Short chain fatty acid profile during in vitro fermentation of Eubacterium rectale on sukuh sweet potato type-iii resistant starch media

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TYPE-III R

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bacterium

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F2407004

RICULTUR

CULTURA

BOGOR

2011

OFILE DU

rectale

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46

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N SUKUH

RCH MED

CHNOLOG

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SHORT CHAIN FATTY ACID PROFILE DURING IN VITRO

FERMENTATION OF

Eubacterium rectale

ON SUKUH SWEET POTATO

TYPE-III RESISTANT STARCH MEDIA

Septiana Iswani and Maggy Thenawidjaja Suhartono

Department of Food Science and Technology, Faculty of Agricultural Technology, Bogor Agricultural University, IPB Dramaga Campus,

PO BOX 220, Bogor, West Java, Indonesia

Phone: +62 856 8313810, e-mail: septiana.iswani@gmail.com

ABSTRACT

Type-III resistant starch is an indigestible starch fraction which is mainly retrograded amylose formed during cooling of gelatinized starch which will further undergo bacterial fermentation in the colon. The main fermentation products are the short chain fatty acid (SCFA) acetate, propionate, and butyrate. Butyrate has been implicated in providing protection against cancer; it is also the preferred energy substrate of the colonocytes. The objective of this research was to study short chain fatty acid (acetate, propionate, and butyrate) profile during in vitro fermentation (370C, anaerobic condition) of Eubacterium rectale 17629 in medium containing Sukuh sweet potato type-III resistant starch. The result showed that amylose content of Sukuh sweet potato starch was 29.35 ± 0.67%. In this study, pullulanase enzyme (177,222.11 IU/ml) at 5% starch weigh was used to hydrolyze the α -1,6-glucosidic bonds of amylopectin and the incubation was carried for 24 hours at 500C, stored at 40C for 24 hour, and then dried by spray drier to produce type-III resistant starch. Yield of product and resistant starch content were 11.38 ± 0.33% and 30.83 ± 2.44%. Fermentation was carried out using 20 g/L Sukuh type-III resistant starch and 5 g/L glucose as fermentation substrate for Eubacterium rectale 17629’s growth. Sampling was done at 6, 12, 24, 36, and 48 hours to determine bacterial growth (turbidimetry method), pH value, and SCFA production in Experiment I. The analysis of fermentation showed that the bacterial growth increased, followed by pH reduction as the bacteria started to produce SCFA. Molar ratio of acetate, propionate, butyrate were 0 mM : 25.17 mM :50.97 mM ( 0:1:2) at 36 hours fermentation. Experiment II was designed to increase SCFA (butyrate) production. It was carried out using 10 g/L RS3 and 5g/L glucose as substrate fermentation. Molar ratio of SCFA (acetate:propionate:butyrate) were 500.40 mM: 476.28 mM: 477.97mM (1:1:1) at 36 hours and 215.21 mM: 281.10mM: 343.12 mM (1:1.3:1.6) at 48 hours fermentation. This result indicated that fermentation using 10 g/L RS3 and 5 g/L glucose at 36 hours incubation produce the highest butyrate which is potential for cancer prevention.

Keyword: Resistant starch, short chain fatty acid, butyrate, Sukuh, Eubacterium rectale, sweet potato, fermentation

Septiana Iswani. F24070046. Short Chain Fatty Acid Profile during In Vitro Fermentation of Eubacterium rectale on Sukuh Sweet Potato Type-III Resistant Starch. Supervised by Maggy Thenawidjaja Suhartono. 2011.

SUMMARY

Colorectal cancer (CRC) is the fourth most common cause of cancer-related mortality in the world. Within Europe, North America, Australia, and New Zealand, it is the second most common cancer after lung or breast and in general, the incidence and mortality of the disease are increasing. Evidence suggests that diet plays a significant role in the aetiology of CRC. This condition leads to pursue basic research on the development of type-III resistant starch as one of the functional food ingredient with potential cancer prevention ability.

Type-III resistant starch represents retrograded starch, which form crystalline structure of amylose through hydrogen bond in double helices structure. It is not digested by human starch degrading enzyme and will thus undergo bacterial fermentation in the colon. The main fermentation products are short chain fatty acid (SCFA) acetate, propionate, and butyrate. Butyrate plays a key role in the energy metabolism of the colonic epithelial cells and thought to be important in the maintenance of colonic health in humans, that is potential of inhibiting cancer cell.

The objective of this research was to study short chain fatty acid profile during in vitro fermentation (370C, anaerobic condition) of Eubacterium rectale 17629 on Sukuh sweet potato type-III resistant starch media. This research was divided into two steps: (1) production of resistant starch from Sukuh’s sweet potato (chemical characteristic of Sukuh’s starch, preparation enzyme, production process, and analysis of product RS content) and (2) in vitro fermentation process by Eubacterium rectale 17629 (medium preparation, fermentation process, and SCFA profile analysis of fermentation product)

In this study, type-III resistant starch was produced from Sukuh sweet potato starch. Sukuh sweet potato was chosen because it has high productivity (25-30 ton/ha) and made it potential to be developed for food industries. Sukuh’s sweet potato starch solution (10%) was gelatinized by heating

for 30 minutes at 1200C. Afterwards, the starch solution was hydrolyzed by incubation with

pullulanase (Promozyme®) debranching enzyme (177,222.11 IU/ml) at 5% (w/w) starch for 24 hours at 500C. Pullulanase has been applied to produce a simple linier, low molecular-weight and re-crystallizable polymer chains by hydrolyzing α-1,6-glucosidic bonds. The debranched starch was then stored at 40_{C for 24 hours to induce retrogradation then dried by spray drier and yield the type III} resistant starch.

Proximate analysis showed that Sukuh starch extract has moisture content of 16.45%, ash content of 0.20%, crude fat content of 0.65%, protein content of 0.69%, and carbohydrate content of 82.01%. The low content of protein and fiber reflected the purity of starch. Amylose content of Sukuh starch was 29.35 ± 0.67%. Yield of resistant starch was 11.36 ± 0.36% and the RS content was 30.83 ± 2.44%.

In vitro fermentation of RS3 was divided into 2 experiments. Experiment I was design to evaluate the growth of Eubacterium rectale 17629, SCFA production pattern and optimum time that produce the highest butyrate concentration. Glucose (5 g/l) (0.5%) and RS3 from Sukuh sweet potato at 20 g/l (2%) were added into medium. Fermentation reached the highest absorbance at 660 nm (0.193) at 36 hours incubation and at 48 hours incubation (0.198). It showed that Eubacterium rectale 17629 could use resistant starch substrate from Sukuh sweet potato as the energy source to grow. The

lowest pH value of 4.16 was achieved at 48 hours fermentation. Based on SCFA profile of Experiment I, it could be concluded that the optimum time for production of highest SCFA (mainly butyrate) by in vitro fermentation of Sukuh type-III resistant starch was 36 hours incubation, where the molar ratio of acetate: propionate: butyrate were 0.0 mM: 25.17 mM: 50.97mM (0:1:2).

In experiment II, fermentation was carried out using glucose at 5 g/L and RS3 at 10 g/l (1%). Incubation was performed at 370_{C for 36 hours and 48 hours. Experiment II showed that molar ratio} of acetate: propionate: butyrate were 500.40 mM: 476.28 mM: 477.97 mM (1: 1: 1). In Experiment II, acetate was the most dominant of SCFA compare with propionate and butyrate while in Experiment I butyrate was the most dominant among two others. This result indicated that fermentation using 10 g/L RS3 and 5 g/L glucose at 36 hours incubation produce the highest butyrate which provide potential cancer prevention.

SHORT CHAIN FATTY ACID PROFILE DURING IN VITRO

FERMENTATION OF

Eubacterium rectale

ON SUKUH SWEET

POTATO TYPE-III RESISTANT STARCH MEDIA

BACHELOR THESIS

In the partial fulfillment of the requirement for SARJANA TEKNOLOGI PERTANIAN at the Department of Food Science and Technology

Faculty of Agricultural Technology Bogor Agricultural University

By :

SEPTIANA ISWANI

F24070046

FACULTY OF AGRICULTURAL TECHNOLOGY

BOGOR AGRICULTURAL UNIVERSITY

BOGOR

2011

Title : Short Chain Fatty Acid Profile during In Vitro Fermentation of Eubacterium rectale

on Sukuh Sweet Potato Type-III Resistant Starch

Name : Septiana Iswani

Student ID : F24070046

Approved by, Academic Advisor,

(Prof. Dr. Ir. Maggy Thenawidjaja Suhartono) NIP. 19530507 197701 2 001

Acknowledged by,

Plt. Head of Department of Food Science and Technology,

(Dr. Ir. Nurheni Sri Palupi, M.Si) NIP. 19610802 198703 2 002

STATEMENT LETTER OF THESIS AND

SOURCE OF INFORMATION

Hereby I genuinely stated that the bachelor thesis entitled Short Chain Fatty Acid Profile during In Vitro Fermentation of Eubacterium rectale on Sukuh Sweet Potato Type-III Resistant Starch is an authentic work of mine under supervision of academic counselor and never being presented in any forms and universities. All the informations taken and quoted from published or unpublished works of other writers had been mentioned in the texts and attached in refrences at the end of the bachelor thesis.

Bogor, July 2011

The undersigned,

Septiana Iswani

© Created right owned by Septiana Iswani, 2011 All right reserved

This thesis may not be translated or copied in whole or in part without the written permission of the publisher (Bogor Agricultural University) in any forms or by any means, electronic, mechanical,

AUTHOR BIOGRAPHY

Septiana Iswani was born in Jambi, 9 September 1989. The author is the eldest daughter in her family with Drs. H. Ishak Muhammad, M.Pd as her father, Hj. Siti Zainah Wahyuni as her mother, and Wina Maryana as her little sister. The author studied in TK Adhyaksa I Jambi (1994-1995), SD Adhyaksa I Jambi (1995-2001), SMPN 7 Kota Jambi (2001-2004), and SMA N 1 Kota Jambi (2004-2007). Then, she was accepted in Bogor Agricultural University with USMI (Undangan Seleksi Mahasiswa IPB) pathway at Department of Food Science and Technology, Faculty of Agricultural Technology in 2007. During her studied, the author was active in organization and campus activity. The author actively joined in BEM TPB IPB (Badan Esekutif Mahasiswa TPB) in 2007-2008 as staff in entrepreneurship division, the she was accepted as a secretary of communication and information division in BEM FATETA IPB (2009-2010). Then, the author was active as a member in Himpunan Mahasiswa Teknologi Pangan IPB and Himpunan Mahasiswa Jambi. In campus activity, the author was active as a supporting committee in TPB Sehat (2007), BEM TPB Entrepreneurship Seminar (2008), MPKMB Patriot 45 (2008), PLASMA (Pelatihan Sistem Manajemen Pangan Halal), Techno-F 2009 (PAK division), BAUR 2009 (consumption division), and leader in Techno-Fateta Gathering 2009-2010. The author got some achievement in Biology Olympiad of Senior High School Level in Jambi Province (2006-2007) and finalist of Poster Competition in International Symposium Go Organic Thailand. In 2011, the author got experience as an assistant lab work of Food Analysis in Food Science and Technology Department. The author began her research for her bachelor thesis “Short Chain Fatty Acid Profile during In Vitro Fermentation of Eubacterium rectale on Sukuh Sweet Potato Type-III Resistant Starch Media supervised by Prof. Dr. Ir. Maggy Thenawidjaja Suhartono.

vi

PREFACE

First of all, the praise and very gratefulness is delivered to The Almighty, Allah SWT, The Greatest Creator and The Best Motivator to His everlasting and mercy, keeping me tough during my research and achieve my bachelor degree. Shalawat is also delivered to the most honorable prophet, Nabi Muhammad SAW and his family. This bachelor thesis is based upon research during October 2010 to April 2011 with entitled “Short Chain Fatty Acid Profile during In Vitro Fermentation of

Eubacterium rectale on Sukuh Sweet Potato Type-III Resistant Starch.” The writer would like to thank and greatly appreciation to:

1. My beloved father Drs. H. Ishak Muhammad, M.Pd., my mother, Hj. Siti Zaenah Wahyuni,

and my little sister, Wina Maryana, for love, support, pray, and motivation almost every moment in my life

2. Prof. Dr. Maggy Thenawidjaja Suhartono as my academic supervisor for her valuable

guidance, excellence advice, and warm support from the beginning to the completion of my thesis

3. Dr. Ir. Slamet Budijanto, M.Agr and Antung Sima Firlieyanti, STP, M.Sc. as my examiner

for suggestion and constructive advice in completing my thesis

4. Endang Yuli Purwani, for her guidance, kindness, help and advice for providing all my

research requirements at BB Padi Sukamandi and BB Pasca Panen

5. Mrs. Ika Malikha, Mr. Marwan Wahyudi, Ms. Desi Awalina for their sympathetic help and

advice during research in laboratory

6. Lectures, laboratory assistant, and staff in Food Science and Technology Department of

Bogor Agricultural University, Seafast Centre, and BB Pasca Panen

7. My beloved bestfriend since junior high school: Dyah Ayuningtyas, Siti Anindita Farhani, Dayu Dian Perwatasari, Fajriani Kurnia Rosdi, and Firnaliza Rizona for their help, spirit, love, and togetherness along this time.

8. My lovely bestfriend in Department of Food Science and Technology: Amelia Safitri,

Belinda Priska Chentya Dewi, Erlindawati, Indri Putri Handayani, Azizati Fieki Rachmatillah, Rozak Hackiki for their support, laugh, kindness, and togetherness.

9. My partner in research, Michael Devega, for his cooperative support and help during this research

10. My bestfriend in SQ boarding house: Kak Mumpuni, Iia, Ulfa, Nia, Hana, Eni, Dini, Dudu, Fitrah, Orin, Fida, Lina for their support, help, and togetherness

11. My friend in ITP 44: Bu Elmiati, Tiara, Okky, Cherish, Agy, Adi, Kenny, Dinda, Eliana, Reggie, Irsyad, Nadiah, Sarah, Atika, Mumun, Vanya, Ronald, Bertha, Lisa, Iman, Andri, Vendry, Kevin, Reza, Andrew, Khafid, and all of my friend that could not be explained one by one, but always give beautiful moment in the author’s life

12. My friend in Kewirausahaan BEM TPB 44, Danus MPKMB 45, B23, HIMAJA (Yanta, Eko,

Eki, Esi, Wawat), Kominfo Department of BEM FATETA Merah Saga, and my GiBol dormitory’s friend (Resti and Siska).

Finally, the writer hopes that this bachelor thesis will be useful and give real contribution in developing of food science and technology.

Bogor, July 2011

vii

TABLE OF CONTENT

Page

PREFACE ……….. vi 

TABLE OF CONTENT ... vii 

LIST OF TABLE ... ix 

LIST OF FIGURE ... x 

LIST OF APPENDIX ... xi 

I.INTRODUCTION ... 1 

1.1  BACKGROUND ... 1 

1.2  OBJECTIVE OF THE RESEARCH ... 2 

1.3  IMPLICATION OF THE RESEARCH ... 2 

II. LITERATURE REVIEW ... 3 

2.1   SWEET POTATO’S STARCH ... 3 

2.2   RESISTANT STARCH... 4 

2.3  BENEFICIAL OF RESISTANT STARCH ... 6 

2.4  Eubacterium rectale BACTERIA ... 7 

2.5  BENEFICIAL OF SHORT CHAIN FATTY ACID ON COLON CANCER ... 8 

III.METHODOLOGY ... 9 

3.1  MATERIALS AND INSTRUMENT ... 9 

3.2  METHODOLOGY ... 9 

3.2.1  PRODUCTION OF TYPE-III RESISTANT STARCH ... 9 

3.2.1.1  Chemical Characteristic of Sukuh Sweet Potato’s Starch ... 9 

3.2.1.2  Production Process of Type-III Resistant Starch ... 11 

3.2.1.3  Analysis of Resistant Starch Content ... 13 

3.2.2  IN VITRO FERMENTATION ... 14 

3.2.2.1  Peptone Yeast Glucose Preparation ... 14 

3.2.2.2  Activation Eubacterium Rectale 17629 ... 15 

3.2.2.3  In Vitro Fermentation Process ... 15 

3.2.2.4  Analysis of Product Fermentation... 15 

IV.RESULT AND DISCUSSION ... 17 

4.1  CHEMICAL CHARACTERISTIC OF SUKUH’S SWEET POTATO STARCH ... 17 

4.2  PULLULANASE ENZYME ACTIVITY ... 18 

4.3  PRODUCTION OF TYPE-III RESISTANT STARCH... 19 

4.4  ANALYSIS OF FERMENTATION PRODUCT (EXPERIMENT I) ... 21 

4.4.1  Turbidimetry and pH measurement ... 22 

4.4.2  Production of Short Chain Fatty Acid ... 23 

4.5  ANALYSIS OF FERMENTATION PRODUCT (EXPERIMENT II) ... 24 

4.5.1  Turbidimetry and pH measurement ... 24 

viii

V.CONCLUSION AND RECCOMENDATION ... 32 

5.1  CONCLUSION ... 32 

5.2  RECCOMENDATION ... 32 

REFERENCES ... 33 

ix

LIST OF TABLE

Page

Table 1. General characteristic of superior variety of sweet potato in Indonesia ... 4 

Table 2. Classification of type of RS, food sources, and factor affecting their resistance to digestion in the colon. ... 6 

Table 3. Medium composition of PYG for 1000 ml ... 14 

Table 4. Mineral mix solution composition ... 14 

Table 5. SCFA standard mixture for standard curve ... 16 

Table 6. Proximate analysis result of Beauregard, Evangeline, Jago, and Sukuh sweet potato starch (dry bases) ... 17 

Table 7. Yield of type-III resistant starch production ... 19 

Table 8. Proportion of SCFA production by Eubacterium rectale 17629 over 48 h of fermentation at 370C ... 24 

Table 9. Absorbance and pH value over fermentation of Sukuh type-III resistance starch by E.rectale 17629 in experiment II ... 25 

Table 10. Production and proportion of short chain fatty acid by Eubacterium rectale 17629 in Experiment II ... 25 

Table 11. Comparison of SCFA production from different substrate, bacteria, and fermentation time 26  Table 12. Changes in fermentation products for five strains of E.rectale growth for 24 h on YCFA medium containing 0.5% glucose ... 30 

x

LIST OF FIGURE

Page

Figure 1. Sukuh’s sweet potato ... 3

Figure 2. Structure of Resistant Starch ... 5

Figure 3. Flow chart of the research ... 10

Figure 4. Flow chart of type-III resistant starch production ... 12

Figure 5. Yield of Sukuh type-III resistant starch product ... 19

Figure 6. Turbidimetry result of E.rectale 17629 growth over 48 h fermentation at 370C ... 22

Figure 7. Change of pH fermentation by E.rectale 17629 over 48 h at 370C ... 23

Figure 8. SCFA profile produced by Eubacterium rectale 17629 over 48 h of fermentation ... 24

Figure 9. Alternative anaerob fermentative pathway ... 29

xi

LIST OF APPENDIX

Page

Appendix 1. Amylose content analysis ... 41

Appendix 2. Yield of type-III resistant starch derived from Sukuh’s sweet potato ... 42

Appendix 3. Analysis of resistant starch content ... 43

Appendix 4. Pullulanase enzyme activity ... 44

Appendix 5. Analysis of fermentation product (pH value and absorbance) (Experiment I) ... 45

Appendix 6. Calculation of Short Chain Fatty Acid Concentration (Acetic acid, Propionic acid, Butyric acid) (Experiment I) ... 46

Appendix 7. Analysis of fermentation product (pH value and absorbance) (Experiment II) ... 51

Appendix 8. Calculation of Short Chain Fatty Acid Concentration (Acetic acid, Propionic acid, Butyric acid) (Experiment II) ... 52

Appendix 9. SCFA chromatogram of 5µl standard (0.1743 M acetate, 0.1334 M propionate, 0.1084 M butyrate) Experiment I ... 56

Appendix 10. SCFA chromatogram of 10 µl standard (0.3487 M acetate, 0.2667 M propionate, 0.2167 M butyrate) Experiment I ... 57

Appendix 11. SCFA chromatogram of 15 µl standard (0.5230 M acetate, 0.4001 M propionate, 0.3250 M butyrate) Experiment I ... 58

Appendix 12. SCFA chromatogram of 25 µl standard (0.87169 M acetate, 0.6669 M propionate, 0.5418 M butyrate) Experiment I ... 59

Appendix 13. SCFA chromatogram for 6 hours fermentation in Experiment I (2% RS) (1) ... 60

Appendix 14. SCFA chromatogram for 6 hours fermentation in Experiment I (2% RS) (2) ... 61

Appendix 15. SCFA chromatogram for 12 hours fermentation in Experiment I (2% RS) (1) ... 62

Appendix 16. SCFA chromatogram for 12 hours fermentation in Experiment I (2% RS) (2) ... 63

Appendix 17. SCFA chromatogram for 24 hours fermentation in Experiment I (2% RS) (1) ... 64

Appendix 18. SCFA chromatogram for 24 hours fermentation in Experiment I (2% RS) (2) ... 65

Appendix 19. SCFA chromatogram for 36 hours fermentation in Experiment I (2% RS) (1) ... 66

Appendix 20. SCFA chromatogram for 36 hours fermentation in Experiment I (2% RS) (2) ... 67

Appendix 21. SCFA chromatogram for 48 hours fermentation in Experiment I (2% RS) (1) ... 68

Appendix 22. SCFA chromatogram for 48 hours fermentation in Experiment I (2% RS) (2) ... 69

Appendix 23. SCFA chromatogram of 5µl standard (0.1743 M acetate, 0.1334 M propionate, 0.1084 M butyrate) Experiment II ... 70

Appendix 24. SCFA chromatogram of 10 µl standard (0.3487 M acetate, 0.2667 M propionate, 0.2167 M butyrate) Experiment II ... 71

Appendix 25. SCFA chromatogram of 25 µl standard (0.87169 M acetate, 0.6669 M propionate, 0.5418 M butyrate) Experiment II ... 72

Appendix 26. SCFA chromatogram for 36 hours fermentation in Experiment II (1% RS) (1) ... 73

Appendix 27. SCFA chromatogram for 36 hours fermentation in Experiment II (1% RS) (2) ... 75

Appendix 28. SCFA chromatogram for 48 hours fermentation in Experiment II (1% RS) (1) ... 77

CHAPTER I

INTRODUCTION

1.1

BACKGROUND

Colorectal cancer (CRC) is one of the most commonly diagnosed cancers in affluent countries and is a leading cause of cancer-related mortality in the USA and Australia. Change of consumption pattern and lifestyle, full with stress and bad lifestyles cause high prevalence of colorectal cancer case and increase interest of consumer in maintaining healthy gastrointestinal tract. Accumulating evidence from epidemiological and experimental studies suggest that diet is an important environmental factor in the aetilogy of CRC.

Data derived from the Department of Pathology Anatomy, Faculty of Medicine, University of Indonesia, in the period of 1996 to 1999 revealed that there were 35.2% colorectal cancer cases of young age (less than 40 years). While the data of Ministry of Health revealed the incidence of colorectal cancer under 45 years of age in 4 major cities in Indonesia, i.e. 47.85%, 54.5%, 44.3% and 48.2% in Jakarta, Bandung, Makassar, and Padang respectively (Cravo, 1999). Compared to developed countries, there is higher incidence of young colorectal cancer patients in Indonesia (Sudoyo et al, 2010)

Sweet potato (Ipomoea batatas L) is a tuber crops relatively easy to be cultivated in Indonesia. Sweet potato also can be a cheap source of carbohydrate for Indonesian people. Variety of sweet potato has been developed to increase variation of Indonesia agriculture plant. High productivity of sweet potato, supported with appropriate climate and high preference of sweet potato by Indonesian people make it potential to be developed. Innovation of resistant starch based of sweet potato as a component of functional food can be an alternative to increase added value of the sweet potato starch.

Sukuh is one of the superior variety which is released by Research Institute for Legume and Tuber crops, Malang. Sukuh has ellipse and spherical tuber, short stalk tuber, yellow skin color, and white flesh color. Sukuh has highest starch content with 31% from 35% dry matter. Beside that, Sukuh has high productivity around 25-30 ton/ha and 2.0-2.5 months of harvest time (RILET, 2008). However, Sukuh is known as variety which is not sell well in market among other sweet potato that has colorful flesh. High productivity and high starch content make it potential to be developed as ingredient for functional food, like resistant starch.

Topping and Clifton (2001) reported that RS intake correlates negatively with colorectal cancer risk. The extensive studies have shown that resistant starch has physiological functions similar to those of dietary fiber (Asp, 1994). In addition, resistant starch also has functional effect as a prebiotic to promote the survival of intestinal microorganism.

Resistant starch defines as the sum of starch and products of starch degradation not absorbed in the small intestine of healthy individuals (EURESTA, 1993). Resistant starch is divided into 4 types, which are type I, II, III, and IV. RS1 is a physically inaccessible starch (e.g. coarsely ground grains and cereals); RS2 represents starch that is in a certain granular form including un-gelatinized starch granules (e.g. green banana, raw potato); RS3 is a re-crystallized or retrograded polymers of starch (e.g. gelatinized and cooled high amylose starch), and RS4 is a chemically modified starch (Englyst et al, 1992).

Resistant starch type III represents retrograded starch, which form the crystalline structure formation of amylose by hydrogen bond in double helices. Resistant starch results from the highly

2 retrograded of amylose fraction, the quantity formed being directly proportional to the amylose content (Annison and Topping, 1994). The degree of resistant starch formation depends on water content of starch suspension, autoclaving temperature, enzymatic reaction, cooling and drying process, and other component (e.g. lipid, protein, glucose) (Eerlingen and Delcour, 1995).

Resistant starch that reaches the large bowel is fermented by colonic microflora as a carbon source for its growth. Butyrate-producing strains of the genus Eubacterium are found in rather high cell counts (Finegold et al, 1983). The products of fermentation include gases (CO2, CH4, H2), short chain fatty acids (acetate, propionate, butyrate), lactate, ethanol (Macfarlane and Cummings, 1991).

Fermentation product of resistant starch depends on fermentation substrate, type of bacteria, and starch degradation enzyme. Short chain fatty acid as a major of fermentation product has been shown to be the preferred energy substrate of the colonocytes (Roediger, 1980) and reduced risk of colon cancer (Hill, 1995). Brouns et al(2002) and Wang et al(1999) proved that fermentation of resistant starch in colon produced the highest of butyrate ratio than fermentation of another dietary fiber (e.g. pectin, oat, bran, non starch polysaccharide, wheat).

One of the SCFA, butyrate, is known play a key role in the energy metabolism of the colonic epithelial cells and thought to be important in the maintenance of colonic health in humans (Mortensen and Clausen, 1996). Butyrate is utilized as an important energy source by the colonocyte (Roediger, 1982). Butyrate also has a range of effects particularly relevant to bowel cancer. Recent study has also focused on the effects of butyrate on induction of apoptosis (e.g. programmed cell death) of cancer cell (Le Leu, 2007).

Estimation of short chain fatty acid in colon is done by in vitro fermentation. In vivo

fermentation to estimate short chain fatty acid as a fermentation product is difficult because the absorption of SCFA is so fast. Beside, in vitro fermentation shows the pattern of bacterial growth in using resistant starch as a fermentation substrate (Khan and Edwards, 2005). Characteristics of resistant starch (e.g. quality, quantity, structure, fraction, and composition) influence proportion and profile of SCFA as the fermentation product (Tovar et al, 1992).

1.2

OBJECTIVE OF THE RESEARCH

The objectives of the research were to study short chain fatty acid profile during in vitro fermentation of Eubacterium rectale 17629 on Sukuh sweet potato type III resistant starch and to determine the effect of concentration of resistant starch to the SCFA profile by Eubacterium rectale 17629.

1.3

IMPLICATION OF THE RESEARCH

This research is expected to get basic information in developing functional food product derived from Sukuh sweet potato’s resistant starch in prebiotic form

.

CHAPTER II

LITERATURE REVIEW

2.1

SWEET POTATO’S STARCH

Sweet potato (Ipomoea batatas) is one of the tropical tuber in Indonesia. Many varieties of sweet potato are member of the morning glory family, Convolvulaceae. In 2005, production area of sweet potato in Indonesia reach 178,336 hectare and production crop reach 1,856,969 ton (BPS, 2006). Research priority should be given to sweet potato to generate a better and more efficient production technology. This will help boost development of sweet potato in Indonesia. However, the Indonesian government still puts higher priority on rice research. Even sweet potato is the secondary food crops after cassava, research priority of sweet potato is lower compared to the legumes such as soybean and peanut.

Sweet potato is source of carbohydrate with variation of skin and flesh color includes white, yellow, orange, red, purple, or brown. Flesh of sweet potato contains fiber and oligosaccharides (stacchiose, verbascosa, and raffinose) which cause flatulence. Classification of sweet potato:

Kingdom :Plantae

Division : Spermatophyta

Subdivision : Angiospermae

Ordo : Concolvulales

Family : Convolvulacea

Genus : Ipomoea

Species : Ipomoea batatas (RILET, 2008)

Figure 1. Sukuh’s sweet potato (RILET, 2008)

There are five superior varieties of sweet potato that are released by Research Institute for Legumes and Tuber crops (RILET) in Malang, that are Sari, Boko, Sukuh, Jago, and Kidal (RILET, 2008). Superior variety of sweet potato is sweet potato which has high productivity and endurance toward pest infection of boleng (Cylas formicarius) and sweet potato’s scabies disease (Sphaceloma batatas) (RILET, 2008). General characteristic of superior variety of sweet potato in Indonesia is outlined in Table 1.

4 Table 1. General characteristic of superior variety of sweet potato in Indonesia

Variety Physic form Chemical content Productivity Harvest

time

Sari

Oval shaped tuber, short stalk tuber, red skin color, yellow flesh, sweet an delicious taste

Dry matter: 28% Starch content: 32% Beta carotene content:

381µg/100 g

30-35 ton / ha 3.5-4

months

Sukuh

Ellipse and spherical tuber, short stalk tuber, yellow skin

color, white flesh color

Dry matter: 35% Starch content: 31% Beta carotene content:

36.59µg/100g

25-30 ton/ha 4.0-4.5

months

Boko

Long and ellipse tuber, very shorter stalk tuber, red skin color, light-yellow flesh color

Dry matter: 32% Starch content: 32% Beta carotene content:

108µg/100g

25-30 ton/ha 4.0-4.5

months

Jago

Spherical tuber, short stalk tuber, white skin color, yellow

flesh color

Dry matter: 33% Starch content: 31% Beta carotene: 85µg/100g

25-30 ton/ha 4.0-4.5

months

Kidal

Spherical tuber, very short stalk tuber, red skin color,

yellow flesh color

Dry matter: 31% Starch content: 32.85% Beta carotene: 345µg/100g

25-30 ton/ha 4.0-4.5

months Source: RILET, 2008

2.2

RESISTANT STARCH

Starch defines as the major dietary source of carbohydrates, is the most abundant storage polysaccharides in plants, and occurs as granules in the chloroplast of green leaves and the amyloplast of seeds, pulses, and tubers (Ellis et al, 1998). Chemically, starches are polysaccharides, composed of a number of monosaccharides or sugar (glucose) molecules linked together with α-D-(1-4) and α -D-(1-6) linkages. The starch consist of 2 main structural components, amylose, which is essentially a linier polymer in which glucose residues are α-D-(1-4) linked typically constituting 15-20% of starch, and amylopectin, which is a larger branched molecule with α-D-(1-4) and α-D-(1-6) linkages and is a major component of starch (BNF,1990)

The term “resistant starch” was first coined by Englyst et al (1982) to describe a small fraction of starch that was resistant to hydrolysis by exhaustive α-amylase and pullulanase treatment

in vitro after 120 minute incubation. However, because starch reaching the large intestine may be more or less fermented by the gut microflora, RS is now defined as the sum of the starch and product of starch degradation which is not absorbed in the small intestine of healthy individuals (Englyst et al, 1992).

There are four type of resistant starch. Type I represents starch that is resistant because it is in a physically inaccessible form, which is locked in the plant cell walls of some foodstuffs, such as partially milled grains, seeds, and legumes (Englyst et al, 1992, Sajilata, 2006). Figure 2 (a) shows microscopic view of the physically inaccessible RS1 in cell or tissue structures of partly milled grains, seeds, and vegetable. Type II is a native starch granules, starch is tightly packed in a radial pattern and is relatively dehydrated. This compact structure limits the accessibility of digestive enzyme and accounts for the resistant nature of RS2 such as, un-gelatinized starch. Figure 2 (b) shows the RS granules of type II resistant starch, that is raw potato, banana, high amylose starch (Sajilata, 2006).

5

a b

c d

Figure 2. Structure of Resistant Starch (a) Structure of type I resistant starch (RS1), (b) Structure of type II resistant starch (RS2), (c) Schematic presentation of enzyme type III resistant starch (RS3) (Micelle model), (d) Preparation of cross-bended starch in modified starch of RS4 (distarch phosphate ester) (Sajilata et al, 2006)

Type III resistant starch is made up retrograded starch or crystalline non-granular starch, like the starch found in cooked and cooled potatoes, bread crust, cornflakes, and high-amylose maize starch (Englyst et al, 1992; Eerlingen and Delcour, 1995). Schematic presentation of RS3 formed in aqueous amylose solutions depicted as micelle model is shown in Figure 2 (c). Type IV refers to specific chemically and thermally modified or re-polymerized starch (Englyst et al, 1992; Eerlingen and Delcour, 1995). Structure of RS4 includes structure of modified starches obtained by chemical treatments like distarch phosphate ester (Figure 2 (d). Table 2 outlines a summary of the different types of RS, their classification criteria, and food sources.

Type-III resistant starch is formed by thermal disruption of the granular structure of the starch in water or gelatinization of the starch and re-crystallization of amylose and amylopectin (retrogradation). Gelatinization will disrupt granular structure by heating starch with an excess of water and amylose leaches from the granules into the solution as a random coil polymer. The gelatinized starch will be re-crystallized by cooling, which is the polymer chains begin to re-associate as double helices, stabilized by hydrogen bonds (Englyst et al., 1992; Eerlingen and Delcour, 1995). Formation is followed by enzymatic debranching of gelatinized starches followed by drying, extrusion, or ion crystallization (addition of salts) (Futch, 2009). Industrial production of resistant starch comes mainly from high amylose maize starches.

6 Table 2. Classification of type of RS, food sources, and factor affecting their resistance to digestion in

the colon

Type of RS Description Food sources Resistance minimized

by

RS1 Physically protected Whole-or partly milled grains

and seeds, legumes Milling, chewing

RS2

Un-gelatinized resistant granules with type B crystallinity, slowly hydrolyzed

by α-amylase

Raw potatoes, green bananas, some legumes, high amylose

corn

Food processing and cooking

RS3 Retrograded starch

Cooked and cooled potatoes, bread, cornflakes, food products with repeated moist

heat treatment

Processing conditions

RS4

Chemically modified starches due to cross-linking with

chemical reagents

Foods in which modified starches have been used (for

example:breads, cakes)

Less susceptible to digestibility in vitro Source: Nugent, 2005

Debranching enzyme using pullulanase has been applied to produce a starch with linier, low-molecular-weight and re-crystallizable polymer chains. Debranching enzymes such as pullulanase rapidly hydrolyze only α-1,6-glucosidic bonds, releasing a mixture of long and shorter unit chains from the parent amylopectin molecule. These fragments are linear polymers containing about 10 to 65 anhydroglucose units linked by α-1,4-glucosidic bond. The debranched starch was then subjected to temperature cycling and incubation at a series of temperature and time to induce retrogradation and yield the resistant starch (Leong et al, 2007)

A study on RS formation showed that as the amylose fraction increased, RS yield increased. The formation was affected by the water content of the starting starch suspension, autoclaving temperature, condition of enzymatic reaction and cooling and drying process, as well as by the presence of other ingredients such as lipids, sugars and salts (Eerlingen and Delcour, 1995). Beside that, formation of resistant starch was also affected by several properties of starch, such as granular structure, crystallinity, amylose and amylopectin ratio, and chain length (Futch, 2009). Cummings et al (1987) also defined some factors associated with RS formation during the processes were the physical state of the food materials (whole or ground), water content, pH, heating time and temperature, composition substrate, number of heating and cooling cycles, freezing methods (slow vs rapid) and drying method.

2.3

BENEFICIAL OF RESISTANT STARCH

The slow hydrolysis of resistant starch makes it useful for the slow release of glucose, which can be especially useful in controlling glycaemic plasma responses (Raben et al, 1994). Some other benefits include increased faecal bulk, lowered faecal pH, and increased excretion of butyrate and acetate (Phillip et al, 1995). Resistance starch was reported has physic character like un-soluble starch but it has function like soluble starch.

Resistant starch has a lower water-holding capacity. It has desirable physicochemical properties such as swelling, viscosity increase, gel formation, and water-binding capacity, making it useful in a variety of foods (Fausto et al, 1997). These properties make it possible to use most

7 resistant starch to replace flour on a 1-for-1 basis without significantly affecting dough handling or rheology (Sajilata, 2006). Beside that, RS is commercially use as a dietary fiber fortification in bread-making, as a texture modifier in baked goods, as a crisping agent, and as a functional ingredient in other food.

2.4

Eubacterium rectale

BACTERIA

Resistant starch can be utilized by microorganism present in the human large intestine. Consequently, the metabolites formed during the fermentation process, i.e., short chain fatty acid, may serve as a main energy source for the colonocytes (Mortensen, et al, 1996), including gases (CO2, CH4, H2), lactate and branched fatty acids (isobutyrate, isovalerate) (Macfarlene et al, 1991) and thus help to maintain colon health. The type and amount of by-product is dictated by the substrate undergoing fermentation. Short chain fatty acids are the principal end-product of colonic fermentation and are produced in the approximate molar ratio of 60:25:15 (acetate, propionate, and butyrate, respectively) (Macfarlene et al, 1991).

Among the genera of colonic bacteria, butyrate producing bacteria such as Eubacterium, Peptostreptococci, Clostridia, Roseburia spp and Butyrofibriofibri- solvens are thought to have beneficial effect on the human host (Purwani and Suhartono, 2009). Eubacterium is an obligately anaerobic gram positive non-spore forming bacteria. It has road (bacilli) shape. The genus

Eubacterium (i.e. Eubacterium rectale) which is the second most common genus in the human intestine,also commonly found in the human fecal (collected from a pilot study), including several known butyrate producers (Schwiertz et al, 2002).

Several colonic bacterial groups produce amylase which hydrolyzes the starch into oligosaccharide (Wang et al, 1999). It may induce the growth of useful bacteria including butyrate producing bacteria. It is known that bacterial butyrate production is stimulated particularly by resistant starches (Wang et al, 1999; Sharp and Macfarlene, 2000). Ability of several bacteria in using starch as a substrate fermentation depend on the existence of degradation enzyme.

Many species of Eubacterium is an obligately anaerobic gram positive non spore-forming. Cells of Eubacterium rectale are slightly curved slender rods of moderate (0.5 x 2.0-5.0 µm) length. Cells are gram positive, but decolorize readily to give a Gram-negative staining reaction. Cells from early to mid-exponential phase cultures are clearly motile when examined by phase-contrast microscopy (Duncan and Flint, 2008).

Eubacterium rectale possesses genes for the production of butyrate that show high similarity to genes from other Clostridia (Mahowald, 2009). Metabolic pathway involves condensation of 2

molecules of acetylCoA to form butyrate and is accompanied by oxidation of NADH to NAD+. In

vitro studies have shown that in the presence of carbohydrate, Eubacterium rectale consumes large amounts of acetate for butyrate (Duncan and Flint, 2008). The last step in Eubacterium rectale’s butyrate production pathway is catalyzed by the butyrylCoA dehydrogenase/electron transfer flavoprotein (Bcd/Etf) complex, and offers a recently discovered additional pathway for energy conservation, via a bifurcation of electrons from NADH to crotonylCoA and ferredoxin (Li F, 2008). Reduced ferredoxin, in turn, can be re-oxidizied via hydrogenases, or via the membrane-bound oxidoreductase, Rnf, which generates sodium-motive force.

Generally, metabolic pathway of Eubacterium rectale has a similarity with

butyrate-producing Clostridia, because it possesses genes for the production of butyrate that show high similarity to genes from other Clostridia (Mahowald, 2009). The reaction involves a lot of enzymatic process and some factors may effects certain product forming. Glucose is metabolized to pyruvate via Embden-Meyerhof-Parnas (EMP) pathway and produces two moles of ATP and NADH, from each

8 mole of glucose. Propionic acid is produced via 2 main pathways: 1) fixation of CO2 to form succinate (the dicarboxylic pathway); and 2) from lactate and acrylate. Pyruvate is an intermediate compound for SCFA production, then can be metabolized to acetate, butyrate, and propionate.

2.5

BENEFICIAL OF SHORT CHAIN FATTY ACID ON COLON

CANCER

Short chain fatty acid is an organic fatty acid with 1 to 6 carbon atoms. It is the principal anion which arised from bacterial fermentation of polysaccharide, oligosaccharide, proteins, peptide, and glycoprotein precursors in the colon (Cumming et al, 1997). Production of SCFA is affected by many factors, including numbers and types of microflora present in the colon (Roberfroid, 2005), substrate source (Cook and Sellin 1998), gut transit time, hexose availability, enzyme production of bacteria, and amount of carbohydrate. Various population data showed that SCFA production in order of acetate:propionate:butyrate in a molar ration of approximately 60:20:20 or 3:1:1, respectively in the proximal and distal colon (Topping and Clifton, 2001)

Short chain fatty acids, the major by-product of fermentation, have several actions that appear relevant to maintaining the health of the large bowel. They are rapidly absorbed by the colonic mucosa, promoting water and sodium absorption, and thereby preventing osmotic diarrhea (Ruppin et al, 1980). Also, together with lactate, they help to acidify the colonic lumen (Macfarlene et al, 1991). A low pH reduces the bacterial conversion of primary bile acids into secondary bile acids (Cummings, 1983). In addition, a low pH can affect other processes including the ionization of SCFA; epithelial proliferation; the balance of bacterial species; bacterial metabolism of ingested carcinogens; bacterial activation of oxygen free radicals; the activity of bacterial enzymes (e.g. β-glucosidase, β -glucuronidase); and faecal water cytotoxicity (Cummings, 1983).

Acetate or acetic acid (C2H4O2) is an organic acid which is colorless and gives vinegar sour taste. Acetate is an essential metabolic fuel in ruminant animal because all glucose reaching the rumen is fermented by the resident bacteria (Cummings, 1997). Acetate is rapidly cleared from the blood with a half life of only a few minutes (Skutches et al, 1979) and is metabolized by skeletal and cardiac muscle (Scheppach et al, 1991). Acetate either orally or intravenously has little effect on glucose metabolism and does not stimulate insulin release in man (Scheppach et al, 1988)

Propionate or propionic acid (CH3CH2COOH) is a clear, colorless liquid with a slightly sweetish odor. Propionate can be found in portal blood, although some may be metabolized in the colonic epithelium and may be a differentiating factor, but with less power than butyrate (Gamet et al, 1992). Some experiment have shown inhibition of hepatic cholesterol synthesis by propionate and redistribution of cholesterol from plasma to liver (Chen et al, 1984)

There is strong evidence that short chain fatty acid butyrate is important for the metabolic welfare of the epithelium of the large bowel. Butyrate is utilized as an important energy source by the colonocyte, the main cell type in the colon (Roediger, 1982). Butyrate also has a range of effects particularly relevant to bowel cancer. It ‘stabilizes’ DNA by inhibiting histone deacetylase (Candido et al, 1978) and increasing methylation of DNA; it induces differentiation; and it reduces the growth rate in a range of mammalian cells, including colorectal cancer cell lines (Whitehead, 1987). Recent interest has also focused on the effects of butyrate on induction of apoptosis (i.e. programmed cell death) of colonic epithelial cells (Hague et al, 1993).

CHAPTER III

METHODOLOGY

3.1

MATERIALS AND INSTRUMENT

Starch sample used in this research was sweet potato Sukuh strain, which was obtained from Indonesian Center of Agricultural Post Harvest Research and Development, Bogor. Starch degrading enzyme was obtained from NOVO Nordisk through the PT Halim Sakti Pratama, Jakarta. The enzyme

was Promozyme® which has standard activity 177, 222.11 IU/ml and density was 1.20 g/ml,

respectively. Strain of colonic butyrate producing bacteria used Eubacterium rectale 17629 was obtained from culture collection of Balitvet, Bogor, West Java.

The material prepared to make basal medium (PYG) for maintaining E.rectale 17629

composed of tryptone, bacteriological peptone, yeast extract, beef extract, glucose, Tween 80, resazurin, CaCl2, MgSO4, K2HPO4, KH2PO4, NaHCO3, NaCl, Vitamin K1,CO2. Beside that, chemical reagent used in this research were ethanol, pure amylose, NaOH, acetic acid, iodine, KI, KCl-HCl buffer, pepsin, Tris-maleat buffer, pancreatic α-amylase, KOH, HCl, citrate buffer, amyloglucosidase, phenol, H2SO4, glucose, HCl, butyrate standard (Fluka), acetate standard (Fluka), propionate standard (Fluka).

The instrument used in this research were gas chromatography (Agilent technologies 7890A), spray drier, aluminum foil, spectrophotometer, shaking water bath, incubator, micropipette, reaction tube, syringe 1µl, centrifugation, centrifuge tube, autoclave, volumetric flask, refrigerator, and fermentation tube.

3.2 METHODOLOGY

This research was divided into two steps: (1) production of type-III resistant starch from Sukuh’s sweet potato (chemical characteristic of Sukuh’s starch, preparation enzyme, production process, and analysis of product RS content) and (2) in vitro fermentation process by Eubacterium rectale 17629 (medium preparation, bacteria activation, fermentation process, and SCFA profile analysis of fermentation product). Generally, step of this research could be seen in Figure 3.

3.2.1 PRODUCTION OF TYPE-III RESISTANT STARCH

3.2.1.1 Chemical Characteristic of Sukuh Sweet Potato’s Starch

Chemical characteristic of sweet potato’s starch of Sukuh variety were known by proximate and amylose content analysis. Proximate analysis included moisture content (oven method) (AOAC, 1995); ash content (dry ash method) (AOAC, 1995); protein content (Kjeldahl method) (AOAC, 1995); fat content (Soxhlet method) (AOAC, 1995); crude fiber content (AOAC, 1995); carbohydrate analysis (by difference method) (AOAC, 1995).

a. Moisture content by Oven Method (AOAC, 1995)

Sample was weighed 1-2 g into a aluminum crucible. The sample was dried into oven at 1000C for 1 hour. Then, it was cooled in desiccators and weighed after reaching room temperature. Sample weighing was repeated until reach constantly weigh. The loss of weigh is used to calculate the moisture content of the sample.

10 Figure 3. Flow chart of the research

b. Ash content by Drying Ashing Method (AOAC, 1995)

Sample was weighed 5-10 g into a tared porcelain crucible. The porcelain crucible was ignited 12-18 hours (or overnight) at about 5500C. Then, muffle furnace was turned off and wait to open it until the temperature has dropped to at least 2500C, preferably lower. Using safety tongs, porcelain crucible was transferred quickly into desiccators with a porcelain plate and desicant, allow it to cool prior to weighing. Sample weighing was repeated until reach constantly weigh. The loss of weigh is used to calculate the ash content of the sample.

c. Fat content by Soxhlet Method (AOAC, 1995)

Sample was weighed 2 g into pre dried extraction thimble and dried 6-18 hours at 1000C. The thimble containing dried sample was placed in soxhlet. Amount of 150 ml hexane was added into soxhlet and extracted at a rate of 5 or 6 drops per second condensation for about 4 hours. Then, flask was removed and the solvent was evaporated by heating. Boiling flask with extracted fat was dried in an air oven 1000C for 1.5-2 hours, then cooled in desiccators and weighed.

d. Protein content by Kjeldahl Method (AOAC, 1995)

Sample of 100-250 mg was placed into Kjeldahl flask. Then 1.0 ± 0.1 g K2SO4, 40 ± 10 mg HgO, and 2.0 ± 0.1 ml H2SO4 were added. The solution was boiled for 1-1.5 hours. At distilattion

Proximate analysis

Analysis of amylose

content Sukuh starch

Production of type-III resistant starch

Preparation of fermentation medium with Sukuh’s sweet potato type III resistant starch

as a substrate

Activation of Eubacterium rectale 17629

culture

Analysis of resistant starch content

In vitro fermentation of Eubacterium rectale 17629 bacteria in medium (an-aerob

condition) Analysis of

Pullulanase enzyme activity

Fermentation product pH

measurement

Absorbance measurement

Short chain fatty acid analysis using gas chromatography

11 stage, a little amount of water is transferred through flask wall and shaken carefully to resform the crystal. Solution was transferred to the destillation ware, rinsed 5-6 times with 1-2 distilled water, followed by adding distilled water to distillation ware and 8-10 ml of 60% NaOH - 5% Na2S2O3. Erlenmeyer was placed under the condencer with 5 ml H3BO3 and 2-4 drops of red-methylene blue added.end of condenser must be soaked in H3BO3 solution. A titration stage, sample solution was diluted into 50 ml, and then titrated with 0.02 N standardized HCl until grey color appears. The borate anions formed in previous stage was titrated with standardized acid (HCl) which is proportional to the amount of nitrogen.

e. Carbohydrate content (by difference method) (AOAC, 1995)

Carbohydrate (%) = (100% - ((protein (%) + water (%) + ash (%) + fat (%))

f. Amylose Content Analysis (Apriyantono et al., 1989)

Determination of amylose content began with make standard curve of pure amylose. The standard curve was made by dissolving 40 mg pure amylose into 1 ml 95% ethanol and 9 ml NaOH 1N solution. The mixture was boiled for 10 minutes to gelatinize the solution. After it was cooled, it was adjusted with distilled water into 100 ml by volumetric flask, prepared series of dilution in the 100 ml of volumetric flask: 1.0; 2.0; 3.0; 4.0; and 5.0 ml of amylose solution, 0.2; 0.4; 0.6; 0.8; and 1.0 ml of acetic acid 1N, respectively. The series of dilution was added 2 ml iodine solution (0.2 g iodine and 2 g KI were diluted into 100 ml distilled water) for each dilution and was added distilled water to adjust into 100 ml. The mixture was incubated for 20 minutes. Intensity of blue color complex for each dilution was measured at 620 nm using Spectrophotometer (Spectronic 20). The data was plotted to make standard curve.

Amylose content was measured by dissolving 100 mg starch sample into reaction tube, added 1 ml ethanol 95% and 9 ml NaOH 1N solution. Sample was boiled for 10 minutes to gelatinize starch solution. After it was cooled, starch paste was adjusted with distilled water into 100 ml by volumetric flask. In the amount of 5 ml of the dilution is moved into 100 ml volumetric flask, added 1 ml acetic acid 1N, 2 ml iodine solution and distilled water to adjust into 100 ml. After incubated for 20 minutes, the absorbance was measured with spectrophotometer at 625 nm. The absorbance of the sample was compared to the standard curve to determine the amylose content.

3.2.1.2 Production Process of Type-III Resistant Starch

a. Analysis of Pullulanase Enzyme Activity (Hartoyo et al., 2009)

The standard curve was made by dissolving 0.2 g glucose and adjusted by adding distilled water in 100 ml of volumetric flask, then prepared series of dilution: 0.0; 0.1; 0.2; 0.3; 0.4; and 0.5 ml glucose solution and added acetate buffer 20 mM pH 5.0: 0.5; 0.4; 0.3; 0.2; 0.1; and 0.0 ml, respectively and mixed well. The mixtures were incubated 250C for 10 minutes. Then, the mixture was added by 0.5 ml DNS solution (3 g NaOH, 34.6 g NaK-tartarat, 3 g DNS were diluted into 300 ml distilled water) for each dilution, then mixed well. The mixture was boiled for 5 minutes. After it was cooled, it was added 2 ml of distilled water and absorbance of the color complex was measured at 550 nm using spectrophotometer (Spectronic 20). The data was plotted to make standard curve.

Substrate (pullulan) solution (2%) was made by dissolving 0.02 g pullulan substrate into 1 ml of distilled water. The mixture was divided into 2 parts:

Sample: Amount of 0.05 ml of the pullulan solution (2%) was dissolved by adding 0.35 ml of acetate buffer then mixed well. The mixture was incubated at 250C for 10 minutes. Then, it was added by 0.1 ml pullulanase enzyme and was incubated at 250C for 10 minutes. Then, the mixture mixed by 0.5 ml

12 of DNS solution then mixed well. The mixture was boiled for 5 minutes. After it was cooled, 2 ml of distilled water added to the mixture and absorbance of the color complex was measured at 550 nm using spectrophotometer (Spectronic 20). The data was plotted to make standard curve.

Control: Amount of 0.05 ml of the pullulan solution (2%) is dissolved by adding 0.35 ml of acetate buffer then mixed well. The mixture was incubated at 250C for 10 minutes. Then, the mixture was added by 0.5 ml DNS of solution and 0.1 ml pullulanase enzyme and mixed well. After that, the mixture was boiled for 5 minutes and mixed with 2 ml distilled water. After it was cooled, absorbance of the color complex was measured at 550 nm using spectrophotometer (Spectronic 20).

b. Production Process of Type-III Resistant (Vatanasuchart et al, 2010)

Figure 4. Flow chart of type-III resistant starch production 20 g Sukuh sweet potato’s starch +

180 g distilled water (10% (w/w))

Adjust the acidity of the starch suspension to pH 5.00

Gelatinization (1200C, 30 min)

Cool down to 500C

Hydrolyze the samples in a shaking water bath controlled 500C; 24 hours

Add 5% pullunase (Promozyme®) (177,222.11 IU/ml) of the starch weight

Deactivation the enzyme the hydrolyze sample by heating (900_{C; 30 min)}

Retrograde the starch by placing in refrigerator (40C; 24 hours)

Homogenization

Centrifugation (4500 rpm;15 min)

Dry the starch using spray drier

13 Starch sample of sweet potato Sukuh strain was obtained from Indonesian Center of Agricultural Post Harvest Research and Development, Bogor. The starch was processed to produce type-III resistant starch by the hydrolysis of pullulanase enzyme Promozyme® (177,122.11 IU/ml). The procedure for type-III resistant starch production is shown in Figure 4.

3.2.1.3

Analysis of Resistant Starch Content (Goni et al, 1996)

Starch sample was weighed 100 mg and put into a 50-ml centrifuge tube and added 10 ml of KCl-HCl buffer pH 1.5 (pH adjustment with 2 M HCl or 0.5 M NaOH). The solution was mixed well and added 0.2 ml of the pepsin solution (1 g pepsin/10 ml buffer KCl-HCl) (445 units/mg solids), mixed well and incubated in a water bath at 400C for 60 minutes with constant shaking. The mixture was added 9 ml Tris-maleate buffer pH 6.9 (pH adjustment with 2 M HCl or 0.5 M NaOH). Then, 1 ml of diluted pancreatic α-amylase (Sigma, 40 mg enzme/ml Tris-maleate buffer) (15.4 units/mg) was added to the mixture, homogenized, and incubated for 16 hours in a shaking water bath at 370C.

After the incubation, sample was centrifuged (3000 rpm, 15 minutes) and supernatant was discarded. Residue was washed at least once with 10 ml of distilled water, centrifuged again and supernatant was discarded. Then, 3 ml of distilled water was added for moistening the sample and added 3 ml KOH 4M, mixed well and left for 30 minutes at room temperature with constant shaking

After that, the mixture was added approximately 5.5 ml of 2 M HCl, 3 ml of 0.4 M sodium

acetate buffer, pH 4.75 (pH adjustment with 2 M HCl or 0.5 M NaOH), and 80µl of diluted

amyloglukosidase (0.1% w/v), homogenized, and incubated at 600C for 45 minutes with constant shaking. Mixture then was centrifuged (15 minutes, 3000g), supernatant was collected. The residue was washed at least once with 10 ml of distilled water, centrifuged again and combined supernatant with that was obtained previously. Volume of the supernatant was measured and adjusted to 25-1000 ml, depending on RS content. Furthermore, resistant starch content was measured using phenol sulphuric acid method (AOAC 1990)

Total Glucose Analysis (Phenol Sulfuric Acid Method) (AOAC, 1990)

Resistant starch would be measured using phenol sulfuric acid method (AOAC, 1990). Glucose curve standard was made by dissolving 50 mg glucose and adjusted with distilled water into 100 ml by volumetric flask, and 5 ml of that solution was adjusted into 50 ml. Series of dilution was prepared from that glucose standard stock: 0.0; 0.2; 0.4; 0.6; 0.8; and 1.0 ml. The series of dilution was adjusted with distilled water into 1.0 ml of total volume for each series. For each series was added by 0.5 ml fenol 5% and 2.5 ml of concentrated H2SO4, it was homogenized and the absorbance was measured using spectrophotometer at 490 nm.

Diluted solution (supernatant) from previous method was taken 0.2 ml and it was adjusted with distilled water into 10 ml by volumetric flask. In the amount of 1 ml of that solution was added by 0.5 ml fenol 5% and 2.5 ml of concentrated H2SO4, it was homogenized and the absorbance was measured using spectrophotometer at 490 nm.

14

3.2.2

IN VITRO FERMENTATION

3.2.2.1

Peptone Yeast Glucose Preparation (Purwani and Suhartono, 2009)

Peptone Yeast Glucose medium composition and mineral mix could be seen in Table 3 and Table 4. All the ingredients, except cystein hydrochloride, haemin solution, and vitamin K solution were mixed. The mixture was boiled to dissolve the ingredient and it was cooled by spraying it with CO2 gas. After that, the medium solution was added by Vitamin K, haemin solution, and Cystein-HCl x H2O. The medium was adjusted to pH 7.2 with NaOH 8 N. The medium was distributed into 20 ml amount into serum bottle flushed with CO2. The bottle was sealed with a butyl rubber septum and autoclaved at 1210_{C for 15 minutes to sterilize. The remaining medium could be store in refrigerator} before next uses.

Table 3. Medium composition of PYG for 1000 ml

Component Amount Unit

Tripticase peptone (trypton) 5.00 g

Peptone 5.00 g

Yeast extract 10.00 g

Beef extract 5.00 g

Glucose 5.00 g

K2HPO4 2.00 g

Tween 80 1.00 ml

Resazurin 1.00 mg

Salt solution / mineral mix solution 40 ml

Distilled water 950 ml

Haemin solution 10 ml

Vitamin Ksolution 0.20 ml

Cystein-HCl x H2O 0.50 g

Vitamin K, haemin solution, Cystein-HCl x H2O was added after it was boiled and cooled by spraying it with CO2 gas. Medium was adjusted to pH 7.2 with NaOH 8 N. The medium was flushed again with CO2 and autoclaved at 1210C for 15 minutes to sterilize.

Table 4. Mineral mix solution composition

Mineral Amount Unit

CaCl2 x 2H2O 0,25 g

MgSO4 x 7H2O 0.50 g

K2HPO4 1 g

KH2PO4 1 g

NaHCO3 10 g

NaCl 2 g

15

3.2.2.2

Activation

Eubacterium Rectale 17629

(Purwani and Suhartono, 2009)

Culture of Eubacterium rectale 17629 needed to be activated to begin fermentation step. Activated was done by inoculating 10 ml of Eubacterium rectale 17629 strain into 10 ml sterilized Peptone Yeast Glucose (PYG) while flushed with CO2 and incubated 370C in anaerobic condition for 24 hours. Stock of bacteria culture was preserved by saving it in cold temperature (50C). Bacteria culture was activated every once a month or before it was used as a fermentative culture.

3.2.2.3

In Vitro Fermentation Process (Purwani and Suhartono, 2009)

Experiment I was design to evaluate the growth of Eubacterium rectale 17629, its

fermentation capability (SCFA production pattern and optimum time that produce the highest butyrate concentration. Glucose (5 g/l) (0.5%) and RS3 (derived from sweet potato of Sukuh variety starch treated with pullulanase) in amount of 20 g/l (2%) were added into medium. The medium with resistant starch as a substrate was distributed into 20 ml of medium and inoculated with 1 ml of 24 hours pre-culture. Incubation was performed at 370C for 6, 12, 24, 36, and 48 hours.

Experiment II was conducted to analyze the effect of resistant starch concentration on short chain fatty acid profile during in vitro fermentation of Eubacterium rectale 17629. Fermentation was carried out using glucose (5 g/l) (0.5%) and RS3 (10 g/l) (1%) into medium 20 ml. The medium was inoculated with 1 ml of 24 hours pre-culture of Eubacterium rectale. Incubation was performed at 370C for 48 hours and also incubated at the optimum time that produced the highest butyrate in the first experiment (36 hours).

3.2.2.4

Analysis of Product Fermentation

a. Measurement of pH value

The pH of the fermentation culture for each sampling was measured using pH-meter. Lower result of pH in the fermentation medium showed higher production of short chain fatty acid by

Eubacterium rectale 17629.

b. Turbidity Measurement (Purwani and Suhartono, 2009)

Sampling which was performed every 6 hours and fermentation product in optimum time was measured the cell growth. Cell growth was determined qualitatively by using turbidimetry method. Absorbance of fermentation product suspense was measured using spectrophotometer at 660 nm. Higher result of absorbance showed higher cell turbidity and higher growth of the bacteria cell in the fermentation medium.

c. Short Chain Fatty Acid Analysis using Gas Chromatography (Purwani and Suhartono, 2009)

Short chain fatty acid (SCFA) production was measured using Gas Chromatography (Agilent technologies 7890A). Culture sample was centrifuged at 10000 rpm for 10 minutes. Supernatant was collected into a 1.5-2 ml effendorf tube for storage at 40C until use. Before injecting sample, 94 µl of sample was spiked 2 µl of acetic acid ALDRICH 71251), 2 µl of propionic acid (SIGMA-ALDRICH 94425), and 2 µl of butyric acid (SIGMA-(SIGMA-ALDRICH 19215). Standard curve for each compound was also made to determine SCFA concentration in the sample. SCFA standard mixture for standard curve was shown in Table 5.

Mixture (sample and SCFA spike) was injected into a high resolution gas chromatography (Agilent Technologies, 7890 GC System) equipped with a flame ionization detector and a HP Innowax 19091-136 column (60 m x 0.250 m). The carrier gas was helium with a flow rateof 1.8 ml/min, and the split ratio was 40:1. The oven temperature was maintained at 900C for 0.5 min, and

16 then increased to 1100C at a rate of 100C/min, increased to 1700C at a rate of 50C/min and finally increased to 2100C at a rate of 200C. Injector and detector temperatures were set into 2750C. Acetate, propionate, and butyrate were used for standard and the result was expressed as mmol/L.

Table 5. SCFA standard mixture for standard curve Acetic

Acid (µl)

Acetic acid conc. (M)

Propionic Acid (µl)

Propionic acid conc.

(M)

Butyric Acid

(µl)

Butyric acid conc. (M)

dH2O (µl)

5 0.1743 5 0.1334 5 0.1084 485

10 0.3487 10 0.2667 10 0.2167 470

15 0.5230 15 0.4001 15 0.3250 455

20 0.6974 20 0.5335 20 0.4334 440

CHAPTER IV

RESULT AND DISCUSSION

4.1

CHEMICAL CHARACTERISTIC OF SUKUH’S SWEET POTATO

STARCH

The moisture content, fat, protein, ash, carbohydrate, crude fiber, and amylose content of Sukuh sweet potato starch are shown in Table 6. Proximate analysis of Jago, Beauregard, and Evangeline starch are shown in Table 6 as the comparison for each other.

Table 6. Proximate analysis result of Beauregard, Evangeline, Jago, and Sukuh sweet potato starch

(dry bases)

Analyte Beauregard (%)* Evangeline (%)* Jago (%)** Sukuh (%)

Moisture 3.97 ± 0.7 1.83 ± 0.4 13.73 16.45

Ash 0.08 ± 0.1 0.07 ± 0.1 0.23 0.20

Crude fat 0.23 ± 0.3 0.11 ± 0.1 0.44 0.65

Protein 0.17 ± 0.0 0.24 ± 0.0 0.56 0.69

Crude Fiber Carbohydrate

0.06 ± 0.1 95.49

0.00 ± 0.00 97.75

0.00 85.04

0.00 82.01

Amylose 23.60 ± 1.20 27.10 ± 0.30 25.83 ± 0.21 29.35 ± 0.67

Source: *Futch, 2004; **Devega, 2011

Sukuh variety is national superior variety of sweet potato with compact structure. It has 4-4.5 month of harvest age. Production of this variety reach 25-30 ton/ha. It has spherical and ellipse form and has short stalk tuber. Sukuh has yellow skin color, white flesh and delicious taste. Sukuh has crude fiber content 0.85%, protein content 1.62%, total sugar content 4.56%, starch content 31.16% (RILET, 2008). Proximate analysis showed that Sukuh extracted starch has moisture content (16.45%), ash content (0.20%), crude fat content (0.65%), protein content (0.69%), carbohydrate content (82.01%), and crude fiber content (0%). Sukuh is potential to be developed in starch and flour industry, include resistant starch industry, because it has high content of starch and flour yield (RILET, 2008). Decreasing of protein and fiber content showed purity of extracted resulted starch.

Starch content of sweet potato depended on the variety as well as age during harvest. Sugar content fell significantly when harvest was done beyond 90 days, but the amylose content was greatly affected by the time of harvest (Antarlina and Kumalaningsih, 1990). Sukuh has harvest age 4-5 month (120 days), but it doesn’t influence the amylose content of the starch.

The fibre content in sweet potato varies to a great extent depending on varietals variation and age of the crop, where the fibre content increases with the maturity. Fibre content in starch derived from tuber extractions may vary to greater extent on the techniques and sieves, used for removal of the fibrous material. Sweet potato flour (containing 2-3% fibre) had different compositions compared to the isolated starch (having 0.1-0.15% fibre) (Moorthy, 2002).

Sukuh and Jago variety are the example of superior variety which is released by Research Institute for Legume and Tuber crops, Malang. Starch extraction of the sweet potato is obtained and it has high purity. It can be shown that the amount of protein and dietary fiber in sweet potato tuber was higher than in extraction starch. High purity of starch is needed to make enzyme hydrolisis effectively.

18 Starch-protein interaction and formation of amylose-lipid complexes reduced yield of resistant starch. Amylose-lipid complexes are enzyme-degradable,however retrogradation of amylose is dificcult to form crystalline structure and reduced yield of resistant starch (Sajilata, 2006).

In 1987, at the Loisiana State University Agricultural Center (LSU AgCenter) Larry Ralston developed the Beaurgard, a new variety of high quality, high yield sweet potato and The Evangeline sweet potato released by the LSU AgCenter in 2007 is a new variety of sweet potato developed by Dr Don LaBonte. (Futch, 2009). The Beauregard sweet potato yielded a starch with 23.6% amylose, while Evangeline, Jago, and Sukuh sweet potato starch contained 27.1%, 25.83%, and 29.35% amylose, respectively.Moorthy (2002) found that sweet potatoes have an amylose content around 20%. Most of chemical content of two variety is similar with Sukuh sweet potato. The quality of chemical content sweet potato starch is affected mainly by the biological and environmental factors. These factors include genotype, soil types, and climatic conditions, which are very different from one crop to another (Katayama et al, 1999).

Differentiation result in proximate analysis appeared in the moisture content between Beauregard and Evangeline with Jago and Sukuh. Both of Beauregard and Evangeline have low content of moisture content compared with Jago and Sukuh. Differentiation in method of starch extraction influence the differentiation of moisture content. In extraction of Beauregard and Evangeline, pulp of sweet potato passed through a 150 µm sieve then it was centrifuge at 3000 g. The precipitate was removed and freeze dried (Futch, 2009). Compared with method of Jago and Sukuh starch extraction, pulp of sweet potato was not sieved through a 150 µm. Pulp of Jago and Sukuh sweet potato was sieved by adding distilled water with the ratio sweet potato : distilled water was 1 : 3 (b/v), then sieved by pore material. Starch was precipitated and washed by distilled water, then it was dried by oven. Water removal by freeze drier resulted lower moisture content on Beauregard and Evangeline starch compared with Jago and Sukuh which were dried by dry oven.

4.2

PULLULANASE ENZYME ACTIVITY

Debranching using pullulanase has been applied to produce a sample with linier, low-molecular-weight and recrystallizable polymer chains. Debranching enzymes such as pullulanase rapidly hydrolize only α-1.6-glucosidic bonds, releasing a mixture of long and shorter unit chains from the parent amylopectin molecule. These fragments are linier polymers containing about 10 to 65 anhydroglucose units linked by α,1-4-glucosidic bonds. The debranched starch was then subjected to temperature cycling and incubation to induce retrogradation and yield the RS (Leong, 2007).

Industrial pullulanase (Promozyme®), rather than highly purified pullulanase was used in this study to establish the feasibility of ultimately developing a process for commercial application. Pullulanase enzyme activity assay measured the amount of reducing sugar formed by the hydrolitic action of the enzyme on starch. The original unit (IU) was defined as the amount of enzyme which produces reducing substances, expressed as glucose, during incubation time (10 minutes) under stated condition; although this is not a true representation of the actual course of the reaction.

Based on this study, pullulanase activity reached 177, 222.11 IU/ml. It was defined as the

amount of enzyme (ml enzyme) which produced µmol glucose as released by debranching enzyme

(Anonim, 1963). While compared with standard activity and density of Promozyme® D2 were 1350 NPUN/g and 1.20 g/ml, respectively. This starch degrading enzyme was obtained from Novo Nordisk through the PT Halim Sakti Pratama, Jakarta.

4

s a b s a s i c g r g o T

4.3

PRO

Gener stage is heatin absorption dur become strong starch granule. amylopectin c starch gel is no in the insoluble Polym critical nuclei) growth and pe rate whereas th generally proc of starch, at ab Produ Table 7. Figur

Production 1 2 3 4 5 6 7 Yi el d (% )

ODUCTION

rally, formatio ng process wh ring heating pr ger than starch . The second st hain. Gelatiniz ot stable and fo e short chain p mer recrystalli ), propagation ( erfection). The

he maturation eeds rapidly w bout 5oC (Gray uction of type-re 5 explains co

Tab Starch Weigh (g 20.01 20.00 20.00 20.01 20.03 20.07 20.02 Figure 10,6 10,8 11 11,2 11,4 11,6 11,8 1 11,76

N OF TYPE

on of type III r

ich will trigge ocess. Water a h molecule affi tage is starch r zed starch is e orm crystal wh polymer and res

sation is a th (crystal growth e nucleation an rate is more t when the incuba

and Bemiller, -III resistant st omparison star

ble 7. Yield of t

g)

Resist Produc

e 5. Yield of Su 2

6 11,75 ₁

E-III RESIST

resistant starch er gelatinizatio absorption is ca

inity inside the retrogradation easier to be di hen cooled (retr sistant to diges hree-stage proc h from the nucl nd propagation temperature de ation temperatu

2003). tarch using me rch weigh and r

type-III resista tant Starch ct Weigh (g)

2.35 2.35 2.34 2.21 2.22 2.22 2.22

ukuh type-III r

3 4

11,71

11,05

Producti

TANT STA

h could be div on. The starch aused by kinet e granule such which cause re igested than th rogradation). S stive enzymes ( cess that invo lei formed) and n rates determi

ependent (Eerli ure is close to t

ethod of Vatan result of type-I

ant starch produ

Yield 11 11 11 11 11 11 11 resistant starch 5 11,08 ₁ on

ARCH

vided into two

granule swell tic energy from h that water wi ecrystallization he raw starch Starch retrograd

(Wasserman et olves nucleatio d maturation (c ine the overall ingen et al., 19 the glass transi

nasuchart (201 III RS product

uction d (%) .76 .75 .71 .05 .08 .05 .09 product 6 7 1,05 11,09 Sukuh's RS

o stages, the fi led due to wat m water molecu ill come into t n of amylose a even though t dation will resu t al, 2007) on (formation

continued crys recrystallisati 995). Nucleati ition temperatu

10) is showed . Mean (%) 11.36 ± 0.36 S 19 rst ter ule the and the ult of tal on on ure in

20 Yield of Sukuh resistant starch product using method of Vatanasuchart (2010) was 11.36 ± 0.36%. Sukuh resistant starch has higher yield of RS III product compared with Jago 10.91 ± 0.63% (Devega, 2011) but lower than Salossa sweet potato (20%) (Evalin, 2011), sago RS III (18%) (Purwani and Suhartono, 2009), cassava RS III dried by extrusion (30%), hot air (21.5%), and spray drier (14.3%). Lower yield of the RS III product caused lost of starch material in each followed step of RS III production. In gelatinization step, starch was moved into plastic then it was moved again into erlenmeyer for hydrolyze sample using pullulanase treatment in shaking waterbath. There was some material which was left in the previous medium when the starch was moved. Beside that, drying using spray drier resulted lower yield of RS III product than using extrusion, hot air, or drum drier (Vatanasuchart, 2010). High temperature of spray drier made high amount of starch was evaporated and resulted in lower yield of RS III product.

Debranching pullulanase enzyme resulted starch consist of debranched amylopectin unit chains and some hydrolyzed amylose fraction which has lower molecular weigh fraction. RS also contained a starch fraction with structural properties similar with amylose which could be soluble in liquid starch mix. Soluble fraction of the starch could be lost along centrifugation step. Centrifugation separated starch fraction and amylose fraction could be soluble in liquid fraction and separated from precipitate of RS III and resulted in lower yield of RS III product.

RS yields in gelatinized starch depend strongly on process condition like gelatinization temperature, enzyme hydrolysis condition (pH, temperature, time), enzyme concentration, retrogradation condition, and drying. Retrogradation time and temperature influence strongly with yield of resistant starch product. Indeed, it was concluded that the formation of highly resistant starch fractions in gelatinized starch can be considered as crystallization of amylose in a partially crystalline polymer system (Eerlingen and Delcour, 1995). Formation of B-type crystals is needed to make type-III resistant starch while at lower temperature (0-50C) it was observed formed. Crystallinity of the resistant starch fraction was comparable with storage time of the starch gel (Eerlingen and Delcour, 1995).

Specific method to determine RS in foods is classified as direct method and indirect method. Direct method quantifies RS in the residues obtained after removing digestible starch (Berry, 1986). Indirect method determines RS as the difference between total starch and digestible starch (Tovar et al., 1990; Englyst et al., 1992). Goni (1996) has been developed a direct method to quantify RS in food and food product. It was derived from Berry (1986) method with essential modifications. The main features of the analytical procedure are: removal protein; removal of digestible starch; solubilization and enzymatic hydrolysis of RS; and quantification of RS as glucose released x 0.9 where stomach and intestine physiological conditions (pH, transit time) are approximately simulated (Goni, 1996).

Resistant starch product derived from Sukuh sweet potato contained higher RS content (30.83 ± 2.44 %) (Table 9) than those from rice starch (21-26%), sago starch (31-38%) (Purwani and Suhartono, 2009), Jago sweet potato starch (28.15) but lower than Salossa sweet potato starch (38.22%) (Evalin, 2011). The difference could have been caused by several factors such as starch and enzyme used and also heating and cooling condition. Purwani (2009) reported that resistant starch content of Sukuh sweet potato was 13.77%. Increasing of resistant starch content happened caused by heating, pullulanase treatment, and retrogradation process of the starch to form crystalline structure of amylose.

Amylose content, RS yield and resistant starch content are positively correlated (Berry, 1986). Increasing of amylose content influence with low starch degradability and increasing the yield of resistant starch (Escarpa et al., 1996). When heated to about 500C, in the presence of water, the

21 amylose in the granule swells; the crystalline structure of the amylopectin disintegrates and the granule ruptures. The polysaccharides chains take up a random configuration, causing swelling of the starch and thickening of the surrounding matrix such as, gelatinization- a process that renders the starch easily digestible. On cooling / drying, recrystallization (retrogradation) occurs. This take place very fast for the amylose moiety as the linier structure facilitates cross linkages by means of hydrogen bonds. Crystallization of amylose in retrogradation process made starch become resistant and difficult to access by enzyme and reduced starch digestibility (Sajilata, 2006).

The rate and extent to which a starch may retrograde after gelatinization essentially depends on the amount of amylose present. Repeated autoclaving of wheat starch may generate up to 10% RS. The level obtained appeared to be strongly related to the amylose content, and the retrogradation of amylose was identified as the main mechanism for the formation of RS that can be generated in larger amounts by repeated autoclaving (Berry, 1986). Sukuh starch has high amylose content (29.35 ± 0.67%). High amylose content in Sukuh starch influence the formation and content of type-III resistant starch.

Compared with Purwani and Suhartono (2009), RS 3 from sago and rice starch were treated with pullulanase, α-amylase, and both of them. Among the three treatment of enzyme applied to the starch, using pullulanase alone resulted least breakdown of the starch. Following pullulanase digestion, the liquid present in the flask appeared clear and odorless. It was contrast with using amylase or enzyme cocktail of amylase and pullulanase. When amylase or its combination with pullulanase was applied, the liquid present in the flask showed brown and sweet smelling.

Formation of resistant starch also depends on the water content and autoclaving temperature. Indeed, as the amylose concentration increase, RS yield increases. A minimum of water, however, is necessary for plasticization of the environment and for the incorporation into the crystal structure (B-type crystal structure indeed contain about 27% water). The influence of the autoclaving temperature varies with starch type. Autoclaving at 1480 C, however, results in crytal melting (Eerlingen, 1995).

Positive effect of starch debranching enzyme also happened on the formation of resistant starch. The hydrolysis of α-1,6 glycosidic bonds would produce more free linear chains in the hydrolyzate. These linear chains, as similar to amylose, could participate in crystal formation by chain elongation and folding. These newly formed crystals could become more perfect during storage of the hydrolizate. Hydrolisis of α-1,6 glycosidic bonds could disentangle, from aylopectin, the double helices and crystallite, which are formed by the re-association of amylopectin A-chains during retrogradation. These disentangled starch entities have unassociated linier chain segments at both ends. Storage at particular temperature and time would like promote association of these free linier chain segments and their close packing. Consequently, the number of perfect starch crystals would increase. Without disentanglement from the amylopectin molecule, the association of the linier segments starch entities and subsequent crystallite formation may be difficult due to lower flexibility of the starch chain segments owing to their closer proximity to the branching points (Leong et al., 2007).

4.4

ANALYSIS OF FERMENTATION PRODUCT (EXPERIMENT I)

In vitro fermentation of RS3 was divided into 2 experiments. Experiment I was design to evaluate the growth of Eubacterium rectale 17629 include fermentation capability (SCFA production pattern and optimum time that produce the highest butyrate concentration. Glucose (5 g/l) (0.5%) and RS3 (derived from sweet potato of Sukuh variety starch treated with pullulanase) in amount of 20 g/l (2%) were added into medium. Concentration of 2% Sukuh type-III resistant starch was chosen

22 referred to Evalin (2011) that reported 2% Salossa sweet potato type-III resistant starch give highest concentration of butyric acid.

In experiment I, the medium with resistant starch as a substrate was distributed into 20 ml of medium and inoculated with 1 ml of 24 hours pre-culture. Incubation was performed at 370C for 6, 12, 24, 36, and 48 hours. Interval time was chosen to simulate physiological condition in the digestive system.

4.4.1

Turbidimetry and pH measurement

Growth of Eubacterium rectale 17629, its fermentation capability (SCFA production pattern and optimum time that produce the highest butyrate concentration was evaluated in Experiment I. Glucose (5 g/l) and RS3 (derived from sweet potato of Sukuh variety starch treated with pullulanase) in amount of 20 g/l (2%) were added into medium. Sampling was done after 6, 12, 24, 36, and 48 hours of incubation at 370C.Turbidimetry result and pH change is presented in Figure 6 and Figure 7, respectively.

Fermentation medium without bacteria culture is used as a blank. Fermentation product for each sampling was measured using spectrophotometry at 660 nm. Higher result of absorbance showed higher cell turbidity and higher growth of the bacteria cell in the fermentation medium. Result showed that the absorbance (cell growth) increased after 6 and 24 hours. Fermentation reached the highest absorbance at 48 hours of fermentation. It showed that Eubacterium rectale 17629 could use resistant starch substrate from Sukuh sweet potato. Eubacterium rectale 17629 could produce amylase to degrade the resistant starch into glucose. They used resistant starch as carbon source for its growth.

Figure 6. Turbidimetry result of E.rectale 17629 growth over 48 h fermentation at 370C 0,186

0,188 0,188

0,193

0,198

0,184 0,186 0,188 0,19 0,192 0,194 0,196 0,198 0,2

0 10 20 30 40 50 60

Absorb

anc

e (660nm)

23 Figure 7. Change of pH fermentation by E.rectale 17629 over 48 h at 370C

Decreasing of pH was observed over the time course of fermentation. The lowest pH value (4.16) was achieved at 48 h fermentation. Reduction of pH value occurred as the bacteria produce short chain fatty acid during fermentation process. SCFA production made the fermentation suspension become acid and decreasing pH value of medium. Lower pH value are believed to prevent the overgrowth of pH-sensitive pathogenic bacteria and lower the production of potentially harmful toxic or carcinogenic products in the colon, including secondary bile acids and protein fermentation products (ammonia and phenols) (Topping et al, 2001).

4.4.2

Production of Short Chain Fatty Acid

Bacteria of Eubacterium rectale is reported to be one of the most abundant bacterial species in human feces both from anaerobic cultivation (Finegold et al., 1983; Moore & Holdeman, 1986) and culture-independent analysis of 16S rRNA sequences (Aminov et al., 2006). The genus Eubacterium

includes many species of obligately anaerobic bacteria, with Eubacterium limosum designated as the type species. Species of the genus Eubacterium produce mixture of organic acids as fermentation product from carbohydrate, including butyric, acetic, lactic or formic acids, but not propionic and succinic acids, as major products (Krumholz & Bryant, 1986). Most also produce hydrogen gas.

The profile and and proportion of SCFA produced by Eubacterium rectale 17629 is

presented in Figure 8 and Table 8. Gas chromatography result showed that acetic, propionic, and butyric acid were produced during 48 hours of fermentation. Chromatogram result is explained in Appendix. Based on chromatogram, peak of acetic acid, propionic acid, and butyric acid is appeared approximately at 8.6; 10.08; and 11.79 minute. Peak of acetic acid firstly appeared at chromatogram because it has higher volatility than propionic and butyric acid. Volatility is influenced by differentiation of fatty acid’s boiled point. Chromatogram result based on fatty acid peak was quantified by fatty acid standard chromatogram. The highest production of butyrate was achieved at 36 hours (50.97 mmol/L) while propionate was achieved at 36 hours (25.17 mmol/L). The value zero was applied for zero or negative concentration based on standard curve calculation.

7,21

5,07 4,79

4,37 _{4,26 4,16}

0 1 2 3 4 5 6 7 8

0 10 20 30 40 50 60

pH

75 Appendix 27. SCFA chromatogram for 36 hours fermentation in Experiment II

77 Appendix 28. SCFA chromatogram for 48 hours fermentation in Experiment II

79 Appendix 29. SCFA chromatogram for 48 hours fermentation in Experiment II