Hidrolisat Kolagen Dari Kulit Ikan Gabus (Channa Striata) Yang Menunjukkan Aktivitas Penghambatan Ace Antioksidan.
i
ACE INHIBITION AND ANTIOXIDANT ACTIVITIES OF
COLLAGEN HYDROLYSATE FROM SKIN OF
SNAKEHEAD FISH (Channa striata)
SILVIE FEBRIANA WIBAWA
POSTGRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2015
ii
iii
STATEMENT LETTER OF THESIS AND SOURCE OF
INFORMATION
I declare that this thesis entitled ACE Inhibition and Antioxidant Activities
of Collagen Hydrolysate from Skin of Snakehead Fish (Channa striata) is my
own work with guidance of 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derived and quoted from published and unpublished works of other authors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, September 2015
Silvie Febriana Wibawa
F251140666
iv
SUMMARY
SILVIE FEBRIANA WIBAWA. ACE Inhibition and Antioxidant Activities of
Collagen Hydrolysate from Skin of Snakehead Fish (Channa striata). Supervised
by MAGGY THENAWIDJAJA SUHARTONO and PUSPO EDI GIRIWONO.
Snakehead fish (Channa striata) is one of fresh water fish consumed by
Asian people to maintain good health due to its high content of protein, essential
fatty acids and minerals. The skin of this fish which contains collagen
(approximatelly 16.57 %) is a byproduct of albumin supplement production. Thus,
it is potential to become a collagen source which is used widely in food and other
needs including for producing bioactive peptides. The aims of this research were
to produce peptides with Angiotensin-I Converting Enzyme (ACE) inhibition and
antioxidant activities from snakehead fish by using collagenase from local
collagenolytic bacteria such as Bacillus licheniformis F11.4 (isolated from shrimp
waste in Palembang) and Stenotrophomonas sp. (isolated from red oncom in
Bogor).
The protein concentration of acid soluble collagen extracted from skin of
snakehead fish skin was 0.4 mg/mL. SDS-PAGE analysis revealed that this
collagen which was categorized of type I collagen contained -, α1-, αβ-chain
with molecular weights of 267, 152, 136 kDa constitutively. Crude collagenase
from both bacteria used produced peptides with molecular weights of 18-267 kDa.
Semi-purified Stenotrophomonas sp. collagenase generated peptides with lower
molecular weights (14-152 kDa) than peptides generated from semi-purified
Bacillus licheniformis F11.4 collagenase (15-267 kDa). Stenotrophomonas sp.
collagenase was able to hydrolyze -chain of collagen. Collagen hydrolysates had
better ACE inhibition and antioxidant activities that collagen. Stenotrophomonas
sp. collagenase produced collagen peptides that have higher ACE inhibition and
antioxidant activities than peptides generated by Bacillus licheniformis F11.4
Collagenase. Collagen hydrolysate generated from Stenotrophomonas sp. crude
collagenase after 90 minutes of incubation had the highest ACE inhibitory (13.32
% inhibition/µg protein) effect and reducing power among the other collagen
hydrolysate. In addition, collagen hydrolysate generated from semi-purified
Stenotrophomonas sp. collagenase after 90 minutes of incubation showed the
highest ability to scavenge free radical such as DPPH (73.22 % inhibition/50µg
protein). Based on this data, this collagen hydrolysate may become potential for
developing medicine or functional food for the alleviation of hypertension.
Keywords: snakehead fish, acid soluble collagen, collagen hydrolysate, ACE
inhibitor, antioxidant
v
RINGKASAN
SILVIE FEBRIANA WIBAWA. Hidrolisat Kolagen dari Kulit Ikan Gabus
(Channa striata) yang Menunjukkan Aktivitas Penghambatan ACE Antioksidan.
Dibimbing oleh MAGGY THENAWIDJAJA SUHARTONO dan PUSPO EDI
GIRIWONO.
Ikan gabus merupakan salah satu ikan air tawar yang sering dikonsumsi oleh
orang Asia untuk menjaga kesehatan akibat tingginya kandungan protein, asam
lemak esensial, dan mineral pada ikan tersebut. Kulit ikan gabus yang
mengandung sekitar 16.57 % kolagen merupakan limbah dari produksi suplemen
albumin. Oleh karena itu, kulit ikan gabus berpotensi menjadi sumber kolagen
sehingga kolagennya dapat digunakan di industri pangan maupun non-pangan
termasuk untuk memproduksi peptida bioaktif. Tujuan dari penelitian ini adalah
untuk memproduksi peptida dengan aktivitas penghambatan Angiotensin-I
Converting Enzyme (ACE) dan antioksidan dari ikan gabus dengan menggunakan
kolagenase dari Bacillus licheniformis F11.4 (diisolasi dari limbah kulit udang di
Palembang) dan Stenotrophomonas sp. (diisolasi dari oncom merah di Bogor).
Konsentrasi protein dari kolagen larut asam yang diekstrak dari kulit ikan
gabus adalah 0.4 mg/mL. Analisis SDS-PAGE menunjukkan bahwa kolagen yang
ini dapat dikategorikan menjadi kolagen tipe I ini mengandung rantai , α1, αβ
dengan berat molekul 267, 152, 136 kDa berturut-turut. Kolagenase kasar dari
kedua bakteri yang digunakan mampu menghasilkan peptida dengan berat
molekul 18-267 kDa. Kolagenase semi-murni dari Stenotrophomonas sp.
Menghasilkan peptida dengan berat molekul yang lebih rendah (14-152 kDa)
dibandingkan peptida yang dihasilkan oleh kolagenase semi-murni dari Bacillus
licheniformis F11.4 (15-267 kDa). Kolagenase Stenotrophomonas sp. Mampu
menghidrolisis rantai pada kolagen. Kolagen hidrolisat menunjukkan aktivitas
penghambatan ACE dan antioksidan yang lebih baik daripada kolagen.
Kolagenase dari Stenotrophomonas sp. Mampu memproduksi peptida kolagen
dengan aktivitas penghambatan ACE dan antioksidan yang lebih tinggi
dibandingkan peptida yang dihasilkan oleh kolagenase dari Bacillus licheniformis
F11.4. Hidrolisat kolagen yang dihasilkan oleh kolagenase kasar dari
Stenotrophomonas sp. setelah diinkubasi selama 90 menit inkubasi menunjukkan
aktivitas penghambatan ACE (13.32 % penghambatan/µg protein) dan kekuatan
mereduksi yang paling baik diantara hidrolisat kolagen lainnya. Selain itu,
hidrolisat kolagen yang diproduksi oleh kolagenase semi-murni dari
Stenotrophomonas sp. setelah diinkubasi selama 90 menit menunjukkan
kemampuan menetralkan radikal bebas DPPH yang paling tinggi (73.22 %
penghambatan/50µg protein). Berdasarkan data penelitian ini, kolagen hidrolisat
ini berpotensi untuk dikembangkan menjadi obat atau pangan fungsional untuk
penderita penyakit darah tinggi.
Kata kunci: ikan gabus, kolagen larut asam, hidrolisat kolagen, penghambatan
ACE, antioksidan
vi
© Hak Cipta Milik IPB, Tahun 2015
Hak Cipta Dilindungi Undang-Undang
Dilarang mengutip sebagian atau seluruh karya tulis ini tanpa mencantumkan
atau menyebutkan sumbernya. Pengutipan hanya untuk kepentingan pendidikan,
penelitian, penulisan karya ilmiah, penyusunan laporan, penulisan kritik, atau
tinjauan suatu masalah; dan pengutipan tersebut tidak merugikan kepentingan
IPB
Dilarang mengumumkan dan memperbanyak sebagian atau seluruh karya tulis ini
dalam bentuk apa pun tanpa izin IPB
vii
ACE INHIBITION AND ANTIOXIDANT ACTIVITIES OF
COLLAGEN HYDROLYSATE FROM SKIN OF
SNAKEHEAD FISH (Channa striata)
SILVIE FEBRIANA WIBAWA
Thesis
in the partial fulfillment of the requirement for degree of
Master of Science
at
Food Science Master Program
POSTGRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2015
viii
Examiner in Thesis Examination:
Dr. Siti Nurjanah, STP, MSi
ix
Title
: ACE Inhibition and Antioxidant Activities of Collagen Hydrolysate
from Skin of Snakehead Fish (Channa striata)
Name
: Silvie Febriana Wibawa
Student ID : F251140666
Approved by
Advisory Committee
Date of Thesis Examination:
September 17th, 2015
Date of Graduation:
x
PREFACE
Praise to The Lord Jesus Christ for His mercy, blessings, and guidance
throughtout the research and finishing this thesis. The research entitled “ACE
inhibition and antioxidant activities of collagen hydrolysate from skin of
snakehead fish (Channa striata)” was carried out in Bogor Agricultural University
from September 2014 until April 2015.
By completion of this research and thesis, the author would like to express
great appreciation and sincere thanks to Prof. Dr. Ir. Maggy T. Suhartono and
Puspo Edi Giriwono, PhD as supervisors for their guidances, help, and advices
during completing this research and thesis. The author would like to express her
thanks to her examiner in thesis examination, Dr. Siti Nurjana STP, MSi, for her
time, corrections, and input in improving this thesis.
Sincere thankes is also expressed to Dean of Bogor Agricultural University
Postgraduate School and his staff, Head of Food Science Master Program and her
staff, Bu Ika Malikha as laboratory technician in Laboratory of Microbiology and
Biochemistry PAU IPB, and friends from IPN 2013 and 2014. Thanks to Nesya,
Mutiara Pratiwi, Florentina, Kak Diana, Mbak Ino, Kak Novan, and Kak Wenny
for the support, advice, and help during the study and completing this research.
Thanks to DIKTI for supporting the study through fresh graduate scholarship
program.
Last but not least, I would like to say a big thanks to her parents and sister
for their support during the study of Food Science, the reseach, and thesis
completion. Hopefully this thesis is useful for the readers and gives a contribution
in food science development.
Bogor, September 2015
Silvie Febriana Wibawa
xi
TABLE OF CONTENT
LIST OF TABLES
xii
LIST OF FIGURES
xiii
LIST OF APPENDICES
xiv
1 INTRODUCTION
Background
Problems
Objectives
Significance
1
1
2
2
2
2 LITERATURE STUDY
Snakehead fish (Channa striata)
Collagen
Collagenase and Collagenolytic Bacteria
Bioactive Peptides
3
3
4
6
8
3 RESEARCH METHODOLOGY
Time and Place
Material and Instruments
Methods
Statistical Analysis
13
13
13
13
18
4 RESULT AND DISCUSSION
Collagen and Collagen Hydrolysate Profile
Inhibition of Angiotensin-I Converting Enzyme (ACE)
Antioxidant Activity
19
19
22
24
5 CONCLUSION AND RECOMMENDATION
Conclusion
Recommendation
Acknowledgement
28
28
28
28
BIBLIOGRAPHY
29
APPENDICES
36
AUTHOR BIOGRAPHY
48
xii
LIST OF TABLES
1. Table 1 Peptides with ACE inhibition derived from fish
2. Table 2 Peptides with antioxidant activities derived from fish
3. Table 3 Protein concentration and percentage of peptides produced of
10
12
collagen and its hydrolysates
4. Table 4 ACE inhibition (%) by collagen hydrolysates using crude
collagenase (C) and semi-purified collagenase (SP) from Bacillus
licheniformis (F11.4) and Stenotrophomonas sp. (Isolate 11), n=2,
different letters indicate significance at p
ACE INHIBITION AND ANTIOXIDANT ACTIVITIES OF
COLLAGEN HYDROLYSATE FROM SKIN OF
SNAKEHEAD FISH (Channa striata)
SILVIE FEBRIANA WIBAWA
POSTGRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2015
ii
iii
STATEMENT LETTER OF THESIS AND SOURCE OF
INFORMATION
I declare that this thesis entitled ACE Inhibition and Antioxidant Activities
of Collagen Hydrolysate from Skin of Snakehead Fish (Channa striata) is my
own work with guidance of 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, September 2015
Silvie Febriana Wibawa
F251140666
iv
SUMMARY
SILVIE FEBRIANA WIBAWA. ACE Inhibition and Antioxidant Activities of
Collagen Hydrolysate from Skin of Snakehead Fish (Channa striata). Supervised
by MAGGY THENAWIDJAJA SUHARTONO and PUSPO EDI GIRIWONO.
Snakehead fish (Channa striata) is one of fresh water fish consumed by
Asian people to maintain good health due to its high content of protein, essential
fatty acids and minerals. The skin of this fish which contains collagen
(approximatelly 16.57 %) is a byproduct of albumin supplement production. Thus,
it is potential to become a collagen source which is used widely in food and other
needs including for producing bioactive peptides. The aims of this research were
to produce peptides with Angiotensin-I Converting Enzyme (ACE) inhibition and
antioxidant activities from snakehead fish by using collagenase from local
collagenolytic bacteria such as Bacillus licheniformis F11.4 (isolated from shrimp
waste in Palembang) and Stenotrophomonas sp. (isolated from red oncom in
Bogor).
The protein concentration of acid soluble collagen extracted from skin of
snakehead fish skin was 0.4 mg/mL. SDS-PAGE analysis revealed that this
collagen which was categorized of type I collagen contained -, α1-, αβ-chain
with molecular weights of 267, 152, 136 kDa constitutively. Crude collagenase
from both bacteria used produced peptides with molecular weights of 18-267 kDa.
Semi-purified Stenotrophomonas sp. collagenase generated peptides with lower
molecular weights (14-152 kDa) than peptides generated from semi-purified
Bacillus licheniformis F11.4 collagenase (15-267 kDa). Stenotrophomonas sp.
collagenase was able to hydrolyze -chain of collagen. Collagen hydrolysates had
better ACE inhibition and antioxidant activities that collagen. Stenotrophomonas
sp. collagenase produced collagen peptides that have higher ACE inhibition and
antioxidant activities than peptides generated by Bacillus licheniformis F11.4
Collagenase. Collagen hydrolysate generated from Stenotrophomonas sp. crude
collagenase after 90 minutes of incubation had the highest ACE inhibitory (13.32
% inhibition/µg protein) effect and reducing power among the other collagen
hydrolysate. In addition, collagen hydrolysate generated from semi-purified
Stenotrophomonas sp. collagenase after 90 minutes of incubation showed the
highest ability to scavenge free radical such as DPPH (73.22 % inhibition/50µg
protein). Based on this data, this collagen hydrolysate may become potential for
developing medicine or functional food for the alleviation of hypertension.
Keywords: snakehead fish, acid soluble collagen, collagen hydrolysate, ACE
inhibitor, antioxidant
v
RINGKASAN
SILVIE FEBRIANA WIBAWA. Hidrolisat Kolagen dari Kulit Ikan Gabus
(Channa striata) yang Menunjukkan Aktivitas Penghambatan ACE Antioksidan.
Dibimbing oleh MAGGY THENAWIDJAJA SUHARTONO dan PUSPO EDI
GIRIWONO.
Ikan gabus merupakan salah satu ikan air tawar yang sering dikonsumsi oleh
orang Asia untuk menjaga kesehatan akibat tingginya kandungan protein, asam
lemak esensial, dan mineral pada ikan tersebut. Kulit ikan gabus yang
mengandung sekitar 16.57 % kolagen merupakan limbah dari produksi suplemen
albumin. Oleh karena itu, kulit ikan gabus berpotensi menjadi sumber kolagen
sehingga kolagennya dapat digunakan di industri pangan maupun non-pangan
termasuk untuk memproduksi peptida bioaktif. Tujuan dari penelitian ini adalah
untuk memproduksi peptida dengan aktivitas penghambatan Angiotensin-I
Converting Enzyme (ACE) dan antioksidan dari ikan gabus dengan menggunakan
kolagenase dari Bacillus licheniformis F11.4 (diisolasi dari limbah kulit udang di
Palembang) dan Stenotrophomonas sp. (diisolasi dari oncom merah di Bogor).
Konsentrasi protein dari kolagen larut asam yang diekstrak dari kulit ikan
gabus adalah 0.4 mg/mL. Analisis SDS-PAGE menunjukkan bahwa kolagen yang
ini dapat dikategorikan menjadi kolagen tipe I ini mengandung rantai , α1, αβ
dengan berat molekul 267, 152, 136 kDa berturut-turut. Kolagenase kasar dari
kedua bakteri yang digunakan mampu menghasilkan peptida dengan berat
molekul 18-267 kDa. Kolagenase semi-murni dari Stenotrophomonas sp.
Menghasilkan peptida dengan berat molekul yang lebih rendah (14-152 kDa)
dibandingkan peptida yang dihasilkan oleh kolagenase semi-murni dari Bacillus
licheniformis F11.4 (15-267 kDa). Kolagenase Stenotrophomonas sp. Mampu
menghidrolisis rantai pada kolagen. Kolagen hidrolisat menunjukkan aktivitas
penghambatan ACE dan antioksidan yang lebih baik daripada kolagen.
Kolagenase dari Stenotrophomonas sp. Mampu memproduksi peptida kolagen
dengan aktivitas penghambatan ACE dan antioksidan yang lebih tinggi
dibandingkan peptida yang dihasilkan oleh kolagenase dari Bacillus licheniformis
F11.4. Hidrolisat kolagen yang dihasilkan oleh kolagenase kasar dari
Stenotrophomonas sp. setelah diinkubasi selama 90 menit inkubasi menunjukkan
aktivitas penghambatan ACE (13.32 % penghambatan/µg protein) dan kekuatan
mereduksi yang paling baik diantara hidrolisat kolagen lainnya. Selain itu,
hidrolisat kolagen yang diproduksi oleh kolagenase semi-murni dari
Stenotrophomonas sp. setelah diinkubasi selama 90 menit menunjukkan
kemampuan menetralkan radikal bebas DPPH yang paling tinggi (73.22 %
penghambatan/50µg protein). Berdasarkan data penelitian ini, kolagen hidrolisat
ini berpotensi untuk dikembangkan menjadi obat atau pangan fungsional untuk
penderita penyakit darah tinggi.
Kata kunci: ikan gabus, kolagen larut asam, hidrolisat kolagen, penghambatan
ACE, antioksidan
vi
© Hak Cipta Milik IPB, Tahun 2015
Hak Cipta Dilindungi Undang-Undang
Dilarang mengutip sebagian atau seluruh karya tulis ini tanpa mencantumkan
atau menyebutkan sumbernya. Pengutipan hanya untuk kepentingan pendidikan,
penelitian, penulisan karya ilmiah, penyusunan laporan, penulisan kritik, atau
tinjauan suatu masalah; dan pengutipan tersebut tidak merugikan kepentingan
IPB
Dilarang mengumumkan dan memperbanyak sebagian atau seluruh karya tulis ini
dalam bentuk apa pun tanpa izin IPB
vii
ACE INHIBITION AND ANTIOXIDANT ACTIVITIES OF
COLLAGEN HYDROLYSATE FROM SKIN OF
SNAKEHEAD FISH (Channa striata)
SILVIE FEBRIANA WIBAWA
Thesis
in the partial fulfillment of the requirement for degree of
Master of Science
at
Food Science Master Program
POSTGRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2015
viii
Examiner in Thesis Examination:
Dr. Siti Nurjanah, STP, MSi
ix
Title
: ACE Inhibition and Antioxidant Activities of Collagen Hydrolysate
from Skin of Snakehead Fish (Channa striata)
Name
: Silvie Febriana Wibawa
Student ID : F251140666
Approved by
Advisory Committee
Date of Thesis Examination:
September 17th, 2015
Date of Graduation:
x
PREFACE
Praise to The Lord Jesus Christ for His mercy, blessings, and guidance
throughtout the research and finishing this thesis. The research entitled “ACE
inhibition and antioxidant activities of collagen hydrolysate from skin of
snakehead fish (Channa striata)” was carried out in Bogor Agricultural University
from September 2014 until April 2015.
By completion of this research and thesis, the author would like to express
great appreciation and sincere thanks to Prof. Dr. Ir. Maggy T. Suhartono and
Puspo Edi Giriwono, PhD as supervisors for their guidances, help, and advices
during completing this research and thesis. The author would like to express her
thanks to her examiner in thesis examination, Dr. Siti Nurjana STP, MSi, for her
time, corrections, and input in improving this thesis.
Sincere thankes is also expressed to Dean of Bogor Agricultural University
Postgraduate School and his staff, Head of Food Science Master Program and her
staff, Bu Ika Malikha as laboratory technician in Laboratory of Microbiology and
Biochemistry PAU IPB, and friends from IPN 2013 and 2014. Thanks to Nesya,
Mutiara Pratiwi, Florentina, Kak Diana, Mbak Ino, Kak Novan, and Kak Wenny
for the support, advice, and help during the study and completing this research.
Thanks to DIKTI for supporting the study through fresh graduate scholarship
program.
Last but not least, I would like to say a big thanks to her parents and sister
for their support during the study of Food Science, the reseach, and thesis
completion. Hopefully this thesis is useful for the readers and gives a contribution
in food science development.
Bogor, September 2015
Silvie Febriana Wibawa
xi
TABLE OF CONTENT
LIST OF TABLES
xii
LIST OF FIGURES
xiii
LIST OF APPENDICES
xiv
1 INTRODUCTION
Background
Problems
Objectives
Significance
1
1
2
2
2
2 LITERATURE STUDY
Snakehead fish (Channa striata)
Collagen
Collagenase and Collagenolytic Bacteria
Bioactive Peptides
3
3
4
6
8
3 RESEARCH METHODOLOGY
Time and Place
Material and Instruments
Methods
Statistical Analysis
13
13
13
13
18
4 RESULT AND DISCUSSION
Collagen and Collagen Hydrolysate Profile
Inhibition of Angiotensin-I Converting Enzyme (ACE)
Antioxidant Activity
19
19
22
24
5 CONCLUSION AND RECOMMENDATION
Conclusion
Recommendation
Acknowledgement
28
28
28
28
BIBLIOGRAPHY
29
APPENDICES
36
AUTHOR BIOGRAPHY
48
xii
LIST OF TABLES
1. Table 1 Peptides with ACE inhibition derived from fish
2. Table 2 Peptides with antioxidant activities derived from fish
3. Table 3 Protein concentration and percentage of peptides produced of
10
12
collagen and its hydrolysates
4. Table 4 ACE inhibition (%) by collagen hydrolysates using crude
collagenase (C) and semi-purified collagenase (SP) from Bacillus
licheniformis (F11.4) and Stenotrophomonas sp. (Isolate 11), n=2,
different letters indicate significance at p