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Physicochemical properties of resistant starch type III from sago starch at different palm stages
Materials Today: Proceedings xxx (xxxx) xxx
Contents lists available at ScienceDirect
Materials Today: Proceedings journal homepage: www.elsevier.com/locate/matpr
Physicochemical properties of resistant starch type III from sago starch at different palm stages Rinani Shima Abd Rashid a, Abdul Manan Dos Mohamed a,⇑, Shamini Nair Achudan a, Peter Mittis b a b
Malaysian Institute of Chemical & Bioengineering Technology, Universiti Kuala Lumpur, Vendor City Industrial Park, Alor Gajah 78000, Melaka, Malaysia Craun Research Sdn. Bhd., Lot, 14, Jalan Sultan Tengah, Kuching 93050, Sarawak, Malaysia
a r t i c l e
i n f o
Article history: Received 21 October 2019 Received in revised form 27 January 2020 Accepted 28 January 2020 Available online xxxx Keywords: Sago starch Resistant starch Amylose Autoclaving Swelling power and solubility
a b s t r a c t In this study, resistant starch type III (RS3) was produced from different sections, the top and bottom part of Metroxylon sagu palm at different growth stages namely, Plawei Manit, Bubul and Angau Muda. Sago starch was subjected for two cycles of autoclaving, debranching by pullulanase and cooling for the production of resistant starch. The physicochemical properties of sago RS3 studied were the starch morphology by Scanning Electron Microscopy (SEM), resistant starch content, amylose content, swelling power and solubility as influenced by different palm growth stages and sections. Significant differences in the resistant starch content between 23.89% and 41.62% as well as amylose content which yielded 27.22% to 35.74% from different growth stages were observed (p < 0.05). Granules of resistant starch showed irregular and rough surface structure as compared to smooth granular surface, oval shaped of native sago starch. There are significant variations (p < 0.05) observed in the results of swelling power and solubility from 7.98 g/g to 19.41 g/g for swelling power and from 5.63% to 15.10% for solubility from the different growth stages. Ó 2020 Elsevier Ltd. All rights reserved. Selection and Peer-review under responsibility of the scientific committee of the 4th International Conference on Green Chemical Engineering and Technology: Materials Science.
1. Introduction Having yearly production of 103,600 tonnes, Malaysia is one of the largest sago starch producers in the world [1]. It was well known in Southeast Asia because sago starch was used to prepare variety of traditional cuisine such as puddings, soups, noodle, biscuits and many more dishes. Sago palm grows vastly was somewhat seen ignored by farmers and less attention has been paid to sago palm in its starch content history. Sago starch was extracted from its log. It was found and calculated that a large amount of starch in the trunk of sago palm is about 3 to 4 times higher productivity of starch yield per unit area than wheat, corn, rice and about seventeen times higher than tapioca starch yields [2]. Propagation of sago palm may start from seed and seedlings (suckers). After sucker emergence or planting, it takes about 4–6 years for the sucker to grow to the trunk formation stage before furthering its growth to the flowering stage. The estimation time for the sago palm to grow is 8–15 years from sucker to the flowering stages [3]. Starch will become saturated from the bottom of the stem ⇑ Corresponding author. E-mail address: [email protected] (A.M. Dos Mohamed).
upwards and when it reaches maturity, the trunk was fully piled up with the starch up to the crown [4]. Study by [5] showed that different growth stages of sago palm in peat soil affect the physicochemical properties of the starch. The variations include starch granules size and amylopectin and amylose content found in the base and middle part of the sago palm at different stages of growth. This finding is important as it might become one of the factors of starch inconsistency and low quality as reported by industries apart from processing techniques issues. One of the important applications of sago starch is the resistant starch. Resistant starch (RS) is defined as any starch passed to the large bowel instead because it cannot be digested by small intestine. When flowing down reaching the colon, it feeds the good bacteria that needs. This is called prebiotics. Resistant starch is divided to 5 different types depending on the nature and how to prepare it [6]. They are known as RS because they are bound within the fibrous cell walls. Due to the commercial availability and common application in food processing, type II and type III RS are vastly used in learning the physiological significance of RS. This study was conducted to determine the resistant starch type III (RS3) physicochemical properties, produced from sago starch at different maturity stages and parts of sago palm. Three physiological growth
https://doi.org/10.1016/j.matpr.2020.01.511 2214-7853/Ó 2020 Elsevier Ltd. All rights reserved. Selection and Peer-review under responsibility of the scientific committee of the 4th International Conference on Green Chemical Engineering and Technology: Materials Science.
Please cite this article as: R. S. A. Rashid, A. M. Dos Mohamed, S. N. Achudan et al., Physicochemical properties of resistant starch type III from sago starch at different palm stages, Materials Today: Proceedings, https://doi.org/10.1016/j.matpr.2020.01.511
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R.S.A. Rashid et al. / Materials Today: Proceedings xxx (xxxx) xxx
stages of sago palm i.e. Plawei Muda (full trunk growth), Bubul (bolting) and Angau Muda (flowering) were studied. 2. Materials and methods
(Hitachi SU3500, Tokyo, Japan). The starch sample was coated with gold in sputter coater before observation. The starch was observed at 50 (RS3) and 250 (native sago starch) and were randomly collected. The granule size and starch characteristic were analysed by software for image processing.
2.1. Materials 2.8. Resistant starch content The sago palm log sections were obtained from mineral soil at Mukah, Sarawak, and were collected from two different heights, which is bottom (1 m from the ground) and top (1 m from top of palm tree) heights of the sago trunk and delivered to the Food Technology laboratory, UniKL for the project. The extraction of sago starch from sago log section was prepared according to the [5]. All other chemicals were analytical grade and obtained either from Merck, Darmstadt Co. or Sigma Chemical Co. 2.2. Preparation of sago starch Top sago pith was removed and chopped into 1–2 cm of small cubes. Distilled water was added before the cubes was ground in a blender. The slurry produced was filtered using sieve and squeezed to extract the starch liquid. The starch was washed three times with distilled water until clear starch colour changes to white in colour. The starch cake then was transferred onto the tray and dry in convection oven at 50 °C until 15% or lesser moisture content achieved. Finally, dried starch was ground and sieved through a 150 mm sieve to acquire a fine sago powder. The sago starch was stored at room temperature and ready for further tests.
Resistant starch was determined according to [7]. An amount of 100 mg of sample was used in this analysis. Pepsin solution (400 units/mg, 40 °C, pH 1.5, 60 min) was used to remove protein followed by addition of a-amylase solution (4 units/mg, 37 °C, pH 6.9, 16 hrs) to hydrolyze digestible starch. The method was proceeded by addition 4 M potassium hydroxide (KOH) to solubilize resistant starch. Amyloglucosidase solution was added (0.12 unit/ mg, 60 °C, pH 4.8, 45 min) to hydrolyze resistant starch. Finally, glucose content was determined by using the glucose oxidase and peroxidase reagent according to the formula as follow
Resistant Starch Content ¼
mg glucose dilution factor 0:9 100% sample weight ðmg; dry basisÞ
2.9. Amylose determination
An amount of 20 g of starch from top part of Plawei Manit was suspended in 100 mL of distilled water and was autoclaved at 121 °C for 1hr. The gelatinized starch was let to cool down to 60 °C and pullulanase enzyme was prepared to begin the enzymatic debranching.
The total amylose content was determined according to [8] using colorimetric method after removal of lipids from starch with 120 mL hot 75% n-propanol at 85 °C for 7 h in a Soxhlet extractor. Twenty (20) mg of sample was mix with 8 mL of 90% dimethyl sulfoxide. The mixture was heated (85 °C, 15 min), and the volume was made up to 25 mL with distilled water. One milliliter of diluted solution was mix with 5 mL of iodine solution, top up to 50 mL water and finally measured using a UV-VIS spectrophotometer (Shimadzu Model UV-1800, Kyoto, Japan) at 600 nm. The total amylose content of each sample was inferred from the standard curve prepared from different percentage of pure potato amylose and pure potato amylopectin.
2.4. Enzymatic debranching with pullulanase enzyme
2.10. Swelling power and solubility
An activity of 20 PUN/g of pullulanase enzyme was carefully added to the gelatinized starch. The sample was incubated in incubator shaker at 60 °C for 24 hrs at 150 rpm. In order to stop the enzyme reaction, the starch was heated in a water bath at 80 °C for 15 min.
The analysis was conducted according to [9]. Swelling power and solubility of samples were determined where 100 mg of sample was accurately weighed in a pre-weighed 50 mL centrifuge tube. An amount of 10 mL distilled water was added in to the tube. The water bath was heated to 90 °C and the tube was put inside it for 30 min, then proceeded with centrifugation at 2500 g for 15 min. The sediment in the centrifuge tube was weighed after the supernatant was removed. Five (5) mL of the supernatant was carefully transferred to a pre-weighed moisture dish and oven dried at 110 °C for 12 hrs. The dish was then weighed after being cooled in a dessicator. The swelling power and solubility were calculated by the following calculations:
2.3. Preparation of resistant starch
2.5. Thermal processing and cold storage The sample was autoclaved again at 121 °C for 1 hr. and cooled to room temperature before it was kept in a refrigerator at 4 °C for 24 hrs. 2.6. Drying and grinding The sample was dried in the oven at 50 °C for 24 hrs to obtain moisture content approximately of 13%. It was collected, ground and sieved with a 150 mm siever. The procedure was repeated on all other sago starch samples; bottom part of Plawei Manit stage, top and bottom part of Bubul stage and top and bottom part of Angau Muda stage. The untreated native starch from all stages of palm were served as control. 2.7. Scanning electron microscopy (SEM) The morphology of native sago starch and sago RS was examined using a field emission scanning electron microscopy (SEM)
Swelling powerðg=g Þ ¼
Solubilityð%Þ ¼
weigh of the wet sediment ðg Þ 100 weigh of the dry starchðg Þ
weigh of the supernatantðg Þ 100 weight of the drystarchðg Þ
2.11. Statistical analysis The statistical software, IBM SPSS Statistics 20 (New York, U.S. A) was used to analyze all the data. The differences between samples means of an effect were statistically computed using one-way analysis of variance (ANOVA) using the Duncan’s multiple range tests to compare means. Triplicate analysis were done for each
Please cite this article as: R. S. A. Rashid, A. M. Dos Mohamed, S. N. Achudan et al., Physicochemical properties of resistant starch type III from sago starch at different palm stages, Materials Today: Proceedings, https://doi.org/10.1016/j.matpr.2020.01.511
R.S.A. Rashid et al. / Materials Today: Proceedings xxx (xxxx) xxx
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Fig. 1. SEM of native sago starch at different part and stages, (A) Plawei Manit top, (B) Plawei Manit bottom, (C) Bubul top, (D) Bubul bottom, (E) Angau Muda top, (F) Angau Muda bottom, (G) Native commercial at 250 magnification and (H) Sago resistant starch at 50 magnification.
Please cite this article as: R. S. A. Rashid, A. M. Dos Mohamed, S. N. Achudan et al., Physicochemical properties of resistant starch type III from sago starch at different palm stages, Materials Today: Proceedings, https://doi.org/10.1016/j.matpr.2020.01.511
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show higher resistant starch content than the bottom part with the highest content of 41.62% of RS3 obtained from Plawei Manit top, followed by Bubul and Angau Muda. When starch granules were subjected to high temperature in excess water, the water entered into the granule which leads to granule swelling and size increase. As the granule swells, the amylose may leach out as random coil polymer. Further increment of temperature will completely erupt the granule, liberating the polymer chains. The polymer chains begin to reassociate as double helices and stabilized by hydrogen bonds upon cooling, forming a retrograded starch which is resistant to digestion [10]. As shown in the Fig. 1, the top part of sago palm contains smaller granules than the bottom part. This results might relate with the amylose chain distribution, where more linear amylose chains are occurred in the retrograded starch which proliferate the RS3 formation. Further works should be carry on to confirm the phenomenon.
Table 1 Resistant starch content of sago resistant starch from different sago palm stages and parts. Sample
RS3 content (%)
Control Plawei Manit Top Plawei Manit Bottom Bubul Top Bubul Bottom Angau Muda Top Angau Muda Bottom
31.26 ± 1.04bc 41.62 ± 0.89a 33.89 ± 0.71bc 35.32 ± 1.11b 32.82 ± 2.42bc 27.32 ± 1.41 cd 23.89 ± 1.14d
Results are expressed as means ± standard deviation (N = 3). Mean values in the same column followed by different superscript lower case letters are significantly different (p < 0.05). Table 2 Amylose content of native sago starch and sago resistant starch from different sago palm stages and parts. Sample
Plawei Manit Top Plawei Manit Bottom Bubul Top Bubul Bottom Angau Muda Top Angau Muda Bottom
Amylose (%) Native
RS3
28.30 ± 1.63a 28.53 ± 1.76a 28.49 ± 0.87a 29.21 ± 1.28a 23.77 ± 2.18b 25.36 ± 1.64ab
34.47 ± 2.57a 35.74 ± 2.75a 33.87 ± 0.65a 35.31 ± 0.42a 27.22 ± 1.98b 27.44 ± 2.24b
3.3. Amylose content Amylose content of native sago starch and sago resistant starch from different sago palm stages and parts are presented in Table 2. It is clearly observed that amylose content in sago RS3 are higher than in native sago starch. The highest yield comes from Plawei Manit bottom however it is not significant (p < 0.05) with other RS3 samples except from Angau Muda stage. This result proves that gelatinization of starch during autoclaving resulted in a high amount of amylopectin leaching out from the crystalline region of the granules where pullulanase enzyme had better access on the amylopectin to increase the amylose content. The amylose content in the sample will affect the production of RS3. This was demonstrated by [11] where the analysis of resistant starch content from different amylose content (3%–44%) in the barley bread resulted the highest resistant starch content from 44% of amylose. However, determination on amylose/amylopectin chain length distribution in the starch will give significant meaningful relationships between the amylose distribution and resistant starch yield whereby the linear amylose chains are preferred in the RS3 formation due to the faster rate of retrogradation and more resistant crystallites formation [10]. Apart from amylose content, other properties of starch will also influence the starch digestibility such as size of granules, crystallinity, polymerization degree and nonstarch constituents [12].
Results are expressed as means ± standard deviation (N = 3). Mean values in the same column followed by different superscript lower case letters are significantly different (p < 0.05).
samples while the significance difference was determining at a = 0.05 (95% confidence level). 3. Results and discussion 3.1. Scanning electron microscopy (SEM) The granules structure of native sago starch is shown in Fig. 1 (A–G) and resistant starch granules is shown in Fig. 1(H). Native sago starch was smooth, truncated oval-shaped with average diameter value found to be between 21.53 ± 0.00 mm and 56.56 ± 0.01 mm. It was observed that bottom stage of all stages has bigger size of granules as compared the top part of the sago palm with the biggest granules size was observe on Bubul bottom. The control sample which is a commercial native sago (G) shows a mixture of granules size which lead to the inconsistent results. As for resistant starch (H), the granular structure had disappeared and shows irregular shape with a sponge-like, porous structure due to granule disruption during the processing of RS3.
3.4. Swelling power and solubility Table 3 shows the swelling power and solubility capacity of native sago starch and sago RS3 for all stages and part of sago palm. Swelling power is an evaluation on the amount of water being absorbed in starch granules during gelatinization. Apart from that, swelling power is based on the amylose and amylopectin properties including branching degree, distribution of molecular weight,
3.2. Resistant starch content Table 1 shows the resistant starch contents in all growth stages of sago palm. The results depicted that the top part of all stages
Table 3 Swelling power and solubility of native sago starch and sago resistant starch at different growth stages and parts. Sample
Control Plawei Manit Top Plawei Manit Bottom Bubul Top Bubul Bottom Angau Muda Top Angau Muda Bottom
Solubility at 90 °C (%)
Swelling power (g/g) Native
RS3
Native
RS3
13.14 ± 0.17 g 15.58 ± 0.17d 19.41 ± 0.30a 13.79 ± 0.10f 17.42 ± 0.02c 14.57 ± 0.10e 18.85 ± 0.05b
9.24 ± 1.29ab 7.98 ± 0.86c 10.01 ± 1.15a 8.59 ± 0.67b 9.51 ± 0.66ab 9.88 ± 1.16ab 10.87 ± 0.19ab
5.63 ± 0.06e 6.57 ± 0.12d 7.53 ± 0.25b 7.00 ± 0.17 cd 8.07 ± 0.21a 5.93 ± 0.23e 7.20 ± 0.10bc
7.76 ± 1.68a 15.10 ± 0.02b 9.57 ± 0.58c 13.64 ± 1.21b 14.74 ± 0.27b 8.66 ± 0.35d 7.66 ± 0.49e
Results are expressed as means ± standard deviation (N = 3). Mean values in the same column followed by different superscript lower case letters are significantly different (p < 0.05).
Please cite this article as: R. S. A. Rashid, A. M. Dos Mohamed, S. N. Achudan et al., Physicochemical properties of resistant starch type III from sago starch at different palm stages, Materials Today: Proceedings, https://doi.org/10.1016/j.matpr.2020.01.511
R.S.A. Rashid et al. / Materials Today: Proceedings xxx (xxxx) xxx
and length of branch [13]. Amylopectin content contributes to the swelling properties of starch. The higher amylopectin content in starch, the higher swelling power capacity seen in the sample. From the table, it is clearly shown that native sago starch has higher swelling power between 13.14 and 19.41 g/g compared to sago RS3 in the range of 7.98–10.87 g/g. This relates to the higher amylopectin content in the native sago starch due to ability of amylopectin to swell without being restricted by the amylose. For the sago RS3, Angau Muda bottom part shows higher swelling power to relate higher amylopectin in the sample. Higher amylopectin resulted a lower amylose content as discussed in Section 3.3. High swelling power will positively correlated with solubility [14]. High swelling power indicated that more starch was solubilized and thus increased the solubility capacity. This finding was in line with results obtained from native sago starch where Plawei Manit, Bubul and Angau Muda portrayed the same trend. However, condition of the processing during resistant starch production might influence the solubility of the starch. As shown from the table, the low swelling power will have resulted in a higher solubility of the resistant starch. The solubility was increased with smaller granule size and higher amylose content. The solubility can be influenced by several factors like inter-associated forces, swelling power, the existence of surfactants and other related compounds [15]. 4. Conclusion Differences were observed in the physicochemical properties of sago starch and resistant starch from bottom and top parts of sago palms from different maturity stages. SEM micrographs showed that sago starch from bottom parts of all stages have bigger granule than the top part of the sago palm and relate to the consistency in size. Resistant starch production with starch suspension in distilled water by two cycles of autoclaving at 121 °C for 60 mins before and after the addition of pullulanase debranching of the starch polymers at 60 °C for 24 hrs and cooling at 4 °C for 24 hrs, resulted the highest RS3 of 41.62% from Plawei Manit top. A significant difference was observed in the amylose content, swelling power and solubility between the native sago starch and the sago resistant starch of different growth stages. Starch from Plawei Manit top stage have significant difference on swelling power and solubility compared to the other growth stages. Homogeneity of starch granules can be achieved by extracting the starch based on the position of the palm (top and bottom) and stages of maturity. Thus, determination of the granule size distribution and palm stages is important vital in optimization of RS3 production and give sago starch more advantages for specific applications as food ingredients including prebiotic potential.
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tion, Supervision, Visualization. Shamini Nair Achudan: Writing - review & editing, Software, Validation. Peter Mittis: Data curation. 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. Acknowledgements The authors are grateful to Skim Geran Penyelidikan dan Inovasi MARA (SGPIM)-MARA/UNI:1/33/07/18 (1) and UniKL Research Cluster Grant (Biomaterial-based Products and Technology) for financial assistance. References [1] A.M. Ahmad, S.A. Zaman, S.R. Sarbini, Production of resistant starch type III from native sago starch as a potential prebiotic, J. Agric. Food Dev. 2 (2016) 1– 4. [2] A.A. Karim, A.P.-L. Tie, D.M. Manan, I.S. Zaidul, Starch from the sago (Metroxylon sagu) palm tree - properties, prospects and challenges as a new industrial source for food and other uses, Compr. Rev. Food Sci. Food Saf. 7 (2008) 215–228. [3] Y. Yamamoto, Starch productivity of sago palm and the related factor October 29–31 2011, Proceeding of the 10th International Sago Symposium Sago for food security, Bio-energy, and industry from research to market, IPB International Convention Center, Bogor, 2011. [4] A.T. Pei-Lang, A.M.D. Mohamed, A.A. Karim, Sago starch and composition of associated components in palms of different growth stages, Carbohydr. Polym. 63 (2006) 283–286. [5] A.P.L. Tie, A.A. Karim, D.M.A. Manan, Physicochemical properties of starch in sago palms (Metroxylon sagu) at different growth stages, Starch/Stärke 60 (2008) 418–1416. [6] P. Raigond, R. Ezekiel, B. Raigond, Resistant starch in food: a review, J. Sci. Food Agric. 95 (10) (2014) 1968–1978. [7] I. Goñi, L. García-Diz, E. Mañas, F. Saura-Calixto, Analysis of resistant starch: a method for foods and food products, Food Chem. 56 (4) (1996) 445–449. [8] R. Hoover, W. Ratnayake, Determination of total amylose content of starch, current protocols in food analytical, Chemistry (2001) E2.3.1–E2.3.5. [9] M. Kaur, D.P. Oberoi, D.S. Sogi, B.S. Gill, Physicochemical, morphological and pasting properties of acid treated starches from different botanical sources, J. Food Sci. Technol. 48 (4) (2011) 460–465. [10] X. Li, Resistant Starch and Its Applications, in: Zhengyu Jin (Ed.), Functional Starch and Applications in Food, Springer, Singapore, 2018, pp. 63–90, https:// doi.org/10.1007/978-981-13-1077-5_3. [11] A. Åkerberg, H. Liljeberg, I. Björck, Effects of amylose/amylopectin ratio and baking conditions on resistant starch formation and glycaemic indices, J. Cereal 28 (1) (1998) 71–80. [12] J.A. Mir, K. Srikaeo, J. García, Effects of amylose and resistant starch on starch digestibility of rice flours and starches, Int. Food Res. J. 20 (2013) 1329–1335. [13] U. Uthumporn, N. Wahidah, A.A. Karim, Physicochemical properties of STARCH FROM SAGo (Metroxylon sagu) palm grown in mineral soil at different growth stages, IOP Conf. Ser. Mater. Sci. Eng. 62 (1) (2014) 11. [14] M. Shin, K. Woo, P.A. Seib, Hot-water solubilities and water sorptions of resistant starch at 25 °C, Cereal Chem. 80 (2003) 564–566. [15] S.N. Moorthy, Physicochemical and functional properties of tropical tuber starches: a review, Starch/Stärke 54 (2002) 559–592.
CRediT authorship contribution statement Rinani Shima Abd Rashid: Writing - original draft, Methodology, Investigation. Abdul Manan Dos Mohamed: Conceptualiza-
Please cite this article as: R. S. A. Rashid, A. M. Dos Mohamed, S. N. Achudan et al., Physicochemical properties of resistant starch type III from sago starch at different palm stages, Materials Today: Proceedings, https://doi.org/10.1016/j.matpr.2020.01.511
Contents lists available at ScienceDirect
Materials Today: Proceedings journal homepage: www.elsevier.com/locate/matpr
Physicochemical properties of resistant starch type III from sago starch at different palm stages Rinani Shima Abd Rashid a, Abdul Manan Dos Mohamed a,⇑, Shamini Nair Achudan a, Peter Mittis b a b
Malaysian Institute of Chemical & Bioengineering Technology, Universiti Kuala Lumpur, Vendor City Industrial Park, Alor Gajah 78000, Melaka, Malaysia Craun Research Sdn. Bhd., Lot, 14, Jalan Sultan Tengah, Kuching 93050, Sarawak, Malaysia
a r t i c l e
i n f o
Article history: Received 21 October 2019 Received in revised form 27 January 2020 Accepted 28 January 2020 Available online xxxx Keywords: Sago starch Resistant starch Amylose Autoclaving Swelling power and solubility
a b s t r a c t In this study, resistant starch type III (RS3) was produced from different sections, the top and bottom part of Metroxylon sagu palm at different growth stages namely, Plawei Manit, Bubul and Angau Muda. Sago starch was subjected for two cycles of autoclaving, debranching by pullulanase and cooling for the production of resistant starch. The physicochemical properties of sago RS3 studied were the starch morphology by Scanning Electron Microscopy (SEM), resistant starch content, amylose content, swelling power and solubility as influenced by different palm growth stages and sections. Significant differences in the resistant starch content between 23.89% and 41.62% as well as amylose content which yielded 27.22% to 35.74% from different growth stages were observed (p < 0.05). Granules of resistant starch showed irregular and rough surface structure as compared to smooth granular surface, oval shaped of native sago starch. There are significant variations (p < 0.05) observed in the results of swelling power and solubility from 7.98 g/g to 19.41 g/g for swelling power and from 5.63% to 15.10% for solubility from the different growth stages. Ó 2020 Elsevier Ltd. All rights reserved. Selection and Peer-review under responsibility of the scientific committee of the 4th International Conference on Green Chemical Engineering and Technology: Materials Science.
1. Introduction Having yearly production of 103,600 tonnes, Malaysia is one of the largest sago starch producers in the world [1]. It was well known in Southeast Asia because sago starch was used to prepare variety of traditional cuisine such as puddings, soups, noodle, biscuits and many more dishes. Sago palm grows vastly was somewhat seen ignored by farmers and less attention has been paid to sago palm in its starch content history. Sago starch was extracted from its log. It was found and calculated that a large amount of starch in the trunk of sago palm is about 3 to 4 times higher productivity of starch yield per unit area than wheat, corn, rice and about seventeen times higher than tapioca starch yields [2]. Propagation of sago palm may start from seed and seedlings (suckers). After sucker emergence or planting, it takes about 4–6 years for the sucker to grow to the trunk formation stage before furthering its growth to the flowering stage. The estimation time for the sago palm to grow is 8–15 years from sucker to the flowering stages [3]. Starch will become saturated from the bottom of the stem ⇑ Corresponding author. E-mail address: [email protected] (A.M. Dos Mohamed).
upwards and when it reaches maturity, the trunk was fully piled up with the starch up to the crown [4]. Study by [5] showed that different growth stages of sago palm in peat soil affect the physicochemical properties of the starch. The variations include starch granules size and amylopectin and amylose content found in the base and middle part of the sago palm at different stages of growth. This finding is important as it might become one of the factors of starch inconsistency and low quality as reported by industries apart from processing techniques issues. One of the important applications of sago starch is the resistant starch. Resistant starch (RS) is defined as any starch passed to the large bowel instead because it cannot be digested by small intestine. When flowing down reaching the colon, it feeds the good bacteria that needs. This is called prebiotics. Resistant starch is divided to 5 different types depending on the nature and how to prepare it [6]. They are known as RS because they are bound within the fibrous cell walls. Due to the commercial availability and common application in food processing, type II and type III RS are vastly used in learning the physiological significance of RS. This study was conducted to determine the resistant starch type III (RS3) physicochemical properties, produced from sago starch at different maturity stages and parts of sago palm. Three physiological growth
https://doi.org/10.1016/j.matpr.2020.01.511 2214-7853/Ó 2020 Elsevier Ltd. All rights reserved. Selection and Peer-review under responsibility of the scientific committee of the 4th International Conference on Green Chemical Engineering and Technology: Materials Science.
Please cite this article as: R. S. A. Rashid, A. M. Dos Mohamed, S. N. Achudan et al., Physicochemical properties of resistant starch type III from sago starch at different palm stages, Materials Today: Proceedings, https://doi.org/10.1016/j.matpr.2020.01.511
2
R.S.A. Rashid et al. / Materials Today: Proceedings xxx (xxxx) xxx
stages of sago palm i.e. Plawei Muda (full trunk growth), Bubul (bolting) and Angau Muda (flowering) were studied. 2. Materials and methods
(Hitachi SU3500, Tokyo, Japan). The starch sample was coated with gold in sputter coater before observation. The starch was observed at 50 (RS3) and 250 (native sago starch) and were randomly collected. The granule size and starch characteristic were analysed by software for image processing.
2.1. Materials 2.8. Resistant starch content The sago palm log sections were obtained from mineral soil at Mukah, Sarawak, and were collected from two different heights, which is bottom (1 m from the ground) and top (1 m from top of palm tree) heights of the sago trunk and delivered to the Food Technology laboratory, UniKL for the project. The extraction of sago starch from sago log section was prepared according to the [5]. All other chemicals were analytical grade and obtained either from Merck, Darmstadt Co. or Sigma Chemical Co. 2.2. Preparation of sago starch Top sago pith was removed and chopped into 1–2 cm of small cubes. Distilled water was added before the cubes was ground in a blender. The slurry produced was filtered using sieve and squeezed to extract the starch liquid. The starch was washed three times with distilled water until clear starch colour changes to white in colour. The starch cake then was transferred onto the tray and dry in convection oven at 50 °C until 15% or lesser moisture content achieved. Finally, dried starch was ground and sieved through a 150 mm sieve to acquire a fine sago powder. The sago starch was stored at room temperature and ready for further tests.
Resistant starch was determined according to [7]. An amount of 100 mg of sample was used in this analysis. Pepsin solution (400 units/mg, 40 °C, pH 1.5, 60 min) was used to remove protein followed by addition of a-amylase solution (4 units/mg, 37 °C, pH 6.9, 16 hrs) to hydrolyze digestible starch. The method was proceeded by addition 4 M potassium hydroxide (KOH) to solubilize resistant starch. Amyloglucosidase solution was added (0.12 unit/ mg, 60 °C, pH 4.8, 45 min) to hydrolyze resistant starch. Finally, glucose content was determined by using the glucose oxidase and peroxidase reagent according to the formula as follow
Resistant Starch Content ¼
mg glucose dilution factor 0:9 100% sample weight ðmg; dry basisÞ
2.9. Amylose determination
An amount of 20 g of starch from top part of Plawei Manit was suspended in 100 mL of distilled water and was autoclaved at 121 °C for 1hr. The gelatinized starch was let to cool down to 60 °C and pullulanase enzyme was prepared to begin the enzymatic debranching.
The total amylose content was determined according to [8] using colorimetric method after removal of lipids from starch with 120 mL hot 75% n-propanol at 85 °C for 7 h in a Soxhlet extractor. Twenty (20) mg of sample was mix with 8 mL of 90% dimethyl sulfoxide. The mixture was heated (85 °C, 15 min), and the volume was made up to 25 mL with distilled water. One milliliter of diluted solution was mix with 5 mL of iodine solution, top up to 50 mL water and finally measured using a UV-VIS spectrophotometer (Shimadzu Model UV-1800, Kyoto, Japan) at 600 nm. The total amylose content of each sample was inferred from the standard curve prepared from different percentage of pure potato amylose and pure potato amylopectin.
2.4. Enzymatic debranching with pullulanase enzyme
2.10. Swelling power and solubility
An activity of 20 PUN/g of pullulanase enzyme was carefully added to the gelatinized starch. The sample was incubated in incubator shaker at 60 °C for 24 hrs at 150 rpm. In order to stop the enzyme reaction, the starch was heated in a water bath at 80 °C for 15 min.
The analysis was conducted according to [9]. Swelling power and solubility of samples were determined where 100 mg of sample was accurately weighed in a pre-weighed 50 mL centrifuge tube. An amount of 10 mL distilled water was added in to the tube. The water bath was heated to 90 °C and the tube was put inside it for 30 min, then proceeded with centrifugation at 2500 g for 15 min. The sediment in the centrifuge tube was weighed after the supernatant was removed. Five (5) mL of the supernatant was carefully transferred to a pre-weighed moisture dish and oven dried at 110 °C for 12 hrs. The dish was then weighed after being cooled in a dessicator. The swelling power and solubility were calculated by the following calculations:
2.3. Preparation of resistant starch
2.5. Thermal processing and cold storage The sample was autoclaved again at 121 °C for 1 hr. and cooled to room temperature before it was kept in a refrigerator at 4 °C for 24 hrs. 2.6. Drying and grinding The sample was dried in the oven at 50 °C for 24 hrs to obtain moisture content approximately of 13%. It was collected, ground and sieved with a 150 mm siever. The procedure was repeated on all other sago starch samples; bottom part of Plawei Manit stage, top and bottom part of Bubul stage and top and bottom part of Angau Muda stage. The untreated native starch from all stages of palm were served as control. 2.7. Scanning electron microscopy (SEM) The morphology of native sago starch and sago RS was examined using a field emission scanning electron microscopy (SEM)
Swelling powerðg=g Þ ¼
Solubilityð%Þ ¼
weigh of the wet sediment ðg Þ 100 weigh of the dry starchðg Þ
weigh of the supernatantðg Þ 100 weight of the drystarchðg Þ
2.11. Statistical analysis The statistical software, IBM SPSS Statistics 20 (New York, U.S. A) was used to analyze all the data. The differences between samples means of an effect were statistically computed using one-way analysis of variance (ANOVA) using the Duncan’s multiple range tests to compare means. Triplicate analysis were done for each
Please cite this article as: R. S. A. Rashid, A. M. Dos Mohamed, S. N. Achudan et al., Physicochemical properties of resistant starch type III from sago starch at different palm stages, Materials Today: Proceedings, https://doi.org/10.1016/j.matpr.2020.01.511
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Fig. 1. SEM of native sago starch at different part and stages, (A) Plawei Manit top, (B) Plawei Manit bottom, (C) Bubul top, (D) Bubul bottom, (E) Angau Muda top, (F) Angau Muda bottom, (G) Native commercial at 250 magnification and (H) Sago resistant starch at 50 magnification.
Please cite this article as: R. S. A. Rashid, A. M. Dos Mohamed, S. N. Achudan et al., Physicochemical properties of resistant starch type III from sago starch at different palm stages, Materials Today: Proceedings, https://doi.org/10.1016/j.matpr.2020.01.511
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show higher resistant starch content than the bottom part with the highest content of 41.62% of RS3 obtained from Plawei Manit top, followed by Bubul and Angau Muda. When starch granules were subjected to high temperature in excess water, the water entered into the granule which leads to granule swelling and size increase. As the granule swells, the amylose may leach out as random coil polymer. Further increment of temperature will completely erupt the granule, liberating the polymer chains. The polymer chains begin to reassociate as double helices and stabilized by hydrogen bonds upon cooling, forming a retrograded starch which is resistant to digestion [10]. As shown in the Fig. 1, the top part of sago palm contains smaller granules than the bottom part. This results might relate with the amylose chain distribution, where more linear amylose chains are occurred in the retrograded starch which proliferate the RS3 formation. Further works should be carry on to confirm the phenomenon.
Table 1 Resistant starch content of sago resistant starch from different sago palm stages and parts. Sample
RS3 content (%)
Control Plawei Manit Top Plawei Manit Bottom Bubul Top Bubul Bottom Angau Muda Top Angau Muda Bottom
31.26 ± 1.04bc 41.62 ± 0.89a 33.89 ± 0.71bc 35.32 ± 1.11b 32.82 ± 2.42bc 27.32 ± 1.41 cd 23.89 ± 1.14d
Results are expressed as means ± standard deviation (N = 3). Mean values in the same column followed by different superscript lower case letters are significantly different (p < 0.05). Table 2 Amylose content of native sago starch and sago resistant starch from different sago palm stages and parts. Sample
Plawei Manit Top Plawei Manit Bottom Bubul Top Bubul Bottom Angau Muda Top Angau Muda Bottom
Amylose (%) Native
RS3
28.30 ± 1.63a 28.53 ± 1.76a 28.49 ± 0.87a 29.21 ± 1.28a 23.77 ± 2.18b 25.36 ± 1.64ab
34.47 ± 2.57a 35.74 ± 2.75a 33.87 ± 0.65a 35.31 ± 0.42a 27.22 ± 1.98b 27.44 ± 2.24b
3.3. Amylose content Amylose content of native sago starch and sago resistant starch from different sago palm stages and parts are presented in Table 2. It is clearly observed that amylose content in sago RS3 are higher than in native sago starch. The highest yield comes from Plawei Manit bottom however it is not significant (p < 0.05) with other RS3 samples except from Angau Muda stage. This result proves that gelatinization of starch during autoclaving resulted in a high amount of amylopectin leaching out from the crystalline region of the granules where pullulanase enzyme had better access on the amylopectin to increase the amylose content. The amylose content in the sample will affect the production of RS3. This was demonstrated by [11] where the analysis of resistant starch content from different amylose content (3%–44%) in the barley bread resulted the highest resistant starch content from 44% of amylose. However, determination on amylose/amylopectin chain length distribution in the starch will give significant meaningful relationships between the amylose distribution and resistant starch yield whereby the linear amylose chains are preferred in the RS3 formation due to the faster rate of retrogradation and more resistant crystallites formation [10]. Apart from amylose content, other properties of starch will also influence the starch digestibility such as size of granules, crystallinity, polymerization degree and nonstarch constituents [12].
Results are expressed as means ± standard deviation (N = 3). Mean values in the same column followed by different superscript lower case letters are significantly different (p < 0.05).
samples while the significance difference was determining at a = 0.05 (95% confidence level). 3. Results and discussion 3.1. Scanning electron microscopy (SEM) The granules structure of native sago starch is shown in Fig. 1 (A–G) and resistant starch granules is shown in Fig. 1(H). Native sago starch was smooth, truncated oval-shaped with average diameter value found to be between 21.53 ± 0.00 mm and 56.56 ± 0.01 mm. It was observed that bottom stage of all stages has bigger size of granules as compared the top part of the sago palm with the biggest granules size was observe on Bubul bottom. The control sample which is a commercial native sago (G) shows a mixture of granules size which lead to the inconsistent results. As for resistant starch (H), the granular structure had disappeared and shows irregular shape with a sponge-like, porous structure due to granule disruption during the processing of RS3.
3.4. Swelling power and solubility Table 3 shows the swelling power and solubility capacity of native sago starch and sago RS3 for all stages and part of sago palm. Swelling power is an evaluation on the amount of water being absorbed in starch granules during gelatinization. Apart from that, swelling power is based on the amylose and amylopectin properties including branching degree, distribution of molecular weight,
3.2. Resistant starch content Table 1 shows the resistant starch contents in all growth stages of sago palm. The results depicted that the top part of all stages
Table 3 Swelling power and solubility of native sago starch and sago resistant starch at different growth stages and parts. Sample
Control Plawei Manit Top Plawei Manit Bottom Bubul Top Bubul Bottom Angau Muda Top Angau Muda Bottom
Solubility at 90 °C (%)
Swelling power (g/g) Native
RS3
Native
RS3
13.14 ± 0.17 g 15.58 ± 0.17d 19.41 ± 0.30a 13.79 ± 0.10f 17.42 ± 0.02c 14.57 ± 0.10e 18.85 ± 0.05b
9.24 ± 1.29ab 7.98 ± 0.86c 10.01 ± 1.15a 8.59 ± 0.67b 9.51 ± 0.66ab 9.88 ± 1.16ab 10.87 ± 0.19ab
5.63 ± 0.06e 6.57 ± 0.12d 7.53 ± 0.25b 7.00 ± 0.17 cd 8.07 ± 0.21a 5.93 ± 0.23e 7.20 ± 0.10bc
7.76 ± 1.68a 15.10 ± 0.02b 9.57 ± 0.58c 13.64 ± 1.21b 14.74 ± 0.27b 8.66 ± 0.35d 7.66 ± 0.49e
Results are expressed as means ± standard deviation (N = 3). Mean values in the same column followed by different superscript lower case letters are significantly different (p < 0.05).
Please cite this article as: R. S. A. Rashid, A. M. Dos Mohamed, S. N. Achudan et al., Physicochemical properties of resistant starch type III from sago starch at different palm stages, Materials Today: Proceedings, https://doi.org/10.1016/j.matpr.2020.01.511
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and length of branch [13]. Amylopectin content contributes to the swelling properties of starch. The higher amylopectin content in starch, the higher swelling power capacity seen in the sample. From the table, it is clearly shown that native sago starch has higher swelling power between 13.14 and 19.41 g/g compared to sago RS3 in the range of 7.98–10.87 g/g. This relates to the higher amylopectin content in the native sago starch due to ability of amylopectin to swell without being restricted by the amylose. For the sago RS3, Angau Muda bottom part shows higher swelling power to relate higher amylopectin in the sample. Higher amylopectin resulted a lower amylose content as discussed in Section 3.3. High swelling power will positively correlated with solubility [14]. High swelling power indicated that more starch was solubilized and thus increased the solubility capacity. This finding was in line with results obtained from native sago starch where Plawei Manit, Bubul and Angau Muda portrayed the same trend. However, condition of the processing during resistant starch production might influence the solubility of the starch. As shown from the table, the low swelling power will have resulted in a higher solubility of the resistant starch. The solubility was increased with smaller granule size and higher amylose content. The solubility can be influenced by several factors like inter-associated forces, swelling power, the existence of surfactants and other related compounds [15]. 4. Conclusion Differences were observed in the physicochemical properties of sago starch and resistant starch from bottom and top parts of sago palms from different maturity stages. SEM micrographs showed that sago starch from bottom parts of all stages have bigger granule than the top part of the sago palm and relate to the consistency in size. Resistant starch production with starch suspension in distilled water by two cycles of autoclaving at 121 °C for 60 mins before and after the addition of pullulanase debranching of the starch polymers at 60 °C for 24 hrs and cooling at 4 °C for 24 hrs, resulted the highest RS3 of 41.62% from Plawei Manit top. A significant difference was observed in the amylose content, swelling power and solubility between the native sago starch and the sago resistant starch of different growth stages. Starch from Plawei Manit top stage have significant difference on swelling power and solubility compared to the other growth stages. Homogeneity of starch granules can be achieved by extracting the starch based on the position of the palm (top and bottom) and stages of maturity. Thus, determination of the granule size distribution and palm stages is important vital in optimization of RS3 production and give sago starch more advantages for specific applications as food ingredients including prebiotic potential.
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tion, Supervision, Visualization. Shamini Nair Achudan: Writing - review & editing, Software, Validation. Peter Mittis: Data curation. 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. Acknowledgements The authors are grateful to Skim Geran Penyelidikan dan Inovasi MARA (SGPIM)-MARA/UNI:1/33/07/18 (1) and UniKL Research Cluster Grant (Biomaterial-based Products and Technology) for financial assistance. References [1] A.M. Ahmad, S.A. Zaman, S.R. Sarbini, Production of resistant starch type III from native sago starch as a potential prebiotic, J. Agric. Food Dev. 2 (2016) 1– 4. [2] A.A. Karim, A.P.-L. Tie, D.M. Manan, I.S. Zaidul, Starch from the sago (Metroxylon sagu) palm tree - properties, prospects and challenges as a new industrial source for food and other uses, Compr. Rev. Food Sci. Food Saf. 7 (2008) 215–228. [3] Y. Yamamoto, Starch productivity of sago palm and the related factor October 29–31 2011, Proceeding of the 10th International Sago Symposium Sago for food security, Bio-energy, and industry from research to market, IPB International Convention Center, Bogor, 2011. [4] A.T. Pei-Lang, A.M.D. Mohamed, A.A. Karim, Sago starch and composition of associated components in palms of different growth stages, Carbohydr. Polym. 63 (2006) 283–286. [5] A.P.L. Tie, A.A. Karim, D.M.A. Manan, Physicochemical properties of starch in sago palms (Metroxylon sagu) at different growth stages, Starch/Stärke 60 (2008) 418–1416. [6] P. Raigond, R. Ezekiel, B. Raigond, Resistant starch in food: a review, J. Sci. Food Agric. 95 (10) (2014) 1968–1978. [7] I. Goñi, L. García-Diz, E. Mañas, F. Saura-Calixto, Analysis of resistant starch: a method for foods and food products, Food Chem. 56 (4) (1996) 445–449. [8] R. Hoover, W. Ratnayake, Determination of total amylose content of starch, current protocols in food analytical, Chemistry (2001) E2.3.1–E2.3.5. [9] M. Kaur, D.P. Oberoi, D.S. Sogi, B.S. Gill, Physicochemical, morphological and pasting properties of acid treated starches from different botanical sources, J. Food Sci. Technol. 48 (4) (2011) 460–465. [10] X. Li, Resistant Starch and Its Applications, in: Zhengyu Jin (Ed.), Functional Starch and Applications in Food, Springer, Singapore, 2018, pp. 63–90, https:// doi.org/10.1007/978-981-13-1077-5_3. [11] A. Åkerberg, H. Liljeberg, I. Björck, Effects of amylose/amylopectin ratio and baking conditions on resistant starch formation and glycaemic indices, J. Cereal 28 (1) (1998) 71–80. [12] J.A. Mir, K. Srikaeo, J. García, Effects of amylose and resistant starch on starch digestibility of rice flours and starches, Int. Food Res. J. 20 (2013) 1329–1335. [13] U. Uthumporn, N. Wahidah, A.A. Karim, Physicochemical properties of STARCH FROM SAGo (Metroxylon sagu) palm grown in mineral soil at different growth stages, IOP Conf. Ser. Mater. Sci. Eng. 62 (1) (2014) 11. [14] M. Shin, K. Woo, P.A. Seib, Hot-water solubilities and water sorptions of resistant starch at 25 °C, Cereal Chem. 80 (2003) 564–566. [15] S.N. Moorthy, Physicochemical and functional properties of tropical tuber starches: a review, Starch/Stärke 54 (2002) 559–592.
CRediT authorship contribution statement Rinani Shima Abd Rashid: Writing - original draft, Methodology, Investigation. Abdul Manan Dos Mohamed: Conceptualiza-
Please cite this article as: R. S. A. Rashid, A. M. Dos Mohamed, S. N. Achudan et al., Physicochemical properties of resistant starch type III from sago starch at different palm stages, Materials Today: Proceedings, https://doi.org/10.1016/j.matpr.2020.01.511