Mhd Ramle Sitti Fatimah , Akihiko Kosugi ,
a a b c
Othman Sulaiman , Rokiah Hashim , Satoru Nirasawa , Tanaka Ryohei , Bhadravathi Eswara
d d b b b
Lokesh , Kumar Sudesh , Yoshinori Murata , Masayoshi Saito & Yutaka Mori
a
Division of Bioresource, Paper and Coatings Technology, School of Industrial Technology,
Universiti Sains Malaysia, 11800, Penang, Malaysia
b
Japan International Research Center for Agricultural Sciences (JIRCAS), 1-1 Ohwashi,
Tsukuba, Ibaraki 305-8686, Japan
c
Forestry and Forest Products Research Institute (FFPRI), 1 Matsunosato, Tsukuba, Ibaraki,
305-8687 Japan
d
Ecobiomaterial Research Laboratory, School of Biological Sciences, Universiti Sains
Malaysia, 11800, Penang, Malaysia
Accepted author version posted online: 18 Feb 2015.

To cite this article: Zubaidah Aimi Abdul Hamid, Takamitsu Arai, Mhd Ramle Sitti Fatimah, Akihiko Kosugi, Othman Sulaiman,
Rokiah Hashim, Satoru Nirasawa, Tanaka Ryohei, Bhadravathi Eswara Lokesh, Kumar Sudesh, Yoshinori Murata, Masayoshi Saito
& Yutaka Mori (2015): Analysis of Free Sugar and Starch in Oil Palm Trunks (Elaeis Guineensis Jacq.) from Various Cultivars as a
Feedstock for Bioethanol Production, International Journal of Green Energy, DOI: 10.1080/15435075.2014.910786

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 ACCEPTED MANUSCRIPT

1 ANALYSIS OF FREE SUGAR AND STARCH IN OIL

2 PALM TRUNKS (Elaeis Guineensis Jacq.) FROM
3 VARIOUS CULTIVARS AS A FEEDSTOCK FOR
4 BIOETHANOL PRODUCTION
5 Zubaidah Aimi Abdul Hamid1, Takamitsu Arai2, Mhd Ramle Sitti Fatimah1, Akihiko Kosugi2,
6 Othman Sulaiman1*, Rokiah Hashim1, Satoru Nirasawa2, Tanaka Ryohei3, Bhadravathi Eswara
7 Lokesh4, Kumar Sudesh4, Yoshinori Murata2, Masayoshi Saito2, Yutaka Mori2
1
8 Division of Bioresource, Paper and Coatings Technology, School of Industrial Technology,
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9 Universiti Sains Malaysia, 11800, Penang, Malaysia

2
10 Japan International Research Center for Agricultural Sciences (JIRCAS), 1-1 Ohwashi,
11 Tsukuba, Ibaraki 305-8686, Japan
3
12 Forestry and Forest Products Research Institute (FFPRI), 1 Matsunosato, Tsukuba, Ibaraki,
13 305-8687 Japan
4
14 Ecobiomaterial Research Laboratory, School of Biological Sciences, Universiti Sains Malaysia,
15 11800, Penang, Malaysia

16 Address correspondence to Othman Sulaiman, Division of Bioresource, Paper and Coatings

17 Technology, School of Industrial Technology, Universiti Sains Malaysia, 11800, Penang,
18 Malaysia. E-mail: othman@usm.my.

19 ABSTRACT

20 In this study, oil palm trunks from various plantations in Malaysia were analyzed for their starch

21 content and sugar concentrations in the sap from the trunks as function of storage time as a

22 feedstock for green energy. Glycoside hydrolases of the samples were also measured during

23 storage. Considering three kinds of sugars accumulation in the trunks, the highest increase was

24 from 31 mg/ml to 198 mg/ml after 60 days of storage followed by the second highest from

25 48~78 mg/ml to 109~133 mg/ml after 15~45 days of storage. Accumulation of the third stages

26 was not observed in most types of trunks. The parenchyma in each trunk had different starch

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27 contents, which ranged from 0.33 to 37 wt%. Trunks with higher starch content in the

28 parenchyma cells showed a tendency towards increased sugar concentration in the sap,

29 suggesting that the breakdown of starch within the sap is related to sugar accumulation. Amylase

30 activity in the trunks increased from 0.21 mU/g to 1.54 mU/g during storage. Invertase showed

31 relatively high activity (6.48 mU/g) at around 15 days storage time. Based on the findings in this

32 work, it seems that enzymes played an important role in the breakdown of starch and sucrose

33 during storage of oil palm trunks.

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34 Keywords: Palms; Sugar; Starch; Enzyme activity; Storage

35 INTRODUCTION

36 Oil palm (Elaeis guineensis Jacq.) is widely planted for its edible oil in tropical countries

37 such as Malaysia and Indonesia. The oil palm is perennial plant originated from East Africa that

38 was introduced to Indonesia and Malaysia in the early 1900’s (Mohan Jain 2009). Early studies

39 revealed the presence of three main parents from species Elaeis guineensis, namely Dura,

40 Pisfera, and Tenera (Hartley 1988). The classification of these cultivars is related to the anatomy

41 of the palm fruit. New cultivars such as Dumpy, Yangambi, AVROS, Chemara and H&C

42 produced from the hybrids of these main parents, and research on crossing among selected

43 cultivars have been very promising especially in terms of yield and quality of fruit (Owolarafe et

44 al. 2007).

45 Palm oil production has been rapidly increasing last several decades. In 2006, it overtook

46 soybean oil as the number one plant based oil. The number one producer of palm oil is

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47 Indonesia, and Indonesia and Malaysia together account for about 87% percent of the world’s oil

48 palm supply (USDA www.fas.usda.gov/psdonline/psd

49 Download). The most significant attribute of oil palm trees is its high productivity.

50 Approximately 3.4 to 4.9 tons of palm oil is produced per hectare per year. This is 10 times more

51 than the amount of soybean oil production at the same scale. However, the oil productivity of

52 palm generally diminishes after about 25 years old of age. At that point, they are cut down and
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53 left in fields. In some cases, the trees are withered by injecting chemicals into their roots which

54 can potentially cause environmental pollution.

55 In Malaysia, about twenty to thirty million tons of old non-productive oil palm trees are

56 harvested annually. Presently, they are under-utilized and there is no effective use for these old

57 trees. The outer layers of some of them are remove and used to produce plywood, but 99% of

58 this resource is burned or land filled. Compared with other types of timber, the oil palm tree has

59 very low mechanical properties therefore they are not suitable for lumber manufacture. New

60 attention has been given to a production of bioethanol from oil palm biomass looks promising to

61 use this agricultural crop, due to its availability, lower cost and potential as a source of this

62 bioenergy (Abu-Khader and Speight 2004, Jung et al. 2011, Kosugi et al. 2010, Moon et al.

63 2010, Murai and Kondo 2011, Yamada et al. 2010). Based on the findings from previous studies,

64 it was determined that the felled oil palm trunk contains large quantity of sap, which accounts for

65 approximately 70% of the whole trunk weight (MPOB www.mpob.gov.my). It was also clearly

66 shown that ethanol and lactic acid were efficiently producible from oil palm sap using yeast and

67 lactic acid bacteria. It was found that the concentration of fermentable sugars in the trunk can be

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68 drastically increased within a suitable storage period (Kosugi et al. 2010, Yamada et al. 2010).

69 Although different aspects of oil palm have been studied, there is little or no information on

70 sugar and starch accumulation in the felled oil palm trunks from different cultivars. Therefore,

71 the objective of this work to evaluate the accumulation of sugar and starch in the felled oil palm

72 trunks from major oil palm cultivars planted in Malaysia as a function of storage time. Also the

73 reasons for the variation in sugar accumulation among different cultivars based on their starch

74 contents and enzyme activities with a different duration of storage and cultivars were
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75 investigated within the scope of this study.

76 MATERIALS AND METHODS

77 Materials and sample preparation

78 The oil palm trunks with an average approximate age of 25 years old were obtained from

79 several plantations in Malaysia. The experiments were performed on the five chosen cultivars of

80 oil palm trunk (Elaeis guineensis Jacq.), namely Dura × Pisifera mix (Dura x URT), Deli Dura ×

81 AVROS, Deli Dura × Pisifera × H&C, Deli Dura × Yangambi (URTA or URTC) and Deli Dura

82 × Yangambi.

83 Logs with 2.5 m length were taken from the middle part of the whole logs as described

84 previously (Yamada et al. 2010). They were felled, and stored under a roof at a temperature of 28

85 - 32°C and at a relative humidity around 70 - 80%. To avoid any possible microbial

86 contamination, 5 cm sections from the ends of each trunk were trimmed before the slicing. A

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87 disc with 10 cm thickness was sliced after being stored periodically, namely 0, 1, 3, 7, 15, 30, 45

88 and 60 days. The sliced samples were then divided into two equal parts which is one part being

89 compressed to obtain sap for analysis sugar and enzyme analysis while another part were used

90 for quantitative analysis of starch. The sap was extracted by cold pressing at a pressure of 0.70

91 MPa and the collected sap was placed in freezer at a temperature of -20°C to maintain the

92 freshness of the samples for further analysis. For starch analysis, separated parenchyma samples

93 from different cultivars of oil palm trunk were prepared. Samples were first dried at 80°C before
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94 they were separated manually into parenchyma and vascular bundles. Samples were ground into

95 fine powder and pass through particle size 100 µ/m screen. Starch content in this fine powder

96 was determined.

97 Analysis of sugar and starch in the samples

98 Sugars contained in the sap were determined by using high performance liquid

99 chromatography (HPLC) equipped with a CarboPac PA (Dinoex Corporation, Sunnyvale, CA,

100 USA) with pulsed amperometric detection (HPAEC-PAD). The mobile phase was double

101 distilled water at a flow rate of 0.6 ml min at 80 °C. The carbohydrate kits, CAR 10-1KT from

102 Sigma Aldrich were used to make standard for calibration curve. Starch content of each sample

103 was determined according to Megazyme total starch assay procedure (AOAC method 996.11).

104 The amount of starch in each sample was measured by calibrating the absorption at a wave

105 length of 510 nm employing UV-VIS spectrophotometer (Megazyme International, Co.

106 Wicklow, Ireland).

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107 Enzyme extraction process and derivatives

108 The frozen trunk particles were reduced in smaller size in a grinder mill (Hamilton Beach)

109 and compressed by a press (Sanseki). Water was also added to the residual material before they

110 were and repressed. The extract was centrifuged at 7,000 rpm for 30 min to remove insoluble

111 material. The resulting supernatant was precipitated with 80% saturated ammonium sulfate and

112 then dissolved in 50 mM sodium phosphate buffer (pH 6.5). In the next step and excess salt was
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113 removed with Econo-Pac 10 DG desalting column (Bio-Rad) and the solutions were used for

114 enzyme activity.

115 Each enzyme activity was measured by determining the amount of reducing sugar released

116 from soluble starch, laminarin, carboxymethylcellulose (CMC), oat-spelt xylan and sucrose. The

117 reaction mixture consists of 0.1 ml of 1% each substrate in 50 mM sodium phosphate buffer (pH

118 6.5) and 0.1 ml enzyme. After incubation for 10 min at 40 °C, reducing sugar was determined by

119 using the Somogyi-Nelson method (Nelson 1944). One unit of each enzyme activity was defined

120 as the amount of enzyme removed in 1 μmol of reducing sugar in 1 min under the above

121 conditions. Glucose kit manufactured by Wako Junyaku Inc. was used to measure invertase

122 activity of the samples.

123 Microstructure analysis of the samples

124 Field emission scanning electron microscope (FE-SEM) was employed to determine the

125 morphological structure of starch in the trunk at the harvesting time and after 60 days of storage

126 period. Following the felling and after 60 days of the storage time, micrographs were taken from

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127 cross sections of 0.5 cm by 0.5 cm specimens. The samples were mounted on the base and coated

128 with gold by an ion sputter coater (Polaron SC515, Fisons Instruments, UK). A Field Scanning

129 Electron Microscope LEO Supra 50 Vp, Carl-Zeiss SMT, Oberkochen, Germany was used for

130 analysis of microscopical samples.

131 RESULTS AND DISCUSSION

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132 The sugar content within the trunks during storage

133 It has been previously reported that appropriate storage of the trunks of oil palms, the sugar

134 content within the sap increases substantially (Kosugi et al. 2010) . In this study, it was

135 considered whether there was a difference in the sugar content value within the sap depending on

136 the cultivar. Two trunks of each typical cultivar grown in Malaysia were collected (Figure 1) and

137 labeled as the two logs I and II (Table 1). The collected trunks were stored in the same

138 conditions mentioned previously and the sugar concentration within the sap was measured during

139 the storage period, as shown in Figure 2.

140 It was assumed that the same cultivar of trunks harvested in the same area would have a

141 similar tendency in sugar accumulation. Sample A-I showed an increase in sugar accumulation

142 between the first day of storage and 60 days, from 52.2 to 137.9 mg/ml and from 31.1 to 198.7

143 mg/ml for cultivar A-II, respectively. On the other hand, no accumulation was observed for

144 either trunk of cultivar B. An accumulation in sugar content within the sap for C was recorded,

145 and the sugar content of cultivar C-I started from 44.1 to 77.2 mg/ml on the initial day of storage

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146 and stayed at a similar level next 45 days before it decreased. The cultivar C-II showed the same

147 trend, except the sugar level declined sharply with a value ranging from 63.5 to 85.2 mg/ml after

148 30 days storage. The sugar level in D-II was the lowest among the other cultivars and did not

149 show day accumulation after logging. However, cultivar D-I showed some sugar accumulation in

150 the sap through 30 days and stayed constant for the rest of the storage period, with values

151 ranging from 76 to 109.9 mg/ml. Sample E showed an increase in sugar accumulation, with a

152 mean concentration of 88.6 to 115.74 mg/ml from the first storage day and reaching a maximum
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153 level at 15 days before significantly declining the next day.

154 The sap samples revealed that sugar typically accumulates within the trunk after a certain

155 storage period. The predominating constituent sugars within palm trunks were glucose, sucrose,

156 and fructose (Kosugi et al. 2010). Each trunk had the possibility of having a high or low sugar

157 concentration dependent on the cultivar, suggesting that in these experiments at least, cultivar is

158 not a factor influencing the sugar content in the sap of oil palm trunks. One of the factors that

159 cause a lower concentration of sugar in sap is its depletion. This happens when a tree needs

160 energy for flowering and early fruit development. To support these processes, starch is degraded

161 in the form of a liquid known as sap sugar, which is transported along the trunk. Heavy fruiting

162 will cause a reduction of sugar level. If palm trees were cut during anthesis or after fruit ripening,

163 it would automatically affect the starch and sugar level in the tree trunk (Latiff 2000, Pallardy

164 2010). It was assumed that some of the cultivars were sampled after fruiting, such as cultivars B

165 and E, which showed a lower sugar content even after storage. The sugar concentration of A-I,

166 A-II, C-II, D-I, and E increased as the storage period was extended. Thus, metabolic energy

167 played an important role to sustain the life cycle of living cells in the trunks and indirectly

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168 produce more concentrated sugar content. Degradation of starch also takes place in this process

169 by releasing sucrose from the starch chain and metabolizing it to monosaccharide of glucose and

170 fructose, which cost slight increase in free sugars (Kilonzo et al. 2007, Ting 1982).

171 Changes in the starch content within the parenchyma immediately

172 after felling and starch content within the parenchyma during the storage

period
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173

174 As a monocotyledonous plant, oil palm trunks contain a numerous amount of ground

175 parenchyma embedded with vascular bundles and fibrous strands. Thus it provides a mechanical

176 support and functional as a storage organ for the whole tree. Oil palm trunk also contains a

177 substantial amount of carbohydrate which is stored in the parenchyma cell. Starch is the most

178 important polysaccharide and a major carbohydrate reserve in oil palm tree. Starch is stored

179 generously within the trunk in the parenchyma cells (Husin 2000, Janick and Paull 2008,

180 Killmann and Lim 1987, Murai and Kondo 2011).

181 It seems that the starch accumulates when high sugar content existed. Thus, a comparative

182 study was carried out on the starch content within the trunk parenchyma of each cultivar. Figure

183 3A illustrates the trunk with the most starch content was coming from cultivar A-II. This trunk

184 had a great amount of sugar accumulation during the storage period. In B-I, B-II, and D-II, a

185 little starch was found within the parenchyma, but there was no sugar accumulation within the

186 trunk. The starch content of the parenchyma based on storage time is as shown in Figure 3B. In

187 the trunks where sugar accumulated, the starch content began decreasing from several days from

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188 initial condition, and after 60 days storage, the starch was virtually non-existent. As illustrated in

189 Figure 3B, the starch content decreased with the duration of storage, which immediately caused

190 an increase in sugar content in the trunk.

191 As mentioned previously, there was a relationship between the starch content and sugar

192 accumulation (Yamada et al. 2010). Trunks with higher starch content within the parenchyma are

193 found to be associated with higher sugar concentration within the sap after being stored. This
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194 condition took place when the sugar content are low after being used to support the living cells in

195 the trunk after felling. Thus, stored starch was converted to sugars in order to maintain the

196 survival of these cells. At this time sugar concentration increased especially as in the case of

197 sucrose. Therefore, at the beginning of the storage time high sucrose level was detected.

198 However, without any transportation of food throughout the trunks, life cycle of these living

199 tissues cannot stand long due to the limited amount of stored starch in the felling trunks. After

200 stored starch has been turned into sugars and being used to extend the life span of this tissue, the

201 sugar content began to decline and this process took place after several days of storage time

202 (Ezeagu et al. 2003, Pallardy 2010, Ting 1982).

203 The micrographs taken from the specimens illustrate the starch in the trunk following the

204 harvesting and after 60 days of the storage (Figure 5). Immediately after logging, an abundance

205 of starch granules can be seen. However after 60 days of storage, comparatively fewer starch

206 granules were observed. The starch accumulation within the trunk of palm tree such as sago has

207 been reported to be different according to the cultivation period (Pei-Lang et al. 2006).

208 Therefore, it appears that a similar phenomenon may seen in the trunks of oil palms. As a

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209 monocotyledonous plant, oil palm trunk contain a numerous amount of ground parenchyma

210 embedded with vascular bundle and fibrous strands. It provides a mechanical support and

211 functional as a storage organ for the whole tree. Oil palm trunk also contains an enormous

212 amount of carbohydrate which is certainly stored in the parenchyma cell. Starch is the most

213 important polysaccharide and a major carbohydrate reserve in oil plant tree (Hartley 1988, Latiff

214 2000). It was synthesized in plant tissue and exists in granule form with different shape and

215 densities. Starch are stored in the trunks and when the tree needs energy for growing process it
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216 will be hydrolyzed into simple sugars in the sap form before being transported to every part of

217 palm tree.

218 Enzyme activity within the trunk

219 The breakdown of starch is due to the amylolytic enzymes which exist within the trunk.

220 Thus, protein was extracted from the stored trunks in order to measure glycoside hydrolase

221 activity. The α-Amylase, β-1, 3-glucanase, invertase, cellulase and xylanase activity were found

222 existence within the trunk as presented in Figure 4. The activity increased drastically from the

223 first day after storage, and higher enzyme activity was observed even after 60 days. Amylase

224 activities consist of α- and β- amylase and it was indicated that amylase activity plays a role in

225 the breakdown of starch which attacked amylose by cleaving to disaccharides.

226 Based on observations in this work, all trunks of cultivars had an equal number of amylase

227 activity. It was found that sugar concentration of sap after storage depended on starch

228 concentration before trees were harvested. On the other hand, invertase is an enzyme which is

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229 usually found in as a catalyst for hydrolysis of sucrose. The invertase activity plays an important

230 role in the breakdown of sucrose within the trunk (Ghiena et al. 1993).

231 After harvested of the trees, an increase in protein within the trunk was observed. However,

232 investigation on this concept is currently in progress since more detailed information is needed to

233 clarify such issue. This could be an indication of great stress experienced by the tree, such as the

234 environmental factors and expression of various resultant genes. It has been reported in previous
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235 works that plants accumulated sugar as a result of stress (Chang and Ryan 1987, Hale and Orcutt

236 1987, Maruyama et al. 2009, Wang et al. 2000).

237 CONCLUSIONS

238 It is assumed that other parameters also existed leading accumulation of sugar within the sap.

239 Sugar concentration in the sap is significantly influenced by the starch content within the trunk

240 as well as the cultivation environment. Additionally, the mechanism in which sugar accumulates

241 within the stored trunk after it is harvested has yet to be clarified if there are any other chemical

242 compounds such as fatty acid, organic acid and etc. that may contribute to this findings. For the

243 practical use and a stable production of sap a high concentration of sugar is required. Study on

244 accumulation of starch and sugar concentration from sap of oil palm trees in Malaysia showed no

245 major significant between cultivars. Accumulation of sugar in oil palm sap could be explained by

246 an increase of sugar content inside the sap when starch content decreased and these phenomena

247 occurred after certain storage period. However the storage period for the highest sugars

248 accumulation in each trunk is different. In this experiment 60 days of storage was indicated as

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249 the highest increases of sugars content for most of the trunks followed by 15~45 days for others.

250 It appears that amylolytic enzymes exist in the trunks also play important roles to degrade the

251 starch into sugar. However, specific study on mechanism of sugar accumulation in the trunks

252 should be further investigated to have a better understanding to this concept.

253 FUNDING
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254 This work was supported by the New Energy and Industrial Technology Development

255 Organization (NEDO) of Japan (304/PTEKIND/650568/J118) and JIRCAS

256 (304/PTEKIND/650659/J118). Funding in the form of assistantship to Hamid Z.A.A “Under

257 MyBrain -15 Scholarship Program” is also acknowledged. We would like to thank Kuala

258 Lumpur Kepong (KLK), Malaysia, Malaysia of the Northern Branch (MKC), Malaysia, and

259 Felda Holding Bhd, Malaysia, for their cooperation in providing the oil palm samples.

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320

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321 Table 1 Oil palm trunk samples taken from various plantations in Malaysia.

Cultivars Location Sample

Negeri Sembilan A-II

Dura × Pisifera mix (Dura ×
A (Felda Kelating Jernih, Serting A-II
URT)
Hilir)
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Selangor (Banting B-I

B Deli Dura × AVROS
Dusun Durian) B-II

C-I
C Deli Dura × Pisifera x H&C Selangor (Sg. Buloh Estate)
C-II

Deli Dura × Yangambi (URTA D –I

D Kedah (Ladang Pelam, Kulim)
or URTC) D-II

E Deli Dura × Yangambi Pahang (Jerantut) E-I

322

323

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324 Figure 1 Picture of oil palm cultivars use in this experiment. A: Dura × Pisifera mix (Dura ×
325 URT), B: Deli Dura × AVROS, C: Deli Dura × Pisifera × H&C, D:Deli Dura × Yangambi,
326 URTA or URTC, E: Deli Dura × Yangambi.

327

328 A B

329
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330

331 C

D
332

333
E
334

335

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336 Figure 2 Sugar concentrations in oil palm sap of various cultivars during storage. I and II refer to
337 log number. Sugar concentration is an average of sugar content of inner part, intermediate part
338 and outer part.
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339

340

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341 Figure 3 (A) Starch content of parenchyma at different cultivars, (B) Starch content of
342 parenchyma during storage. I and II refer to log number. Starch concentration is an average of
343 starch content of inner part, intermediate part and outer part.

A
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344
B

345

346

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347 Figure 4 Enzyme activity in the trunk of cultivar A-I (Dura × Pisifera mix (Dura x URT) during
348 storage. A: Amylase, B: β-1,3-glucanase, C: Cellulase, D: Xylanase, E: Invertase.

A B

349
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C D

350

351
E

352

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353 Figure 5 (A) Scanning electron micrographs of cross section of oil palm trunk at 0 day of
354 storage time, (B) 60 days of storage time.

A
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0 day

355

60 days

356

357

358

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