Perubahan Struktur Pati Garut (Marantha Arundinacea L ) Sebagai Akibat Kombinasi Perlakuan Hidrolisis Asam, Debranching, Siklus Autoclaving Cooling, Dan Heat Moisture Treatment (Hmt)
STRUCTURAL CHANGES OF ARROWROOT STARCH
(Marantha arundinacea L.) AS THE IMPACT OF MULTIPLE
TREATMENTS BY ACID HYDROLYSIS, DEBRANCHING,
AUTOCLAVING-COOLING CYCLES, AND
HEAT MOISTURE TREATMENT (HMT)
MUTIARA PRATIWI
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2016
STATEMENT LETTER OF THESIS AND
SOURCE OF INFORMATION*
I declare that this thesis entitled Structural Changes of Arrowroot Starch
(Marantha arundinacea L.) as the Impact of Multiple Treatments by Acid
Hydrolysis, Debranching, Autoclaving-cooling Cycles, and Heat Moisture
Treatment (HMT) is based on my original work, in collaboration with the advisors
and has not been submitted in any form at any college, except Bogor Agricultural
University. Sources of information derived and quoted from published and
unpublished works of other authors mentioned in the text are listed in the
Bibliography at the end of this thesis.
Hereby I transfer the copyright of this thesis to Bogor Agricultural University.
Bogor, February 2016
Mutiara Pratiwi
F251140646
RINGKASAN
MUTIARA PRATIWI. Perubahan Struktur Pati Garut (Marantha arundinacea L.)
sebagai Akibat Kombinasi Perlakuan Hidrolisis Asam, Debranching, Siklus
Autoclaving-Cooling, dan Heat Moisture Treatment (HMT). Dibimbing oleh
HANIFAH NURYANI LIOE sebagai Ketua Komisi Pembimbing dan DIDAH
NUR FARIDAH sebagai Anggota Komisi Pembimbing.
Pati garut (Marantha arundinacea L.) diketahui memiliki karakteristik yang
sesuai sebagai bahan baku pati resisten tipe III (RS3). Penelitian terdahulu
menunjukkan bahwa pati garut yang dimodifikasi dengan menggunakan kombinasi
hidrolisis asam-debranching-autoclaving-cooling menghasilkan rendemen pati
resisten yang tinggi (39.39%), namun masih lebih rendah bila dibandingkan dengan
pati resisten komersial (Novelose 330) (42.68%). Kombinasi perlakuan tersebut
juga menyebabkan terjadinya perubahan struktur pada pati yang dapat diasosiasikan
dengan sifat resistensi pati. Pada penelitian ini, kombinasi modifikasi pada
penelitian terdahulu dikombinasikan kembali dengan teknik HMT pada suhu 121
o
C dan kadar air 20%. Penelitian ini bertujuan untuk mempelajari perubahan
struktur pada pati garut akibat kombinasi modifikasi hidrolisis asam-debranchingautoclaving-cooling-HMT, yang belum dipelajari pada penelitian lainnya,
khususnya terkait dengan sifat resistensi pati. Pada penelitian ini, perubahan
struktur pati dipelajari dengan menggunakan teknik GFC, FTIR, DSC, XRD, dan
SEM.
Pemisahan dengan GFC menunjukkan bahwa modifikasi yang diberikan
menyebabkan perubahan profil distribusi molekul pati, yaitu terjadi penurunan
fraksi amilopektin dan peningkatan fraksi amilosa. Amilosa berperan dalam
retrogradasi yang menjadi salah satu mekanisme pembentukan pati resisten.
Analisis dengan FTIR menunjukkan bahwa kombinasi perlakuan secara umum
meningkatkan daerah amorf dan menurunkan daerah kristalin. Terbukanya struktur
double helix dari molekul amilopektin serta degradasi amilopektin lebih lanjut
merupakan fenomena yang dapat menyebabkan terjadinya penurunan daerah
kristalin. Dugaan tersebut diperkuat dengan hasil analisis dengan GFC yang
menunjukkan adanya penurunan fraksi amilopektin. Analisis dengan DSC
menunjukkan terjadinya peningkatan suhu gelatinisasi (To, Tp, dan Tc), dengan
persentase kenaikan suhu berkisar antara 2.62-17.10%, dan penurunan entalpi
gelatinisasi (∆H) pada pati garut termodifikasi dibandingkan pati alaminya. Nilai
∆H pada pati termodifikasi berkisar antara 7.11-11.20 J/g, sementara pati alaminya
memiliki nilai ∆H sebesar 12.52 J/g. Peningkatan suhu gelatinisasi pati juga
merupakan indikasi adanya peningkatan interaksi amilosa-amilosa, amilosaamilopektin, dan/atau amilopektin-amilopektin serta pembentukan kristal pati yang
lebih sempurna. Peningkatan interaksi amilosa-amilosa, amilosa-amilopektin,
dan/atau amilopektin-amilopektin tersebut diketahui sebagai salah satu mekanisme
pembentukan pati resisten. Sementara itu, penurunan nilai ∆H yang teramati dengan
DSC menunjukkan adanya disosiasi double helix dari molekul amilopektin. Hal ini
konsisten dengan hasil analisis dengan GFC yang menunjukkan adanya degradasi
amilopektin, serta konsisten pula dengan hasil analisis dengan FTIR yang
menunjukkan peningkatan daerah amorf. Analisis dengan XRD menunjukkan
perubahan tipe kristalin dari tipe A pada pati garut alami menjadi tipe B pada
seluruh kombinasi perlakuan, kecuali pada pati yang dihidrolisis asam. Perubahan
tipe kristalin dari tipe A menjadi tipe B mengindikasikan terjadinya rekristalisasi
pati. Pengamatan dengan SEM menunjukkan bahwa perlakuan tunggal HMT tidak
menyebabkan perubahan morfologi granula pati. Akan tetapi, ketika
dikombinasikan dengan hidrolisis asam, debranching, dan autoclaving-cooling,
HMT memicu terbentuknya struktur kristalit yang rapat. Hal ini mengkonfirmasi
dugaan bahwa HMT berperan dalam penyempurnaan kristal pati yang merupakan
salah satu mekanisme pembentukan pati resisten.
Kata kunci: heat moisture treatment, modifikasi pati, pati garut, pati resisten,
struktur pati
SUMMARY
MUTIARA PRATIWI. Structural Changes of Arrowroot Starch (Marantha
arundinacea L.) as the Impact of Multiple Treatments by Acid Hydrolysis,
Debranching, Autoclaving-cooling Cycles, and Heat Moisture Treatment (HMT).
Under the supervision of HANIFAH NURYANI LIOE as the Chairman and
DIDAH NUR FARIDAH as Advisory Committee Member.
Arrowroot starch (Marantha arundinacea L.) has been known to have the
characteristics required for producing resistant starch type III (RS3). In the previous
study, RS3 from arrowroot starch had been produced through the combination of
acid hydrolysis-debranching-autoclaving-cooling, resulting high amount of
resistant starch (39.39%), yet its yield was still lower than that of commercial
resistant starch (Novelose 330) (42.68%). The combination of modification led to
changes of starch structure which was strongly presumed to be associated with the
starch resistance. In this study, the combination of modification applied in the
previous study was combined with HMT at 121 oC and 20% moisture content, as
an additional treatment to the previous combination. This study was aimed to
investigate structural changes of arrowroot starch as the impact of acid hydrolysisdebranching-autoclaving-cooling-HMT, which have not been revealed in other
studies, particularly associated with the starch resistance after the modifications. In
this study, the structural changes of arrowroot starch were studied by using GFC,
FTIR, DSC, XRD, and SEM techniques.
Separation with GFC showed that the modifications led to changes of
molecular distribution profile of arrowroot starch, showing the decrease of
amylopectin fraction, but the increase of amylose fraction. Amylose fraction
contributes to a rapid retrogradation process as a mechanism of resistant starch
formation. Analysis with FTIR showed that in general the combination of
treatments were found to increase the amorphous region and decrease the crystalline
region. Unraveling of amylopectin double helix and/or further degradation of
amylopectin were presumed to cause the decrease of crystalline region. The
assumption was supported by the result of analysis with GFC, showing the decrease
of amylopectin fraction. Analysis with DSC showed the increase of gelatinization
temperature (To, Tp, and Tc), with the increase of temperature ranging from 2.6217.10%, and the decrease of gelatinization enthalpy (∆H) in modified starch,
compared to its native. ∆H of modified arrowroot starch was found to be ranging
from 7.11-11.20 J/g, while its native showed the ∆H of 12.52 J/g. The increase of
gelatinization temperature also suggested the increasing interaction of amyloseamylose, amylose-amylopectin, and/or amylopectin-amylopectin and the perfection
of starch crystallites. The increasing starch chain interaction of amylose-amylose,
amylose-amylopectin, and/or amylopectin-amylopectin was a mechanism of
resistant starch formation. While the decreasing ∆H of modified arrowroot starch
could reflect the disruption of amylopectin double helix. This result was in line with
the results obtained with GFC, showing the degradation of amylopectin, and also
in a good agreement with the result of FTIR, showing the increase of amorphous
region. Analysis with XRD showed that the A-type crystalline of native arrowroot
starch changed into a B-type in the most of treatment combinations applied, except
in acid hydrolyzed starch. The change of crystalline type from A- to B-type
indicated the occurrence of starch recrystallization. The SEM observation showed
that single treatment of HMT did not change the morphology of granules in shape
and size. While in combination with acid hydrolysis, debranching, and autoclavingcooling, HMT led to the formation of smaller crystalline bodies which closely
adhered to each other. This result confirmed the mechanism of HMT in inducing
the perfection of crystallites as a possible mechanism of resistant starch formation.
Keywords: arrowroot starch, heat moisture treatment, resistant starch, starch
modification, starch structure
© Copyright of Bogor Agricultural University, 2016
Protected by the laws
Any unauthorized quotation of all contents or any part thereof is strictly prohibited.
Quotation is only for educational purpose, research, scientific writing, reports
writing, critique and problem analysis; and quotation would not give any
disadvantage on behalf of Bogor Agricultural University.
Any announcement and duplication of all contents or any part thereof without
permission from Bogor Agricultural University are strictly prohibited.
STRUCTURAL CHANGES OF ARROWROOT STARCH
(Marantha arundinacea L.) AS THE IMPACT OF MULTIPLE
TREATMENTS BY ACID HYDROLYSIS, DEBRANCHING,
AUTOCLAVING-COOLING CYCLES, AND
HEAT MOISTURE TREATMENT (HMT)
MUTIARA PRATIWI
Thesis
in fulfillment of the requirement for degree of
Master Science in
Study Program of Food Science
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2016
Examiner in Thesis Examination: Dr. Ir. Sukarno, MSc
PREFACE
All the praise to Allah subhanahu wa ta’ala for all His grace, mercy, and
blessing through all the way to complete my Master study. This study was intended
as requirement to graduate in Master degree in Bogor Agricultural University. This
study entitled “Structural Changes of Arrowroot Starch (Marantha arundinacea L.)
as the Impact of Multiple Treatments by Acid Hydrolysis, Debranching,
Autoclaving-cooling Cycles, and Heat Moisture Treatment (HMT)” was conducted
in Bogor Agricultural University from January-August 2015.
The author would like to express the deepest gratitude to beloved parents and
brother in appreciation of their love and encouragements, especially during the
completion of my Master study. Nothing can reply their life time support. My
sincere appreciation and deepest gratitude is also expressed to the advisors, Dr. Ir.
Hanifah Nuryani Lioe, MSi and Dr. Ir. Didah Nur Faridah, MSi for their intense
supervision and constant support. The valuable help of constructive comments and
suggestions throughout the experimental and thesis work have contributed to the
success of this research. The author also would like to express a sincere gratitude
to Dr. Ir. Sukarno, MSc as the examiner in thesis examination, for his time,
suggestion, and correction for the improvement of this thesis. Sincere thanks is also
expressed to Indonesian Directorate General of Higher Education (DIKTI) for
supporting the study through fresh graduate scholarship program and for supporting
the research through funding incentive of Penelitian Unggulan Dasar (PUD)
program 2014.
A sincere gratitude is also expressed to Dr. Ir. Dahrul Syah, MSc as the Dean
of Postgraduate School and Prof. Dr. Ir. Ratih Dewanti, MSc as the Head of Food
Science Program for the support and help towards my postgraduate affairs. The
author also would like to express her thanks to all the laboratory technicians of
Department of Food Science and Technology: Pak Taufik, Pak Sobirin, Mbak Ari,
Pak Rojak, Mas Edi, Mbak Nurul, Mbak Irin, and Pak Yahya for the technical help
during my research. The appreciation also goes to Mbak Ririn as the laboratory
technician of the department for the guidance and suggestion for the
chromatography column preparation. Special thanks to Fathma, Fitria, Mbak Ratna,
and Anand for being a cooperative partner in conducting the research. The author
also thanks her colleagues, especially Nesya, Silvie, and Florentina for the support,
advice, and help during the study and research completion. Last but not least,
sincere gratitude also goes to Rio for his constant supports and advices from the
beginning until this stage.
Hopefully this thesis could be useful for the readers and contribute in a wider
scope of food science realm.
Bogor, February 2016
Mutiara Pratiwi
TABLE OF CONTENT
LIST OF TABLES
LIST OF FIGURES
LIST OF APPENDIXES
1 INTRODUCTION
Background
Potential
Objectives
Significance
Hypothesis
xviii
xviii
xviii
1
1
3
3
4
4
2 LITERATURE STUDY
Arrowroot (Marantha arundinacea L.)
Starch
Resistant starch
Starch modification
Acid hydrolysis
Debranching
Autoclaving-cooling
Heat Moisture Treatment (HMT)
Combination of modification to produce resistant starch
Changes of molecular structure in starch modification
Gel Filration Chromatography (GFC) profile
Fourier Transform Infra-Red (FTIR) spectra profile
Differential Scanning Calorimetry (DSC) profile
X-ray Diffraction (XRD) pattern
Scanning Electron Microscopy (SEM) microstructure profile
4
4
5
8
9
10
11
12
13
13
14
14
16
18
21
22
3 RESEARCH METHODOLOGY
Time and place
Material and instruments
Methods
23
23
24
24
4 RESULT AND DISCUSSION
Profiles of starch molecular distribution analyzed by GFC
Changes of crystalline and amorphous regions analyzed by FTIR
Gelatinization properties analyzed by DSC
Crystallinity of starch analyzed by XRD
Morphology of starch granules analyzed by SEM
28
28
33
36
39
43
5 CONCLUSION AND RECOMMENDATION
Conclusion
Recommendation
Acknowledgement
46
46
47
47
BIBLIOGRAPHY
APPENDIXES
AUTHOR BIOGRAPHY
48
59
65
LIST OF TABLES
1 Studies of starch molecular distribution by using Gel Filtration
Chromatography (GFC) technique
2 Studies of starch structure by using Fourier Transform Infra-Red
(FTIR) spectroscopy
3 Thermal studies of starches by using Differential Scanning
Calorimetry (DSC) technique
4 Studies of crystallinity in various starches by using X-ray
Diffraction (XRD)
5 Studies of microstructure in various starches by using Scanning
Electron Microscopy (SEM)
6 Condition for analysis of molecular distribution profile of arrowroot
starch by using Gel Filtration Chromatography (GFC)
7 Gelatinization properties of native and modified arrowroot starch
8 X-ray diffraction pattern of native and modified arrowroot starch
15
17
19
21
23
26
36
40
LIST OF FIGURES
1 Arrowroot plant Marantha arundinacea L. (a) and arrowroot tuber
Marantha arundinacea L. (b)
2 Schematic diagram of starch granule architecture
3 Cluster model of amylopectin
4 Typical DSC thermogram with the To, Tp, Tc, and ∆H values
5 GFC profiles of arrowroot starch using Sephacryl S-HR 400 column
(2.6 cm i.d. x 90 cm)
6 GFC profiles of arrowroot starch using Sephadex G-50 column
(2.6 cm i.d. x 135 cm)
7 Changes of crystalline and amorphous regions of native and
modified arrowroot starches analyzed by Fourier Transform
Infra-Red (FTIR) spectroscopy
8 DSC gelatinization curve of native and modified arrowroot starch
9 X-ray diffraction patterns of native and modified arrowroot starch
10 Scanning Electron Microscopy (SEM) micrographs of native and
modified arrowroot starches: 1. native; 2. AH; 3. AC; 4. HMT. (a)
magnification = 500x; (b) magnification = 1500x
11 Scanning Electron Microscopy (SEM) micrographs of native and
modified arrowroot starches: 1. DE-AC; 2. AH-DE-AC; 3. AH-DEAC-HMT (a) magnification = 500x; (b) magnification = 1500x
5
7
7
20
29
31
34
37
41
45
46
LIST OF APPENDIXES
1
2
3
4
5
6
Reagents for making acetate buffer pH 5.2 solution
Reagents for making mobile phase solution
Reagents for total carbohydrate analysis
Reagents for blue value analysis
Sephadex G-50 column preparation
Sephacryl S-HR 400 column preparation
59
60
61
62
63
64
1 INTRODUCTION
Background
Resistant starch (RS) has been known for its potential health benefits and
functional properties (Sajilata et al. 2006). Resistant starch is defined as the fraction
of starch which is not hydrolyzed to D-glucose in the small intestine within 120 min
of being consumed, but which is fermented by bacteria in the colon to short chain
fatty acids and gases (Fuentes-Zaragoza et al. 2010, Topping and Clifton 2001).
Resistant starch is further divided into five distinct classes: RS1, RS2, RS3, RS4
and RS5. RS1 is physically inaccessible starch, which is entrapped within whole or
partly milled grains or seeds. RS2 belongs to some types of raw starch granules.
RS3 is retrograded starch (either processed from unmodified starch or resulting
from food processing applications). RS4 belongs to starches which are chemically
modified to obtain resistance to enzymatic digestion (Ratnayake dan Jackson 2008,
Sanz et al. 2009). RS5 belongs to resistant starch formed as a single-helix complex
of lipid-amylose (Ratnayake and Jackson 2008, Sanz et al. 2009, Fuentes-Zaragoza
et al. 2010, Ai et al. 2013).
Arrowroot starch (Marantha arundinacea L.) has the characteristics which
are suitable for producing RS3. Arrowroot has an A-type crystalline and it has
amylopectin, containing branching chains with degree of polymerization (DP) of
about 9-30 found in a high amount (up to 96%) (Srichuwong 2006). Arrowroot
starch also has a higher density in the helical structure region (Wang et al. 1998),
higher proportion of short branch chain of amylopectin (Hizukuri et al. 1983), and
greater amount of chains found in each cluster, compared to B-type crystalline
(Takeda et al. 2003). Arrowroot starch has been known to contain observable
amount of amylose, reaching 29.39% (Aprianita 2010), which contributes to a rapid
retrogradation. Besides, arrowroot starch also has low content of fat (0.68%) which
can minimize the possibility of fat-amylose formation that may reduce
retrogradation to some extent. This will enable higher amount of amylose which
can bind with another amylose or amylopectin during retrogradation (Faridah 2011).
RS3 can be obtained through heating and cooling cycles of starch which
respectively involves a gelatinization and retrogradation of starch. Acid
modification and enzymatic debranching of starch are two methods known to
increase short chain amylose which contributes in more rapid retrogradation
(Sandoval et al. 2008, Milasinovic et al. 2010).
Faridah (2011) combined starch modification consisting of acid hydrolysis,
debranching, and autoclaving-cooling to optimize formation of RS3. The
combination applied was found to produce high amount of RS3, up to 39.39%, yet
its yield was still lower than that of the commercial one, Novelose 330, reaching
42.68% (Faridah 2011). Heat Moisture Treatment (HMT) has been known as an
effective technique to increase resistant starch from various sources of starch such
as mung bean, corn, pea, and lentil starch (Chung et al. 2009, Li and Gao 2010). In
this research, HMT was applied to increase RS3 from arrowroot starch. HMT is a
physical modification technique that involves treatment of starch granules at low
moisture levels (
(Marantha arundinacea L.) AS THE IMPACT OF MULTIPLE
TREATMENTS BY ACID HYDROLYSIS, DEBRANCHING,
AUTOCLAVING-COOLING CYCLES, AND
HEAT MOISTURE TREATMENT (HMT)
MUTIARA PRATIWI
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2016
STATEMENT LETTER OF THESIS AND
SOURCE OF INFORMATION*
I declare that this thesis entitled Structural Changes of Arrowroot Starch
(Marantha arundinacea L.) as the Impact of Multiple Treatments by Acid
Hydrolysis, Debranching, Autoclaving-cooling Cycles, and Heat Moisture
Treatment (HMT) is based on my original work, in collaboration with the advisors
and has not been submitted in any form at any college, except Bogor Agricultural
University. Sources of information derived and quoted from published and
unpublished works of other authors mentioned in the text are listed in the
Bibliography at the end of this thesis.
Hereby I transfer the copyright of this thesis to Bogor Agricultural University.
Bogor, February 2016
Mutiara Pratiwi
F251140646
RINGKASAN
MUTIARA PRATIWI. Perubahan Struktur Pati Garut (Marantha arundinacea L.)
sebagai Akibat Kombinasi Perlakuan Hidrolisis Asam, Debranching, Siklus
Autoclaving-Cooling, dan Heat Moisture Treatment (HMT). Dibimbing oleh
HANIFAH NURYANI LIOE sebagai Ketua Komisi Pembimbing dan DIDAH
NUR FARIDAH sebagai Anggota Komisi Pembimbing.
Pati garut (Marantha arundinacea L.) diketahui memiliki karakteristik yang
sesuai sebagai bahan baku pati resisten tipe III (RS3). Penelitian terdahulu
menunjukkan bahwa pati garut yang dimodifikasi dengan menggunakan kombinasi
hidrolisis asam-debranching-autoclaving-cooling menghasilkan rendemen pati
resisten yang tinggi (39.39%), namun masih lebih rendah bila dibandingkan dengan
pati resisten komersial (Novelose 330) (42.68%). Kombinasi perlakuan tersebut
juga menyebabkan terjadinya perubahan struktur pada pati yang dapat diasosiasikan
dengan sifat resistensi pati. Pada penelitian ini, kombinasi modifikasi pada
penelitian terdahulu dikombinasikan kembali dengan teknik HMT pada suhu 121
o
C dan kadar air 20%. Penelitian ini bertujuan untuk mempelajari perubahan
struktur pada pati garut akibat kombinasi modifikasi hidrolisis asam-debranchingautoclaving-cooling-HMT, yang belum dipelajari pada penelitian lainnya,
khususnya terkait dengan sifat resistensi pati. Pada penelitian ini, perubahan
struktur pati dipelajari dengan menggunakan teknik GFC, FTIR, DSC, XRD, dan
SEM.
Pemisahan dengan GFC menunjukkan bahwa modifikasi yang diberikan
menyebabkan perubahan profil distribusi molekul pati, yaitu terjadi penurunan
fraksi amilopektin dan peningkatan fraksi amilosa. Amilosa berperan dalam
retrogradasi yang menjadi salah satu mekanisme pembentukan pati resisten.
Analisis dengan FTIR menunjukkan bahwa kombinasi perlakuan secara umum
meningkatkan daerah amorf dan menurunkan daerah kristalin. Terbukanya struktur
double helix dari molekul amilopektin serta degradasi amilopektin lebih lanjut
merupakan fenomena yang dapat menyebabkan terjadinya penurunan daerah
kristalin. Dugaan tersebut diperkuat dengan hasil analisis dengan GFC yang
menunjukkan adanya penurunan fraksi amilopektin. Analisis dengan DSC
menunjukkan terjadinya peningkatan suhu gelatinisasi (To, Tp, dan Tc), dengan
persentase kenaikan suhu berkisar antara 2.62-17.10%, dan penurunan entalpi
gelatinisasi (∆H) pada pati garut termodifikasi dibandingkan pati alaminya. Nilai
∆H pada pati termodifikasi berkisar antara 7.11-11.20 J/g, sementara pati alaminya
memiliki nilai ∆H sebesar 12.52 J/g. Peningkatan suhu gelatinisasi pati juga
merupakan indikasi adanya peningkatan interaksi amilosa-amilosa, amilosaamilopektin, dan/atau amilopektin-amilopektin serta pembentukan kristal pati yang
lebih sempurna. Peningkatan interaksi amilosa-amilosa, amilosa-amilopektin,
dan/atau amilopektin-amilopektin tersebut diketahui sebagai salah satu mekanisme
pembentukan pati resisten. Sementara itu, penurunan nilai ∆H yang teramati dengan
DSC menunjukkan adanya disosiasi double helix dari molekul amilopektin. Hal ini
konsisten dengan hasil analisis dengan GFC yang menunjukkan adanya degradasi
amilopektin, serta konsisten pula dengan hasil analisis dengan FTIR yang
menunjukkan peningkatan daerah amorf. Analisis dengan XRD menunjukkan
perubahan tipe kristalin dari tipe A pada pati garut alami menjadi tipe B pada
seluruh kombinasi perlakuan, kecuali pada pati yang dihidrolisis asam. Perubahan
tipe kristalin dari tipe A menjadi tipe B mengindikasikan terjadinya rekristalisasi
pati. Pengamatan dengan SEM menunjukkan bahwa perlakuan tunggal HMT tidak
menyebabkan perubahan morfologi granula pati. Akan tetapi, ketika
dikombinasikan dengan hidrolisis asam, debranching, dan autoclaving-cooling,
HMT memicu terbentuknya struktur kristalit yang rapat. Hal ini mengkonfirmasi
dugaan bahwa HMT berperan dalam penyempurnaan kristal pati yang merupakan
salah satu mekanisme pembentukan pati resisten.
Kata kunci: heat moisture treatment, modifikasi pati, pati garut, pati resisten,
struktur pati
SUMMARY
MUTIARA PRATIWI. Structural Changes of Arrowroot Starch (Marantha
arundinacea L.) as the Impact of Multiple Treatments by Acid Hydrolysis,
Debranching, Autoclaving-cooling Cycles, and Heat Moisture Treatment (HMT).
Under the supervision of HANIFAH NURYANI LIOE as the Chairman and
DIDAH NUR FARIDAH as Advisory Committee Member.
Arrowroot starch (Marantha arundinacea L.) has been known to have the
characteristics required for producing resistant starch type III (RS3). In the previous
study, RS3 from arrowroot starch had been produced through the combination of
acid hydrolysis-debranching-autoclaving-cooling, resulting high amount of
resistant starch (39.39%), yet its yield was still lower than that of commercial
resistant starch (Novelose 330) (42.68%). The combination of modification led to
changes of starch structure which was strongly presumed to be associated with the
starch resistance. In this study, the combination of modification applied in the
previous study was combined with HMT at 121 oC and 20% moisture content, as
an additional treatment to the previous combination. This study was aimed to
investigate structural changes of arrowroot starch as the impact of acid hydrolysisdebranching-autoclaving-cooling-HMT, which have not been revealed in other
studies, particularly associated with the starch resistance after the modifications. In
this study, the structural changes of arrowroot starch were studied by using GFC,
FTIR, DSC, XRD, and SEM techniques.
Separation with GFC showed that the modifications led to changes of
molecular distribution profile of arrowroot starch, showing the decrease of
amylopectin fraction, but the increase of amylose fraction. Amylose fraction
contributes to a rapid retrogradation process as a mechanism of resistant starch
formation. Analysis with FTIR showed that in general the combination of
treatments were found to increase the amorphous region and decrease the crystalline
region. Unraveling of amylopectin double helix and/or further degradation of
amylopectin were presumed to cause the decrease of crystalline region. The
assumption was supported by the result of analysis with GFC, showing the decrease
of amylopectin fraction. Analysis with DSC showed the increase of gelatinization
temperature (To, Tp, and Tc), with the increase of temperature ranging from 2.6217.10%, and the decrease of gelatinization enthalpy (∆H) in modified starch,
compared to its native. ∆H of modified arrowroot starch was found to be ranging
from 7.11-11.20 J/g, while its native showed the ∆H of 12.52 J/g. The increase of
gelatinization temperature also suggested the increasing interaction of amyloseamylose, amylose-amylopectin, and/or amylopectin-amylopectin and the perfection
of starch crystallites. The increasing starch chain interaction of amylose-amylose,
amylose-amylopectin, and/or amylopectin-amylopectin was a mechanism of
resistant starch formation. While the decreasing ∆H of modified arrowroot starch
could reflect the disruption of amylopectin double helix. This result was in line with
the results obtained with GFC, showing the degradation of amylopectin, and also
in a good agreement with the result of FTIR, showing the increase of amorphous
region. Analysis with XRD showed that the A-type crystalline of native arrowroot
starch changed into a B-type in the most of treatment combinations applied, except
in acid hydrolyzed starch. The change of crystalline type from A- to B-type
indicated the occurrence of starch recrystallization. The SEM observation showed
that single treatment of HMT did not change the morphology of granules in shape
and size. While in combination with acid hydrolysis, debranching, and autoclavingcooling, HMT led to the formation of smaller crystalline bodies which closely
adhered to each other. This result confirmed the mechanism of HMT in inducing
the perfection of crystallites as a possible mechanism of resistant starch formation.
Keywords: arrowroot starch, heat moisture treatment, resistant starch, starch
modification, starch structure
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STRUCTURAL CHANGES OF ARROWROOT STARCH
(Marantha arundinacea L.) AS THE IMPACT OF MULTIPLE
TREATMENTS BY ACID HYDROLYSIS, DEBRANCHING,
AUTOCLAVING-COOLING CYCLES, AND
HEAT MOISTURE TREATMENT (HMT)
MUTIARA PRATIWI
Thesis
in fulfillment of the requirement for degree of
Master Science in
Study Program of Food Science
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2016
Examiner in Thesis Examination: Dr. Ir. Sukarno, MSc
PREFACE
All the praise to Allah subhanahu wa ta’ala for all His grace, mercy, and
blessing through all the way to complete my Master study. This study was intended
as requirement to graduate in Master degree in Bogor Agricultural University. This
study entitled “Structural Changes of Arrowroot Starch (Marantha arundinacea L.)
as the Impact of Multiple Treatments by Acid Hydrolysis, Debranching,
Autoclaving-cooling Cycles, and Heat Moisture Treatment (HMT)” was conducted
in Bogor Agricultural University from January-August 2015.
The author would like to express the deepest gratitude to beloved parents and
brother in appreciation of their love and encouragements, especially during the
completion of my Master study. Nothing can reply their life time support. My
sincere appreciation and deepest gratitude is also expressed to the advisors, Dr. Ir.
Hanifah Nuryani Lioe, MSi and Dr. Ir. Didah Nur Faridah, MSi for their intense
supervision and constant support. The valuable help of constructive comments and
suggestions throughout the experimental and thesis work have contributed to the
success of this research. The author also would like to express a sincere gratitude
to Dr. Ir. Sukarno, MSc as the examiner in thesis examination, for his time,
suggestion, and correction for the improvement of this thesis. Sincere thanks is also
expressed to Indonesian Directorate General of Higher Education (DIKTI) for
supporting the study through fresh graduate scholarship program and for supporting
the research through funding incentive of Penelitian Unggulan Dasar (PUD)
program 2014.
A sincere gratitude is also expressed to Dr. Ir. Dahrul Syah, MSc as the Dean
of Postgraduate School and Prof. Dr. Ir. Ratih Dewanti, MSc as the Head of Food
Science Program for the support and help towards my postgraduate affairs. The
author also would like to express her thanks to all the laboratory technicians of
Department of Food Science and Technology: Pak Taufik, Pak Sobirin, Mbak Ari,
Pak Rojak, Mas Edi, Mbak Nurul, Mbak Irin, and Pak Yahya for the technical help
during my research. The appreciation also goes to Mbak Ririn as the laboratory
technician of the department for the guidance and suggestion for the
chromatography column preparation. Special thanks to Fathma, Fitria, Mbak Ratna,
and Anand for being a cooperative partner in conducting the research. The author
also thanks her colleagues, especially Nesya, Silvie, and Florentina for the support,
advice, and help during the study and research completion. Last but not least,
sincere gratitude also goes to Rio for his constant supports and advices from the
beginning until this stage.
Hopefully this thesis could be useful for the readers and contribute in a wider
scope of food science realm.
Bogor, February 2016
Mutiara Pratiwi
TABLE OF CONTENT
LIST OF TABLES
LIST OF FIGURES
LIST OF APPENDIXES
1 INTRODUCTION
Background
Potential
Objectives
Significance
Hypothesis
xviii
xviii
xviii
1
1
3
3
4
4
2 LITERATURE STUDY
Arrowroot (Marantha arundinacea L.)
Starch
Resistant starch
Starch modification
Acid hydrolysis
Debranching
Autoclaving-cooling
Heat Moisture Treatment (HMT)
Combination of modification to produce resistant starch
Changes of molecular structure in starch modification
Gel Filration Chromatography (GFC) profile
Fourier Transform Infra-Red (FTIR) spectra profile
Differential Scanning Calorimetry (DSC) profile
X-ray Diffraction (XRD) pattern
Scanning Electron Microscopy (SEM) microstructure profile
4
4
5
8
9
10
11
12
13
13
14
14
16
18
21
22
3 RESEARCH METHODOLOGY
Time and place
Material and instruments
Methods
23
23
24
24
4 RESULT AND DISCUSSION
Profiles of starch molecular distribution analyzed by GFC
Changes of crystalline and amorphous regions analyzed by FTIR
Gelatinization properties analyzed by DSC
Crystallinity of starch analyzed by XRD
Morphology of starch granules analyzed by SEM
28
28
33
36
39
43
5 CONCLUSION AND RECOMMENDATION
Conclusion
Recommendation
Acknowledgement
46
46
47
47
BIBLIOGRAPHY
APPENDIXES
AUTHOR BIOGRAPHY
48
59
65
LIST OF TABLES
1 Studies of starch molecular distribution by using Gel Filtration
Chromatography (GFC) technique
2 Studies of starch structure by using Fourier Transform Infra-Red
(FTIR) spectroscopy
3 Thermal studies of starches by using Differential Scanning
Calorimetry (DSC) technique
4 Studies of crystallinity in various starches by using X-ray
Diffraction (XRD)
5 Studies of microstructure in various starches by using Scanning
Electron Microscopy (SEM)
6 Condition for analysis of molecular distribution profile of arrowroot
starch by using Gel Filtration Chromatography (GFC)
7 Gelatinization properties of native and modified arrowroot starch
8 X-ray diffraction pattern of native and modified arrowroot starch
15
17
19
21
23
26
36
40
LIST OF FIGURES
1 Arrowroot plant Marantha arundinacea L. (a) and arrowroot tuber
Marantha arundinacea L. (b)
2 Schematic diagram of starch granule architecture
3 Cluster model of amylopectin
4 Typical DSC thermogram with the To, Tp, Tc, and ∆H values
5 GFC profiles of arrowroot starch using Sephacryl S-HR 400 column
(2.6 cm i.d. x 90 cm)
6 GFC profiles of arrowroot starch using Sephadex G-50 column
(2.6 cm i.d. x 135 cm)
7 Changes of crystalline and amorphous regions of native and
modified arrowroot starches analyzed by Fourier Transform
Infra-Red (FTIR) spectroscopy
8 DSC gelatinization curve of native and modified arrowroot starch
9 X-ray diffraction patterns of native and modified arrowroot starch
10 Scanning Electron Microscopy (SEM) micrographs of native and
modified arrowroot starches: 1. native; 2. AH; 3. AC; 4. HMT. (a)
magnification = 500x; (b) magnification = 1500x
11 Scanning Electron Microscopy (SEM) micrographs of native and
modified arrowroot starches: 1. DE-AC; 2. AH-DE-AC; 3. AH-DEAC-HMT (a) magnification = 500x; (b) magnification = 1500x
5
7
7
20
29
31
34
37
41
45
46
LIST OF APPENDIXES
1
2
3
4
5
6
Reagents for making acetate buffer pH 5.2 solution
Reagents for making mobile phase solution
Reagents for total carbohydrate analysis
Reagents for blue value analysis
Sephadex G-50 column preparation
Sephacryl S-HR 400 column preparation
59
60
61
62
63
64
1 INTRODUCTION
Background
Resistant starch (RS) has been known for its potential health benefits and
functional properties (Sajilata et al. 2006). Resistant starch is defined as the fraction
of starch which is not hydrolyzed to D-glucose in the small intestine within 120 min
of being consumed, but which is fermented by bacteria in the colon to short chain
fatty acids and gases (Fuentes-Zaragoza et al. 2010, Topping and Clifton 2001).
Resistant starch is further divided into five distinct classes: RS1, RS2, RS3, RS4
and RS5. RS1 is physically inaccessible starch, which is entrapped within whole or
partly milled grains or seeds. RS2 belongs to some types of raw starch granules.
RS3 is retrograded starch (either processed from unmodified starch or resulting
from food processing applications). RS4 belongs to starches which are chemically
modified to obtain resistance to enzymatic digestion (Ratnayake dan Jackson 2008,
Sanz et al. 2009). RS5 belongs to resistant starch formed as a single-helix complex
of lipid-amylose (Ratnayake and Jackson 2008, Sanz et al. 2009, Fuentes-Zaragoza
et al. 2010, Ai et al. 2013).
Arrowroot starch (Marantha arundinacea L.) has the characteristics which
are suitable for producing RS3. Arrowroot has an A-type crystalline and it has
amylopectin, containing branching chains with degree of polymerization (DP) of
about 9-30 found in a high amount (up to 96%) (Srichuwong 2006). Arrowroot
starch also has a higher density in the helical structure region (Wang et al. 1998),
higher proportion of short branch chain of amylopectin (Hizukuri et al. 1983), and
greater amount of chains found in each cluster, compared to B-type crystalline
(Takeda et al. 2003). Arrowroot starch has been known to contain observable
amount of amylose, reaching 29.39% (Aprianita 2010), which contributes to a rapid
retrogradation. Besides, arrowroot starch also has low content of fat (0.68%) which
can minimize the possibility of fat-amylose formation that may reduce
retrogradation to some extent. This will enable higher amount of amylose which
can bind with another amylose or amylopectin during retrogradation (Faridah 2011).
RS3 can be obtained through heating and cooling cycles of starch which
respectively involves a gelatinization and retrogradation of starch. Acid
modification and enzymatic debranching of starch are two methods known to
increase short chain amylose which contributes in more rapid retrogradation
(Sandoval et al. 2008, Milasinovic et al. 2010).
Faridah (2011) combined starch modification consisting of acid hydrolysis,
debranching, and autoclaving-cooling to optimize formation of RS3. The
combination applied was found to produce high amount of RS3, up to 39.39%, yet
its yield was still lower than that of the commercial one, Novelose 330, reaching
42.68% (Faridah 2011). Heat Moisture Treatment (HMT) has been known as an
effective technique to increase resistant starch from various sources of starch such
as mung bean, corn, pea, and lentil starch (Chung et al. 2009, Li and Gao 2010). In
this research, HMT was applied to increase RS3 from arrowroot starch. HMT is a
physical modification technique that involves treatment of starch granules at low
moisture levels (