Short chain fatty acid (SCFA) profile produced by Clostridium butyricum grown on medium containing type 3 resistant starch (RS3) of sweet potato

i SHORT CHAIN FATTY ACID (SCFA) PROFILE PRODUCED

BY Clostridium butyricum GROWN ON MEDIUM CONTAINING TYPE 3 RESISTANT STARCH (RS3) OF SWEET POTATO

BACHELOR THESIS

MICHAEL DEVEGA F24070118

FACULTY OF AGRICULTURAL ENGINEERING AND TECHNOLOGY BOGOR AGRICULTURAL UNIVERSITY

BOGOR 2011

ii SHORT CHAIN FATTY ACID (SCFA) PROFILE PRODUCED

BY Clostridium butyricum GROWN ON MEDIUM CONTAINING TYPE 3 RESISTANT STARCH (RS3) OF SWEET POTATO

Michael Devega and Maggy Thenawidjaja Suhartono

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

PO BOX 220, Bogor, West Java, Indonesia

Phone: +62 819 0415 1448, e-mail: miqael_devega@yahoo.co.id ABSTRACT

RS type 3 (RS3) is retrograded starch, which is not digested by human starch degrading enzyme and will undergo bacterial fermentation in the colon. The main fermentation products are the short chain fatty acid (SCFA) acetate, propionate and butyrate. Butyrate arising from microbial fermentation is important for the energy metabolism of colonic epithelial cells and has a mainly protective role in relation to colonic disease. The purpose of this research was to study short chain fatty acid profile produced by Clostridium butiricum BCC B2571 isolated from human faeces during fermentation in medium containing RS3 from sweet potato (Ipomoea batatas). Pullulanase enzyme (34,779 U/mgprotein) at 5% starch weight was used to hydrolyze α-1,6-glucosidic bonds and the incubation was carried out at 24 hours at 50oC, stored at 4oC at 24 hours to trigger retrogradation, and then dried by spray drier to produce resistant starch type 3. RS3 was fermented in reinforced clostridial medium by Clostridium butyricum BCC B2571 isolated from human faeces. In vitro fermentation resulted in short chain fatty acid (SCFA) at different levels depending on the RS and the concentrations of glucose in the medium. Fermentation of 20 g/l (RS3) and 1 g/l (glucose) resulted in SCFA with the molar ratio of acetate, propionate, and butyrate of : 50 mM : 37 mM : 68 mM (1.3:1:1.8) at 12 h fermentation whereas fermentation of 10 g/l (RS3) and 5 g/l (glucose) resulted in SCFA with the molar ratio of acetate, propionat, butyrate of :453 mM : 248 mM : 225 mM (2:1.1:1) at 48 h fermentation. This result indicated that longer fermentation time and higher glucose concentration could increase butyric acid level but there was metabolic shift to higher formation of acetic acid.

iii Michael Devega. F24070118. Short Chain Fatty Acid (SCFA) Profile Produced By Clostridium butyricum Grown On Medium Containing Type 3 Resistant Starch (RS3) of Sweet Potato. Supervised by Maggy Thenawidjaja Suhartono 2011.

SUMMARY

Gastrointestines is an important part of body metabolism which affects human health. One of the deseases which attack gastrointestines is colorectal cancer (CRC) which can be prevented by proper lifestyle and consumption of healthy functional food. SCFA are the main products of anaerobic microbial fermentation in the large intestine and affect colonic health by providing energy to the epithelial cells. The organic acids produced in the colon are acetic acid, propionic acid, and butyric acid. Interest in butyrate role as a possible protective agent has arisen from its anti-proliferative effects on cells in vitro including colon tumour cell lines.

Resistant starch (RS), is retrogradaded starch that escapes digestion in the small intestine and can be fermented in colon by anaerobic bacteria producing SCFA which contributes to the gastrointestinal health, and thus can be classified as funtional food. The crystalline non-granular starch (RS3) can be obtained from many starchy sources, such as rice, corn, maize, wheat, sago, banana, potato, and sweet potato. In this study, jago sweet potato used contain 31% starch content. Jago sweet potato is usually consumed indirectly as the taste is not preferable.

The objective of the research was to study short chain fatty acid profile produced by

Clostridium butiricum BCC B2571 isolated from human faeces during fermentation of type 3 resistant starch of sweet potato (Ipomoea batatas). This reserach was devided into two stages, (1) chemical analysis and (2) fermentation of type three resistant starch by Clostridium butyricum.

The result of proximate analysis showed that jago sweet potato starch contained water 13.73%, protein 0.44%, ash 0.23%, fat 0.56%, and carbohydrate 85.04%. Amylose content of jago sweet potato starch was 25.83%. The resistant starch was made by applying pullulanase enzyme (34,779 U/mg protein) to hydrolyze the α-1,6-glucosidic bonds and the incubation was carried out at 24 hours at 50oC, stored at 4oC at 24 hour to trigger retrogradation, and then dried by spray drier to produce resistant starch type 3. Resistant starch content of jago sweet potato treated with pullulanase enzyme was 28.15%. In vitro fermentation of jago sweet potato resistant starch by Clostridium butyricum

BCC B2571 resulted in short chain fatty acid (SCFA) at different levels depend on the RS and the concentration of glucose in the medium. Fermentation of 20 g/l (RS3) and 1 g/l (glucose) resulted in SCFA with the molar ratio (acetate:propionate:butyrate) 50 mM : 37 mM : 68 mM (1.3:1:1.8) at 12 h fermentatition, whereas fermentation of 10 g/l (RS3) and 5 g/l (glucose) resulted in SCFA with the highest molar ratio (acetate:propionate:butyrate) 453 mM : 248 mM : 225 mM (2:1.1:1) at 48 h fermentation. This result indicated that longer fermentation time and higher glucose concentrations could increase butyric acid levels but there was metabolic shift to higher formation of acetic acid.

iv SHORT CHAIN FATTY ACID (SCFA) PROFILE PRODUCED

BY Clostridium butyricum GROWN ON MEDIUM CONTAINING TYPE 3 RESISTANT STARCH (RS3) OF SWEET POTATO

BACHELOR THESIS

In the partial fulfillment of the requirement of degree of SARJANA TEKNOLOGI PERTANIAN at the Department of Food Science and Technology Faculty of Agricultural Engineering and Technology

Bogor Agricultural University

By :

MICHAEL DEVEGA F24070118

FACULTY OF AGRICULTURAL ENGINEERING AND TECHNOLOGY BOGOR AGRICULTURAL UNIVERSITY

BOGOR 2011

v Title : Short Chain Fatty Acid Profile (SCFA) Profile Produced by Clostridium butyricum

Grown On Medium Containing Type 3 Resistant Starch (RS3) of Sweet Potato Name : Michael Devega

Student ID : F24070118

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

vi

STATEMENT LETTER OF THESIS AND SOURCE OF

INFORMATION

Hereby I genuinely stated that the bachelor thesis entitled Short Chain Fatty Acid (SCFA) Profile Produced by Clostridium butyricum Grown On Medium Containing Type 3 Resistant Starch (RS3) of Sweet Potato is an authentic work of mine under supervision of academic advisor 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 the references at the end of the bachelor thesis.

Bogor, July 2011 The undersigned,

Michael Devega

vii © Created right owned by Michael Devega, 2011

All rights reserved

This thesis may not be translated or copied in whole or in part without written permission from Bogor Agricultural University in any forms or by any forms,print, photocopy, microfilm, etc.

viii

AUTHOR BIOGRAPHY

The author was born in Bandar Lampung, on November 14th 1989 to Hendrik Devega and Marijam couple. He is the eldest son with one sister and one brother; Ivonnie Devega and Kevin Devega. He studied in SD Fransiskus II Bandar Lampung (1995-2001), SLTP Xaverius I Bandar Lampung (2001-2004), and SMA Regina Pacis Bogor (2004-2007). In 2007, the author continued his further study at IPB with SPMB (Seleksi Penerimaan Mahasiswa Baru) path and he was accepted as Food Science and Technology student in the Faculty of Agriculture Technology.

During his study, he joined organizations such as KEMAKI (Keluarga Mahasiswa Katolik), HIMITEPA (Himpunan Mahasiswa Teknologi Pangan) and he was active in activities such as LCTIP XVI (2008) and XVII (2009), IFOODEX (2009), HACCP VII (2009), and BAUR (2009). The author has work expriences as General Sociology assistant in 2009 to 2010, Food Technique Principal laboratory assistant in 2010, and physics private teacher in 2008 in Forces and Express. The achievements that the author get included First Runner Up in Indonesian Food Bowl Quiz Competition (2011), five research grants and one entrepreneur grant in Pekan Kreativitas Mahasiswa (PKM) held by DIKTI (2011). In October 2010, he started his research entitled “Study of Short Chain Fatty Acid Profile Produced by Clostridium butyricum During Fermentation of Type 3 Resistant Starch of Sweet Potato (Ipoema batatas)” supervised by Prof. Dr. Ir. Maggy Thenawidjaja Suhartono.

ix

PREFACE

First and foremost, Praise to my Lord Jesus Christ for giving me strength to accomplish my research and thesis. All Your blessing motivated my self to achieve my bachelor degree. This bachelor thesis which is entitled “Short Chain Fatty Acid Profile Produced by Clostridium butyricum Grown on Medium Containing Type 3 Resistant Starch (RS3) of Sweet Potato”, was based on research conducted from October 2010 to April 2011. The writer would like to thank :

1. My father Hendrik Devega, my mother Marijam, my sister Ivonnie Devega, my brother Kevin Devega. Thank you for all your love, support, and pray.

2. Prof. Dr. Ir. Maggy Thenawidjaja Suhartono as my academic supervisor for her enormous help both academically and financially throughout the completion of my thesis. I am eternally grateful for her inspiring advice, warm support and encouragement, and also the flexibility he gave me in conducting my research.

3. Dr. Ir. Slamet Budijanto, M.Agr and Dr. Didah Nur Faridah, STP, M.Si as my thesis examiners, whose criticism and suggestions would help me accomplish my thesis.

4. Endang Yuli Purwani for providing all my research requirements, all her guidances, availability whenever required, and all accommodations during research at Sukamandi.

5. Miss Ika Malikha, Mr Marwan Wahyudi, and Ms Desi Awalina for their help in the laboratory. I am grateful for their helpful suggestions and advices.

6. Lecturers of Department of Food Science and Technology for the knowledge that enable me to do this research.

7. My beloved friends in ITP 44 : Reggie, Adi, Melia, Oni, Eliana, Kurnia, Trancy, Mumun, Mba Mus, Renny, Amel, Lisa, Marki, Cherish (my eternal rival), Dimas, Bertha, Ronald, Chyntia, Wima, Punjung, Puji, Icank, Septi, Marvin, Danil, Irsyad, Chandra, Ashari, Fitri,.

8. My Yochan and Yobichan Team : Desir, Alya, Lia, Elvita, Tami, Ni Putu, Meiada, Ulfa, Aisyah, Annisa, Tiara, Lukman.

9. Member of Zeamaysindo : Mitha, Hanna, Sarah, Khafid, Ricky, Uli, Riffi, Hanna, Uswah, Arif, Vendry, Laylia, Adel, Nida, Canov, Irwan, Indri, Fieki, Sindu, Nidya and (alm) Rina.

10. Laboratory assistants in Food Science and Technology Department and SEAFAST Centre. 11. Staff of UPT Food Science and Technology Department : Bu Novi, Mba Anie, Bu Kokom. It is truly hoped that this bachelor thesis will give a worthy addition to the existing knowledge on food science technology area and the readers will get useful informations.

Bogor, July 2011

x

TABLE OF CONTENT

Page

PREFACE ... ix 

TABLE OF CONTENT ... x 

LIST OF TABLE ... xi 

LIST OF FIGURE ... xii 

LIST OF APPENDIX ...xiii 

I.  INTRODUCTION ... 1 

A. BACKGROUND ... 1 

B. OBJECTIVE ... 2 

II.  LITERATURE REVIEW ... 3 

A. SHORT CHAIN FATTY ACID (SCFA) ... 3 

B. RESISTANT STARCH ... 4 

C. Clostridium butyricum ... 5 

D. SWEET POTATO ... 6 

III.  MATERIAL AND METHOD ... 8 

A. MATERIALS AND EQUIPMENTS ... 8 

B. METHODS ... 8 

IV. RESULT AND DISCUSSION ... 14 

A. CHEMICAL ANALYSIS ... 14 

B. RESISTANT STARCH PRODUCTION ... 15 

C. FERMENTATION OF RS3 by Clostridium butyricum ... 16 

V. CONCLUSION AND RECOMMENDATION ... 25 

A. CONCLUSION ... 25 

B. RECOMMENDATION ... 25 

REFERENCE ... 26 

xi

LIST OF TABLE

Page

Table 1. Resistant Starch Classification ...……... 4

Table 2. Starch Content in Sweet Potato... 6

Table 3. Total Production of Sweet Potato in Indonesia... 6

Table 4. RCM Composition……... 12

Table 5. SCFA Standard Mixture for Standard Curve... 13

Table 6. Major Chemical Composition of Jago Sweet Potato Starch...……... 14

xii

LIST OF FIGURE

Page

Figure 1. Jago Sweet Potato...………... 7

Figure 2. Two Stages of Research………... 8

Figure 3. Resistant Starch Yield... 16

Figure 4. Changes of pH and Absorbances Medium over 48 h Fermentation 2% RS... 17

Figure 5. Profile of SCFA after 48 h Fermentation 2% RS ... 18

Figure 6. Changes of pH and Absorbances Medium over 48 h Fermentation 1% RS... 19

Figure 7. Profile of SCFA after 48 h Fermentation 1% RS ... 20

xiii

LIST OF APPENDIX

Page

Appendix 1. Amylose Content Analysis ………... 30 Appendix 2. RS Content Analysis………... 31 Appendix 3. Absorbance and pH Value during Fermentation………... 32 Appendix 4. Acetic, Propionic, and Butyric Acid Standard Curve for Fermentation of 2%

RS ... 33 Appendix 5. GC Area Result for Fermentation of 2% RS... 34 Appendix 6. Acetic, Propionic, and Butyric Acid Standard Curve for Fermentation of 1%

RS... 35 Appendix 7. GC Area Result for Fermentation of 1% RS..…………...……. 36 Appendix 8. SCFA Chromatogram…………....……...…. 37

1

I.

INTRODUCTION

A. BACKGROUND

Gastrointestinal is an important part of body metabolism which affect human health. Human realize that bad lifestyles and behaviors, such as smoking, heavy alcohol consumption, stress, high carbohydrate diet, inadequate consumption of fruits and vegetables can affect their gastrointestinal health. One of the deseases which attack gastrointestinal is colorectal cancer (CRC). Global cancer data showed that colon and rectum cancers (also known as colorectal cancer/CRC) accounted for about 1 million new casesin 2002 (9.4% of the world total). In termsof incidence, colorectal cancers rank fourth in frequency inmen and third in women in the world. Around 15% of CRC is inherited (direct and indirect), the others is acquired. Up to 80% of CRC cases have been attributed to diet. This fact shows that colorectal cancer can be prevented through healthy food and lifestyle.

Colorectal cancer can be prevented by the presence of short chain fatty acid (SCFA) in colon, especially butyric acid. SCFA are organic fatty acids with 1 to 6 carbon atoms which arise from bacterial fermentation of polysaccharide, oligosaccharide, proteins, peptide, and glycoprotein precursors in the colon. SCFA are the main products of anaerobic microbial fermentation in the large intestine and affect colonic health by providing energy to the epithelial cells. The organic acids produced in the colon are acetic acid, propionic acid, and butyric acid. The concentrations of SCFA in the colon are affected by composition of the diet, type and quantity of substrates that survive in the large intestine, and microorganism inside the intestine.

Interest in butyrate role as a possible protective agent has arisen from its anti-proliferative effects on cells in vitro including on colon tumour cell lines. In particular, butyrate is preferred as the energy source for the colonic mucosa and it has been implicated in the protection against colitis and colorectal cancer. Butyric acid also inhibit cancer cell growth by stimulating cancer cell to kill itself (apoptosis) and inhibit DNA repairing enzyme activity. Approximately 95% of the butyrate produced by colonic bacteria is transported across the epithelium, but concentrations in portal blood are usually undetectable as a result of rapid utilisation. Butyrate spesific benefit can be achieved by an intake of resistant starch, oat bran, and wheat bran that result in a good fermentation properties.

Nowdays, people pay more attentions to functional foods. Functional food is defined as a food which consumed not only to fulfill nutrition requirement but also give health benefit to our body. Many foods can be classified as functional food, one of them is resistant starch. Resistant starch is complex of undigest carbohydrate which can give benefit effect to our intestinal health. Resistant starch can be obtained from many sources, such as rice, corn, maize, wheat, sago, banana, potato, and sweet potato.

Resistant starch (RS) is starch that escapes digestion in the small intestine and can be fermented in colon by anaerobic bacteria resulting in the SCFA formation which contribute to the gastrointestinal health. Resistant starch (RS) can be classified into four main types, of which the first three may occur in a typical human diet. RS1 includes physically entrapped starch within whole plant cells and food matrices (e.g. coarsely milled grain). RS2 consists of native starch granules that are highly resistant to digestion by α-amylases (e.g. green banana, high amylose maize starch). RS3 comprises retrograded starches, formed when starchy foods are cooked and cooled. RS4 comprises chemically modified starches (e.g. esterified starches) where the modification interferes with the amylolytic activity of digestive enzymes.

Some carbohydrates, such as sugars and most starch, are rapidly digested and absorbed as glucose into the body through the small intestine and subsequently used for short-term energy needs

2 or stored. Resistant starch, on the other hand, resists digestion and passes through to the large intestine where it acts like dietary fiber. In large intestine, resistant starch fermented by microorganism resulting SCFA. Prevoius research showed that dominant microflora in human intestinal was

Clostridium, Eubacterium, and Fusobacterium that include butyrate producing species.

Sweet potato (Ipomoea batatas) is one of staple food in Indonesia because contains high carbohydrate level as energy source. Sweet potato can be consumed directly as food or processed further become sweet potato flour or sweet potato starch. Unfortunately, utilization of sweet potato is not optimum, however Indonesia is the biggest country which produce sweet potato in the after China. Total production of sweet potato was 2.057.913 ton per year. High carbohydrate level is correlated to high starch level, so it can be utilized as resistant starch source. In recent years, a considerable number of studies have focused on the importance of type 3 resistant starch (RS) as a substrate for colonic fermentation. Thus, sweet potato is very potential to develop as resistant starch source.

B. OBJECTIVE

The objective of the research were to study short chain fatty acid profile produced by

Clostridium butiricum BCC B2571 during fermentation of type 3 resistant starch of sweet potato and to determine the effect of concentration of resistant starch to the SCFA formation by Clostridium butiricum BCC B2571.

3

II.

LITERATURE REVIEW

A. SHORT CHAIN FATTY ACID (SCFA)

Human colonic bacteria ferment resistant starch (RS) and non-starch polysaccharides (NSP, major components of dietary fiber) to short chain fatty acids (SCFA), mainly acetate, propionate, and butyrate. Polymer substrate will be hydrolized into monomer like glucose, galactose, xylose, which then fermented through glycolisis pathway into pyruvate acid. These pyruvate acid is changed into short chain fatty acid and gases.

Short chain fatty acid is an organic fatty acid with 1 until 6 carbon atoms and is the principal anion which arise from bacterial fermentation of polysaccharide, oligosaccharide, proteins, peptide, and glycoprotein precursors in the colon (Cumming et al. 1991). The primary end products beside SCFA are gases (CO2, CH4, and H2) and heat. The general reaction of SCFA production and overall stoichiometry has been summarized for a hexose as follows :

59 C6H12O6 + 38 H2O → 60 CH3COOH (acetate) + 22 CH3CH2COOH (propinonic) + 18 CH3CH2 CH2COOH (butyrate) + 96 CO2 + 268 H+ + Heat.

The production of SCFA is determined by many factors, including the numbers and types of microflora present in the colon (Roberfroid. 2005), substrate source (Cook and Sellin, 1998), gut transit time, hexose availability, enzyme production by bacteria, and amount of carbohydrate. Fermentation involves a variety of reactions and metabolic processes in the anaerobic microbial breakdown of organic matter, yielding metabolizable energy for microbial growth and maintenance and other metabolic end product for host use. Various population data show that SCFA production in order of acetate>propionate>butyrate in a molar ratio of approximately 60:20:20 or 3:1:1, respectively in the proximal and distal colon (Topping and Clifton. 2001).

In ruminants and other herbivores, SCFA are absorbed and transported via the portal vein to the liver and it can be used as for maintanance, growth, and lypogenesis. The fraction not absorbed is distributed to the other body organs and tissues for metabolism. The SCFA produced by fermentation have a very important effect on the host by locally providing energy to the epithelial cells of the colon, decreasing the pH, improving the absorption of calcium, iron and magnesium and beneficial influences of the glucose and lipid metabolism in the liver.

Acetic acid (C2H4OH) is an organic acid which is colorless and gives vinegar sour taste. Acetate acid is absorbed and metabolized in liver, muscle, and brain tissue. Acetic acid is utilized by the liver where it is converted into Acetyl-CoA, which can act as a precursor for lipogenesis, but also stimulates gluconeogenesis (Remesy et al.1992). Low concentrations of acetic acid can also be detected in venous blood in peripheral tissues (Scheppach et al. 1991). Propionic acid is a clear, colorless liquid with a slightly sweetish odor. It has CH3CH2COOH chemical formula. Propionic acid is mainly metabolized in the liver and has been shown to inhibit gluconeogenis and increase glycolysis in rat hepatocytes. It has also been proposed that propionic acid may lower plasma cholesterol concentrations by inhibiting hepatic cholesterogenesis. Butyric acid is a carboxylic acid with the structural formula CH3CH2CH2-COOH and a product of anaerobic fermentation. Butyric acid is metabolized in colon ephitalium cell, it has role to regulate cell growth so it can maintain mucose cell and can reduce tumor cell proliferation. Butyrate is the main fuel for the colonocytes and thus exerts a trophic effect. Butyrate also appears to reduce cell differentiation and stimulate apoptosis in tumour cell lines (Douglas et al. 2006). Furthermore, it also regulates gene expression leading to

anti-4 inflammatory and anti-carcinogenic effects (Hamer et al., 2008). Total SCFA and regional differences in SCFA concentration are implicated in colon desease, especially in cancer and gastrointestinal disorders. Therefore, an increased SCFA production and higher delivery of SCFA distally, especially butyrate, may have a role in prevention these desease.

B. RESISTANT STARCH

Starch is a major reserve polysaccharide in plants. It is found in high levels in roots, tubers, cereal grains and legumes and is present as intracellular granules with different sizes and shapes depending on the starch source. In the diet of mankind and many animals, it is one of the most important carbohydrates. It consists mainly of two glucose polymers: amylose and amylopectin.

Resistant starch (RS) is defined as starch and products of starch degradation that cannot be absorbed in the small intestine of healthy individuals and, hence, might be fermented in the colon (Haralampu, 2000). There are four types of RS. Type I (RS1) represents physically inaccessible starch, which is locked in the plant cell walls of some foodstuffs, such as partially milled grains, seeds and legumes. Type II (RS2) is native granular starch found in food containing uncooked starch, such as bananas, raw potatoes and beans. Type III resistant starch (RS3) is made up of retrograded starch or crystalline non-granular starch, like the starch found in cooked and cooled potatoes, bread crust, cornflakes and retrograded, high-amylose maize starch. Type IV (RS4) refers to specific chemically and thermally modified or repolymerized starches (Englyst et al.,1992). Table 2 shows classification of resistant starch.

RS is not digested in the small intestine, because of that it has a lower energy content than digestible carbohydrates. The consequence of significant amounts of RS reaching the large intestine is the potential for fermentation by colonic micro-organisms. Short chain fatty acids (SCFA) produced in response to fermentation of RS are thought to be responsible for much of the intestinal and systemic effects reported for this fiber. (Englyst et al. 1992). Some factors associated with RS formation during these processes are the physical state of the food material (whole or ground) water content, pH, heating temperature and time, feed composition, number of heating or cooling cycles, freezing methods (slow vs rapid) and drying (Cummings, 1987).

Table 1. Resistant Starch Classification

Class Description Example of Source

RS1

Starch that escapes digestion in the small intestine due to physical protection by the food mixture (i.e., hull, shell, seed casing)

Whole grains, seeds, legumes

RS2

Raw starch granules (ungelatinized) with compact structure which limits accessibility of digestive enzymes

Green banana, raw potato, high-amylose corn starch, raw whole grain flours

RS3

Retrograded starch in which parts of the starch chain can crystallize into components that are less digestible. Most often, this occurs by cooking and cooling starch containing food

Cooked and cooled starch - corn, potato, rice, pasta

RS4

Not found naturally in foods. Starch that has been chemically modified to introduce bonds that are not digestible by human enzymes.

Does not occur in nature. Ingredient source of modified produced from wheat and tapioca are available

5 Some benefits of resistant starch are the slow hydrolysis of RS makes it useful for the slow release of glucose, which can be especially useful in controlling glycaemic plasma responses, increase faecal bulk, lower faecal pH, and increase excretion of butyrate and acetate (Philip et al. 1995). Besides physiological benefits in human, RS has been reported to have potential as a unique ingredient that can yield high-quality foods. For example, application tests of RS showed improvement of crispiness and expansion in certain products and better mouthfeel, colour and flavour as compared with products produced with traditional, insoluble fibres.

The process of making resistant starch is consisted of gelatinizing a slurry of the starch, treating the gelatinized starch with a debranching enzyme, deactivating the enzyme, cooling and isolating the starch product (Schmiedel et al., 2003). Gelatinization process is purposed to make debranching enzyme easier to hydrolize α-1,6 glicosidic bond. Cooling process will stimulate retrogradation which form crystalline structures. The crystalline structure of granules may cause starch to be resistant to enzyme hydrolysis. Starch product can be isolated by hot air dyring, freeze drying, and spray drying.

Pullulanase (pullulan 6-glucanohydrolase, EC 3.2.1.41), an important debranching enzyme in starch processing, can cleave α-1,6 linkages in pullulan, amylopectin and other related polysaccharides (Lin et al. 2006). Debranching of amylopectin will provide an increased opportunity to molecule alignment or aggregation, to form crystalline structures, and is, hence, helpful in RS formation. The pullulanase enzyme preferably reacts with a pH from 4.5 to 5.5 at temperature of 40°C to 60°C. Berry (1986) reported a substantially increased RS3 content during monitoring the debranching effect of pullulanase on potato amylopectin, and attributed this effect to an increase in linear starch chains resulting from debranching.

C. Clostridium butyricum

The bacterial population of the human cecum and colon is numerically large with at least 1.0 x 103 cfu/g, which, with an estimated mass of 250–750 g of digesta, gives a calculated total of 1.0 x 103 cfu in the whole hindgut (Hill, 1995). More than 50 genera and over 400 species of bacteria have been identified in human faeces. Colonic microflora predominantly comprises facultative anaerobes (e.g.

Enterobacteria, Streptococci, Staphylococci, Lactobacilli, Propionibacteria and Bacilli) in the upper part of the colon but lower down these change to strict anaerobes (e.g. Bacteroides, Bifidobacterium, Eubacterium, Peptococci, Fusobacterium and Clostridium) (Roberfroid, 2001).

One of colon bacteria is Clostridium butyricum which has characteristics such as spore former, positive gram bacteria, obligate anaerob and lives in human or animal colon and soil. It has rod shaped with the dimension of 0.5-1.7 x 2.4-7.6µm (Mitsuoka, 1990). Clostridium butyricum plays important role in supporting other beneficial bacteria in gastrointestinal like Bacillus bifidus, Acidophilous bacterium, and Streptococcus faecium. There are some benefits using Clostridium butyricum in the fermentation, these bacteria needs simple growth medium, produces high metabolite, and easy to isolate. Beside that, Clostrium butyricum also has butyrogenic activity, it means these bacteria produce more butyric acid than acetic acid and propionic acid. Bacteria from genus Clostridium and

Bifidobacterium can ferment high amylose starch, but bacteria from genus Clostridium has higher activity in fermentation (Wang, 1999).

Several factors which influence bacteria growth are temperature, oxygen, nutrition, and pH. In general, the medium pH not only affects cell growth and fermentation rate, but also changes final product yield and purity. Changing the medium pH also may induce a metabolic shift (Zhu, 2004). Several studies were performed to determine the optimal cultivation conditions for the butyric acid production by Clostridium butyricum: temperature 35-37oC, pH 4.5-7 and atmosphere of pure CO2 or

6 pure N2 or N2 and CO2 in ratio of 1 : 9; temperature 37oC, pH 6-7.5, medium flushed with oxygen-free nitrogen gas (Zigova & Sturdik 2000).

D. SWEET POTATO

Sweet potato is classified into Planatae kingdom, Spermatophyta division, Angiospermae

subdivision, Concolvulales ordo, Convolvulacea family, Ipomoea genus, Ipomoea batatas species. Sweet potato can grow in tropical and sub-tropical climate and it has characteristics such as thin bark, variation shape, contains fiber in variation amount depend on the variety (Mucthadi dan Sugiyono, 1989). The stem is yellow, green, or orange, whereas the root is long and round. The leaf has round shaped with serration or wavy in the leaf side. The color of flower is white and has bell shaped, but most of it does not has flower. Sweet potatos is divided into two groups, sweet potato which has soft corm because contains more water and sweet potato which has hard corm because contains more starch (Lingga et al. 1989). Sweet potato can be consumed directly or processed further become sweet potato flour or sweet potato starch. Table 2 shows starch content in several variety of sweet potato.

Table 2. Starch Content in Sweet Potato Variety Starch Content (%) Jago Sukuh Sari Boko Kidal 31 31 32 32 33 Source : Departemen Pertanian (2009)

Sweet potato has many advantages such as short period harvest, good adaptation in the lack of soil, and can be alternative food in the crop failure or disaster area. Because has good adaptation, sweet potato plays important role as a source of food in Asia and Pacific region. At about 94% total production of sweet potato around the world is produced from this region. Production of sweet potato is predicted about 98.6 milions ton a year. Indonesia is the second country, after China, which produce the most sweet potato in the world (Christian, 2005). Table 3 shows the total production of sweet potato in Indonesia.

Table 3. Total Production of Sweet Potato in Indonesia

Year Production (ton)

2005 2006 2007 2008 2009 1.856.969 1.854.238 1.886.852 1.880.977 2.057.913 Source : Departemen Pertanian (2009)

One of sweet potato variety is Jago. This variety is semi-compact plant, has productivity until 25-30 ton/ha, and it can be harvested in 4-4.5 months. It has round shape corm, short stem, white bark and flesh and also resistant with boleng pest. This variety has high yields, high starch content, and suitable to be used in flour and starch production. Figure 1 shows jago variety of sweet potato.

7 Figure 1. Jago Sweet Potato

8

III.

MATERIAL AND METHOD

A. MATERIALS AND EQUIPMENTS

1. MATERIALS

The materials used for this research was Jago variety of sweet potato starch, which was obtained from Indonesian Center of Agricultural Post Harvest Research and Development, Bogor, West Java. Fermented bacteria used was Clostridium butyricum BCC B2571, obtained from Culture Collection of Balitvet, Bogor, West Java.

Material used in this research were yeast extract, beef extract powder, peptone, glucose, soluble starch, sodium chloride, sodium acetate, cystein hydrochloride, sodium hydroxide, sodium-potassium tartarate, dinirosalisilate, CO2 gas, K2SO4, HgO, H2SO4 pekat, NaOH 1N, NaOH 0.5 M, HCl 2 M, KOH 4 M, acetic acid 1N, ethanol 95%, concentrated H2SO4, iodine solution, KI, buffer fosfat pH 6.5, citrate buffer 0.05 M pH 4.5 KCl-HCl buffer 0.1 M pH 1.5, tris-maleate buffer 0.1 M pH 6.9, pullulanae, α-amylase, amyloglucosidase, pepsin solution, phenol, pure amylose, acetic acid standard (SIGMA-ALDRICH 71251), propionic acid standard (SIGMA-ALDRICH 94425), butyrate acid standard (SIGMA-ALDRICH 19215), and distillated water,

2. EQUIPMENTS

The equipments used in this research were gas chromatography (Agilent Technoligies 7890 A GC system), analytical scales, incubator, Kjeldahl flask, micropipette, spray dryer, spectrophotometer, reaction tube, volumetric pipette, centrifuge, centrifuge tube, aluminium dish, oven, desiccator, porcelain dish, cuvette and shaking water-bath.

B. METHODS

This reserach was devided into two stages. First stage was chemical analysis consist of proximate analysis, amylose content analysis, and resistant starch content analysis. Second stage was fermentation of type three (3) resistant starch by Clostridium butyricum and analysis the result of fermentation. The stages in this research are shown in Figure 2.

Figure 2. Two Stages of Research First Stage

Chemical Analysis 1. Proximate Analysis 2. Amylose content Analysis 3. Resistant Starch Content Analysis

Second Stage

Fermentation of Type 3 Resistant Starch by Clostridium butyricum

1. pH Measurement 2. Turbidity Measurement

9

1. Chemical Analysis

Chemical analysis in this research including amylose analysis and proximate analysis. Proximate analysis were done for moisture, ash, fat, protein, and carbohydrate.

a. Moisture by Air Oven Method

(AOAC 1995)

Dry metal dish and cover at 100oC, cool in desiccators, and weigh soon after reaching room temperature. Accurrately weigh 2 g well-mixed sample. Uncover sample. Put dry dish, cover, and sample in oven provided with opening for ventilation and maintained at 100oC for 1 h. Cover dish while still in oven, transfer to desiccators and weigh soon after reaching room temperature. Repeat until consistent wight is obtained. Loss in weight is reported as moisture (indirect method).

b. Ash by Direct Method (AOAC 1995)

Weigh 3-5 g well mixed sample into ashing dish that has been ignited, cooled into desiccators and weighed soon after reaching room temperature. Ignite in furnace at 550oC (dull red) until light gray ash result, or to constant weight. Cool in desiccators and weigh soon after reaching room temperature. The remaining weight of sample is ash weight.

c. Fat by Soxhlet Extraction Method (AOAC 1995)

Transfer 5 g sample to defatted extraction thimble and dry 6-18 hours in small beaker at 100oC. Place cotton plug over paper. Add few defatted antibumping chips to 250 ml Erlenmeyer and dry 1 hour at 100oC. Cool to room temperature in dessicator and weigh. Place thimble containig dried sample in soxhlet, supporting it with spiral or glass beads. Add 150 ml hexane to the soxhlet or until thimble is fully submerged. Reflux digested sample 4 hours adjusting heat so that extractor siphon 30 times. Remove flask, and evaporate solvent on steam bath. Dry flask at 100-101oC to constant weight (1.5-2 hours). Cool in desiccator to room temperature and weigh. Constant weight is attained when successive 1 hour drying periods show additional loss of < 0.05% fat. Duplicate determinations should agree within 0.1% fat.

% Fat = g fat x 100/g sample = x 100%

Notes :

a = flask and final sample mass (g) b = empty flask mass (g)

c = initial sample mass (g)

d. Protein by Kjeldahl Method (AOAC 1995)

Sample of 100-250 mg was put into Kjeldahl flask. Then 1.0 ± 0.1 g K2SO4, 40 ± 10 mg HgO, and 2.0 ± 0.1 ml H2SO4 were added. Boiling stones around 2-3 items were put and boil the solution for 1-1.5 hours. At distilattion stage, a little amount of water is transferred step by stage 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 rinsing 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

10 Amylose (%) = [Amylose] x V x FP x 100%

Sample weight

titration stage, sample solution was diluted into 50 ml, and then titrated with 0.02 N standardized HCl until grey color appears. Volume of HCl for titration was then reported.

% N =

e. Carbohydrate Content (by difference) (AOAC 1995)

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

f. Amylose Analysis (Apriyanto et al. 1989)

First step of amylose analysis was to determine standard curve. Amylose standard curve was made by dissolving 40 mg pure amylose into 1 ml 95% ethanol and 9 ml NaOH 1 N solution. The mixture was boiled for 10 minutes until gelatinized, cooled, and moved to volumetric flask. Then prepared series mixture in the test tube: 0, 0.2, 0.4, 0.6, 0.8 and 1 ml previous solution were diluted until 1 ml then add 1 ml acetic acid 1 N and 2 ml Iodine solution (0.2 g iodine and 2 g KI were diluted into 100 ml distillated water). The mixtures were incubated for 20 minutes. Spectrophotometer (Spectronic 21) was used to measure the absorbance of the sample at 625 nm and the data was plotted to make a standard curve.

Amylose content was determined by dissolving 100 mg sweet potato starch into 1 ml ethanol 95% and 9 ml NaOH 1 N solution. Sample solution then boiled for 10 minutes and cooled. Put 5 ml sample solution into 100 ml volumetric flask and add 1 ml acetic acid 1 N and 2 ml Iodine solution. The mixture was incubated for 20 minutes. Measured the absorbance of the sample at 625 nm and the absorbance was compared to the standard curve to determined amylose content. Amylose content was determined by:

2. Production of Type 3 Resistant Starch

a. Production of Type 3 Resistant Starch (Vatanasuchart

et al.

2010)

Weigh 20 g sweet potato starch in erlenmeyer and add distilled water until reach 200 g of total weight to obtain 10% starch suspension (w/w). Adjust the acidity of starch suspension to pH 5 with addition of HCl or NaOH solution. Starch suspension then was autoclaved at 121oC under 15 psi for 15 minutes. The suspension was cooled to 50oC and was debranched by adding 1 ml pullulanase enzyme (34,779 U/mg protein). Place the suspension in a shaking water bath at 50oC during 24 hours. After that, deactivated enzyme by heating the suspension at 90oC for 30 minutes. Slurry starch was stored at 4oC for 24 hours to perform retrogradation. Hydrolyzed starch was centrifuged at 4500 rpm for 15 minutes at room temperature and collect the sediment. The sediment then would be dried using spray dryer. The resulted product was referred as type 3 resistant starch (RS3).

11

b. Resistant Starch Analysis (Goni et al. 1996)

Starch sample was weighed 100 mg into a 50-ml centrifuge tube and mixed with 10 ml KCl-HCl buffer 0.1 M pH 1.5 (pH adjustment with KCl-HCl 2 M or NaOH 0.5 M). The solution was homogenized and added 0.2 ml pepsin solution (1 g pepsin/10 ml buffer KCl-HCl) (445 units/mg solid), mixed well and incubated in water bath 40oC for 60 minutes with constant shaking. The mixture was mixed 9 ml Tris-maleate buffer 0.1 M 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 enzyme/ml Tris-maleate buffer) (15.4 units/mg) was added to the mixture, homogenized, and incubated in water bath shaker 55oC, 120 rpm, for 16 hours. After the incubation, the sample was centrifuged (3000 rpm, 15 minutes) and supernatant was discarded. Residue was washed at least once with 10 ml distilled water, centrifuged again and supernatant was discarded. Then 3 ml distilled water and 3 ml KOH 4 M were added into the centrifuge tube, mixed well, and left for 30 minutes at room temperature with constant shaking. Next, 5.5 ml HCl 2 M, 3 ml citrate buffer 0.05 M pH 4.5 and 100 μl of diluted amyloglucosidase (0.1% w/v) (154000 U) 1% were added into the centrifuge tube, homogenized, and incubated 55oC for 45 minutes. Mixture then was centrifuged (3000 rpm, 15 minutes), supernatant was collected and saved in a 100 ml volumetric flask. Residue was washed with 10 ml distillated water, centrifuged, and supernatant was combined with that obtained previously. Supernatant was adjusted to 100 ml. Resistant starch would be measured using phenol sulphuric acid method (AOAC 1995).

c. Glucose Analysis by Phenol Sulphuric Acid Analysis (AOAC 1995)

Diluted solution from previous method was taken for the total sugar measurement using phenol-sulphuric acid method. 50 μl of solution was pipetted into a test tube and diluted until 1 ml. Then 0.5 ml phenol 5% and 2.5 ml concentrated H2SO4 were added into the test tube. The mixture was homogenized and the absorbance was measured using spectrophotometer at 490 nm. The absorbance was compared with glucose curve standard (10, 20, 30, 40, 50, 60, 65, 80, and 85 ppm) which was made by same method. Resistant starch content was obtained by calculating with formula:

.

3. Fermentation of Type 3 Resistant Starch by

Clostridium butyricum

Fermentation was carried out in two different conditions. First condition was designed to evaluate production pattern of SCFA where resistant starch (20g/L) and glucose (1g/L) were added into the medium. Second condition was designed in order to increase production of SCFA, especially butyric acid, where resistant starch (10g/L) and glucose (5g/L) was added into medium.

a. Fermentation Preparation (Purwani et al. 2009)

Fermentation preparation was devided into 4 steps. The steps include preparation of Reinforced Clostridial Medium (RCM) as growing medium for Clostridium butyricum, culture refresh of

Clostridium butyricum, making of growth medium containing resistant starch, and fermentation type 3 resistant starch by Clostridium butyricum.

The first step was preparation of Reinforced Clostridial Medium (RCM). All of the ingredients were dissolved in 100 ml distilled water in erlenmeyer. The mixture was adjusted to pH 6.8 with NH4OH or HCl and placed the mixture into butyl rubber bottle and sterilizied at 121oC for 15 minutes. The remaining medium was stored in refrigerator for the next uses. Medium composition could be

Resistant Starch (%) = mg glucose x 0.9 x V (ml) x dilution factor x 100 % mg sample

12 seen in Table 3. Second step was culture refresh of Clostridium butyricum. 5 ml Reinforced Clostridial Medium (RCM) sterile and 5 ml of pure culture of Clostridium butyricum were added into tube and flushed with CO2 to keep anaerob condition. The mixture was incubated at 37oC for 24 hours. The presence of turbidity and sediment showed the growth of Clostridium butyricum. The third step was making of growth medium containing type 3 resistant starch. Medium composition used was similar like Reinforced Clostridial Medium (RCM), but the difference was that the soluble starch was replaced with resistant starch at 1% (w/v). All the ingredients were mixed and adjusted to pH 6.8 with NH4OH or HCl and sterilized at 121oC for 15 minutes. The fourth step was fermentation of type 3 resistant starch by Clostridium butyricum. A 24-hours culture of Clostridium butyricum was inoculated into sterilized Reinforced Clostridial Medium (RCM) while flushed with CO2 and incubated 37oC in anaerobic condition.

Table 4. RCM Composition

Source : (Dewi, 2009)

b. Fermentation Analysis

(1) pH Measurement (Purwani et al. 2009)

pH measurement is conducted to observe the changing of pH during fermentation. pH of cultures is determined using pH meter.

(2) Turbidity Measurement (Purwani et al. 2009)

Measurement was conducted to observe the turbidity of cultures caused by bacterial growth. Turbidity of cultures is determined using spectrophotometer on 660 nm.

4.

Short Chain Fatty Acid (SFCA) Analysis (Purwani et al. 2009)

Short chain fatty acid profile (SCFA) was analyzed using Gas Chromatography (Agilent Technoligies 7890 A). Sample (result of fermentation) were centrifuged at 13000 rpm for 10 min. Then supernatant was filtered through a 0.2 μm filter into a 1.5 ml eppendorf tube for storage at 4oC until use. Before injecting the samples, 94 μl of sample was spiked by adding 2 μl of acetic acid ALDRICH 71251), propionic acid ALDRICH 94425), and butyric acid (SIGMA-ALDRICH 19215). Standard curve for each compound was also made to determine SCFA concentration in the sample (Table 5).

Sample of 1 μl were injected into a high resolution gas chromatography (Agilent Technologist, 7890A GC System) equipped with a flame ionization detector and a HP Innowax 19091N-136 column (60 m x 0.250 mm). The carrier gas was helium with a flow rate of 1.8 ml/min, and the split ratio was 40:1. The oven temperature was maintained at 90oC for 0.5 min, and then increased to 110oC at a rate

Ingredients Concentration (g/L)

Yeast Extract Beef Extract Powder

Peptone Glucose Soluble Starch Sodium Chlorida Sodium Acetate Cystein Hidrocloride 3.0 10.0 5.0 1.0 2.0 5.0 3.0 0.5

13 of 10oC/min, increased to 170oC at a rate of 5oC/min and finally increased to 210oC at a rate of 20oC/min. Injector and detector temperatures were set into 275oC. Acetate, propionate and butyrate were used for standard and the result was expressed as mmol/L.

Table 5. SCFA standard mixture for standard curve Concentration

(μl / ml)

Acetic acid (μl)

Propionic acid (μl)

Butyric Acid (μl)

H2O (μl)

0 0 0 0 0

10 5 5 5 485

20 10 10 10 470 30 15 15 15 455 40 20 20 20 440 50 25 25 25 425

14

IV. RESULT AND DISCUSSION

A. CHEMICAL ANALYSIS

The amylose and proximate analysis was done to identify amylose content and identify its water, ash, protein, fat, and carbohydrate content of sweet potato starch. Table 6 shows the amylose and proximate content.

Table 6. Major Chemical Composition of Jago Sweet Potato Starch Chemical Component Sweet Potato Starch (%)

Water Ash Protein Fat Carbohydrate Amylose 13.73 0.23 0.44 0.56 85.04 25.83

The water content of sweet potato starch is 13.73%, protein content is 0.44%, ash content is 0.23%, fat content is 0.56%, and carbohydrate content is 85.04%. Sweet potato is classified as tubers group so it contains high carbohydrate. The variation of water content of the sweet potato starch depend on drying method, drying time, and storage condition. Sweet potato starch also contains protein, fat, and ash even though the contents are low. High purity of the starch is very important to keep hydrolisis enzyme runs smoothly.

Starch which is resistant to digestive enzyme in the small intestine is not only affected by processing but also but its chemical structure. Starch which contains more amylose have higher ability to form amorphous structure because of its hydrogen bond intensiveness. The consequence is starch which can not be swelled or gelatinized better during cooking time will slowly digested (Panlasigui et al. 1991). Therefore, information about amylose content in food is important to produce optimum yield of resistant starch.

Principal of amylose analysis is iodine bound in the spiral shaped starch molecule which yields blue color then measured the absorbance by spectophotometer at 620 nm (Winarno, 1997). The result of amylose analysis showed that amylose content of jago sweet potato starch was 25.83%. It was higher than wheat starch (25%) and potato starch (20%), but lower than corn starch (26%) (Pomeranz, 1992). Collado and Corke (1997) reported that amylose content in sweet potato starch have variation in range 15-27%, depend on variety.

Generally, sweet potato pasta characteristic have tendency to easily retrogradated which showed by pasta viscosity in cooling phase or classified as type C based on classification by Srichuwong (2005). The other characteristics are the very viscous pasta, stable, and it doe not show a peak in heating phase. Increasing viscosity in cooling phase indicate that retrogradation process will be undertaken easily. Intensive of retrogradation level is required in RS3 formation. (Purwani et al. 2009).

Based on amylose content analysis, it can be concluded that ingredient to produce resistant starch in this research can be classified as high amylose starch and it was expected that jago sweet potato can be potential ingredient to produce high yield resistant starch.

15

B. RESISTANT STARCH PRODUCTION

This research used sweet potato starch from Jago variety which is obtained from Indonesian Center of Agricultural Post Harvest Research and Development, Bogor. Sweet pootato starch then modified into type 3 resistant starch using pullulanase enzyme according to Vatanasuchart et al. (2010).

. According to Wasserman et al (2007), making type 3 resistant starch (RS3) can be divided into two stages, the first stage is heating process which will trigger gelatinitation. The starch granule will swell due to water absorption during heating process. Water absorption is caused by kinetic energy from water molecule become stronger than starch molecule affinity inside the granule such that water will come into the starch granule. The second stage is starch retrogradation which cause recrystallization of amylose and amylopectin chain. Gelatinized starch is easier to be digested than the raw starch even though the starch gel is not stable and form crystal when cooled (retrogradation). Starch retrogradation will result in the insoluble short chain polymer and resistant to digestive enzymes.

Polymer recrystallisation is a three-stage process that involves nucleation (formation of critical nuclei), propagation (crystal growth from the nuclei formed) and maturation (continued crystal growth and perfection). The nucleation and propagation rates determine the overall recrystallisation rate whereas the maturation rate is more temperature dependent (Eerlingen et al., 1993). Nucleation generally proceeds rapidly when the incubation temperature is close to the glass transition temperature of starch, at about 5oC (Gray & Bemiller, 2003).

Formation of RS3 involves recrystallisation of amylose in a partially crystalline system in a process that is influenced by the incubation temperature and time (Eerlingen et al., 1993a; Haralampu, 2000). In this research, pullulanase enzyme (5%) was used at 55oC, 24 h incubation hours in pH 5.0. The pullulanase enzyme preferably reacts with pH from 4.5 to 5.5 at a temperature of 40°C to 60°C (Vatanasuchart et al. 2010). Incubation time used was 24 h which refer to Onyago et al (2006) who reported that maximum yields of RS3 of cassava were obtained after 24 h incubation. Pullulanase is debranching enzyme for hydrolysing α-1,6 glucosidic bond of the starch. In the present study, debranching using pullulanase was applied to produce linear, low molecular weight and recrystallizable polymer chains. Debranching enzymes such as pullulanase rapidly hydrolyze only a-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 a-1,4-glucosidic bonds. The debranched starch was then subjected to temperature cycling and incubation at a series of temperature and time to induce retrogradation and formation of the RS (Leong et al. 2007).

After incubating at 55oC 24 h, the starch slurry was heated at 90oC to inactivate the pullulanase enzyme. Starch slurry was then stored in 4oC during 24 h to trigger retrogradation. Onyago et al (2006) reported that retrogradation temperature (4oC) will favour a high nucleation rate and therefore high yields of RS3 (in this study : cassava). The resulting retrograded starch or RS3 was dried by spray dryer. Spray drying method was choosen because of the high efficiency result. Production of RS3 was conducted seven times in this research. Each production used 20 gram sweet potato starch as raw ingredient and resulted in 2.00±0.16 gram RS3 or 10% yield. Yield of RS3 production is presented in Figure 3.

16 Figure 3. Resistant Starch Yield

Resistant starch analysis was conducted according to Goni et al. 1996. The main features of the analytical procedure are removal of 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. The removal of protein was introduced to enhance amylase accessibility avoiding starch-protein associations. Moreover, this step is advisable for a better simulation of physiological conditions (proteolytic digestive enzymes, acidic pH). The removal of digestible starch is done to avoid positive mistake when glucose is released.

Purwani et al (2009) reported that resistant starch content of jago sweet potato was 13.77%. The analysis showed that resistant starch content of jago sweet potato treated with pullulanase was 28.15%, which was lower than Salosa sweet potato starch treated with pullulanase enzyme that contained 38.22% RS (Evalin, 2011). Treatment with pullulanase and retrogradation process were proved could increase resistant starch content of jago sweet potato starch. Purwani et al (2009) reported that sago resistant starch treated with pullulanase contained 31-38% RS and rice resistant starch treated with pullulanase contained 21-26% RS, whereas Zhao (2009) reported that maize resistant starch treated with pullulanase contained 24.5-32.4% RS. It showed that Jago sweet potato starch treated with pullulanase contained higher RS content than rice starch treated with pullulanase enzyme, but lower than sago resistant starch and maize resistant starch which also treated with similar enzyme. The difference could have been caused by several factors such as starch and enzyme used, amylose content, heating and cooling condition, drying method, etc.

C. FERMENTATION OF RS3 by

Clostridium butyricum

In vitro fermentation of RS3 was divided into 2 stages. Stage I was carried out to evaluate production pattern of SCFA to determine optimum fermentation time of Clostridium butyricum to produce high butyric acid. Stage II was carried out to further increase production of SCFA, especially butyric acid content.

17 Two different fermentation conditions were performed in stage I and II. In stage I, concentration of RS3 was 20 g/l (2%) and glucose was 1 g/l (0.1%). Interval time during fermentation was 6 h, 12 h, 24 h, 36 h, and 48 h. This interval was chosen to simulate physiological condition in the digestive system. Concentration of RS used in stage I referred to Evalin (2011) who reported that concentration of 2% RS3 gived highest concentration of butyric acid in Salosa sweet potato RS fermentation. Result of fermentation is presented in Figure 4.

Figure 4. Changes of pH and absorbances medium during 48 h fermentation of 2% RS

A decrease of pH was observed after 6 h of fermentation and it remained until 48 h fermentation, but pH value was not decreased significantly. The lowest pH value (4.74) was achieved at 48 h fermentation. The pH in medium indicated that short chain fatty acid (SCFA) were formed during bacterial growth (Robertfroid, 2001). It was in agreement with Sayar et al (2007) who stated that carbohydrate fermentation result in pH degradation in the colon, caecum, and faeces. pH degradation plays important role in calsium and magnesium absorption, reduce solubility of secondary bile acid, and inhibit pathogenic bacterial growth.

Increased in cell growth was observed during 48 h fermentation. A significant absorbance increase was observed after 6 h fermentation, absorbance began to increase constantly after 12 h fermentation. The highest absorbance was achieved at 36 h fermentation, but after that absorbance was decreased. This implied that during fermentation, Clostridium butyricum grew rapidly. The result showed that the environment condition was suitable for bacteria to grow. Absorbance between 12 h and 36 h increased slightly because of competition of bacteria to get nutrition for their growth. After 36 h fermentation, absorbance began to decrease due to lack of nutrient and medium pH was too low and thus inhibited the bacterial growth.

Absorbance change implied that there are bacterial growth during fermentation.

Clostridium butyricum is one of the colon bateria which can utilize carbohydrate as carbon source. These bacteria can be isolated from human or animal colon and soil, and can be grown in Reinforce Clostridial Medium (RCM) medium. In this research, soluble starch as carbon source was replaced with sweet potato resistant starch.

Resistant starch fermentation by Clostridium butyricum resulted in formation of short chain fatty acid such as acetate acid, propionic acid, and butyric acid. Wang et al (1999) reported that resistant starch has butyrogenic activity, it means that resistant starch can induce butyric acid

18 formation better than other substrates. In this reserach, SCFA profile was determined with Gas Chromatography (Agilent Technoligies 7890 A GC system). The profile of SCFA produced by

Clostridim butyricum in stage I is presented in Figure 5.

Figure 5. Profile of SCFA during 48 h fermentation of 2% RS

Result of SCFA analysis showed that compared to other acids, butyric acid was the most dominant of the short chain fatty acid produced by Clostridium butyricum. The production of acetic acid only occured at 6 to 12 h fermentation, up to 24 h the concentration of acetic acid decreased dramatically. Concentration of acetic acid, propionic and butyric acid had similar pattern profile while concentration of SCFA was increased at 12 h, decreased at 24 h, and increased at 36 h. By 48 h fermentation, all of SCFA was not detectable. Apperently the SCFA content in the medium was too low and could not be detected.

From the SCFA profile above, it can be concluded that the optimum time of fermentation of sweet potato resistant starch to produce butyric acid was 12 h, where the molar ratio of acetic acid, propionic acid, and butyric acid were 50 mM : 37 mM : 68 mM (1.3:1:1,8), but this level is considered as low and can be increased.

Stage II was designed to increase the production of SCFA, especially the butyric acid. The concentration of RS3 was decreased to 10g/L (1%) and glucose was increased to 5g/L (0.5%). This fermentation condition referred to Purwani and Suhartono (2009) who reported that 1% RS and 0.5% glucose increased butyric acid concentration in the fermentation of sago starch. The fermentation time used at this stage was 12 h (referred to Stage I) and 48 h (referred to Purwani and Suhartono, 2009).

19 Figure 6. Changes of pH and absorbances medium during 48 h fermentation of

1% RS

From Figure 6, a decrease of pH was observed after 12 h of fermentation and pH decreased further at 48 h fermentation. Compared to the pH value in stage I after 12 h fermentation, the pH with 1% RS (5.01) was higher than pH using 2% RS (4.80) even though pH after 48 h fermentation with 1% RS (4.69) was lower than pH with 2% RS (4.74). Different pH will affect the distribution of acids, cell membrane transport behavior, and cell lysis. Relative high pH value (6.0) is beneficial for cell growth and butyric acid biosynthesis, especially in Clostridium butyricum (He et al. 2005). Medium pH also affects the specific growth rate, butyric acid production rate, and consumption of sugars.

The higher pH observed in 1% RS medium after 12 h fermentation maybe due to more glucose being available as the carbon source in the growth medium such that the bacterial grew and the consequence was higher pH achieved because of decreased of the SCFA production. This explanation was supported by higher medium absorbance which showed that total bacteria in the medium was increased higher than the previous fermentation. After 48 h fermentation pH in 1% RS medium was lower, the bacteria can produce SCFA and as the consequence, there was higher concentration SCFA which lower the pH. Both fermentations showed similar pattern of absorbance change, while absorbance was increased at the beginning of fermentation and was decreased in the end of fermentation. Decreasing absorbance indicates that the total bacteria was declined because of the metabolics produced by the bacteria, pH degradation, and competition between bacteria to get nutrition which inhibit bacterial growth. The profile of SCFA produced by

20 Figure7. Profile of SCFA after 48 fermentation of 1% RS

Profile of SCFA above showed that after 48 h fermentation, the molar ratio of acetic acid, propionic acid, and butyric acid were 453 mM : 248 mM : 225 mM (2:1.1:1). It was higher than the result at 12 h fermentation, where the molar ratio were 226 mM : 162 mM : 192 mM (1.4:1:1.2). Compared to previous fermentation, 1% RS medium fermentation resulted in higher level of SCFA. Acetic acid was the most dominant of SCFA compared with propionic acid and butyric acid. This result was different than previous fermentation where butyric acid was the most dominant. It was in agreement with Purwani and Suhartono (2009) who reported that high butyrate level was produced after 48 h fermentation of sago resistant starch where the molar ratio of acetic acid, propionic acid, and butyric acid were 83 mM : 47 mM : 46 mM (2:1:1), but it was not in agreement with Evalin (2011) who reported that no more butyrate was produced after 48 h fermentation of Salosa sweet potato resistant starch where the molar ratio of acetic acid, propionic acid, and butyric acid were 591 mM : 0 mM : 0 mM.

Different level of glucose in medium also influence the profile of SCFA produced by

Clostridium butyricum. Higher concentration of glucose (5 g/l) in the Stage II resulted in higher medium turbidity than in the Stage I (1 g/l). It presumably indicated that more glucose as the carbon source in the medium was more useful for Clostridium butyricum to grow than resistant starch as the carbon source. Glucose is more useful for Clostridium butyricum to grow because it can be utilized directly as the carbon source whereas the resistant starch have to be hydrolyzed first to utilize the carbon source. The consequence if the medium contains more glucose is the

21 Table 7. Comparison of SCFA Production from Several RS by Clostridium butyricum

RS Source

Fermentation

time (h) [RS] (%)

[Glucose] (%) Acetic acid (mM) Propionic acid (mM) Butyric acid (mM) Acetic : propionic : butyric Jago Sweet Potato

6 2 0.1 34.31 38,13 52.79 1 : 1.1 : 1.5

12 1 0.5 226.79 162.28 192.25 1 : 1.4 : 1.2

2 0.1 50.13 37.83 68.03 1.3 : 1 : 1.8

24 2 0.1 0.55 16.80 18.81 1 : 30 : 38

36 2 0.1 0 29.73 40.57 -

48 1 0.5 453.65 248.64 225.37 2 : 1.1 : 1

2 0.1 0 0 0 -

Salosa Sweet Potato (Evalin

2011)

6 2 0.1 85.25 60.00 73.51 1.4 : 1 : 1.2

12 2 0.1 0 0 0 -

24 2 0.1 13.45 16.70 120.33 1 : 1.2 : 9

36 2 0.1 939.12 0 14.81 -

48 2 0.1 591.84 0 0 -

Sukuh Sweet Potato (Iswani

2011)

6 2 0.5 0 10.29 13.57 -

12 2 0.5 0 0 15.32 -

24 2 0.5 0 0.84 4.01 -

36 1 0.5 500.40 476.28 477.97 1.1 : 1 : 1

2 0.5 0 25.17 50.97 -

48 1 0.5 215.21 281.10 343.12 1 : 1.3 : 1.6

2 0.5 0 0 22.83 -

Rice (Purwani et al. 2009)

24 0.5 0.5 86.79 1.09 5.74 80 : 1 : 5

48 1 0.1 71.37 1.42 15.89 50 : 1 : 11

Sago (Purwani et al. 2009)

24 0.5 0.5 79.32 0.37 6.99 214 : 1 : 19

48 1 0.1 72.74 3.06 11.12 24 : 1 : 3

Corn (Goni et al. 2000)*

5 1 - 59.70 27.00 13.30 4.5 : 2 : 1

10 1 - 60.90 25.70 13.50 4.5 : 2 : 1

24 1 - 61.80 27.00 11.20 5.5 : 2.5 : 1

48 1 - 62.30 25.90 11.80 5.5 : 2.5 : 1

* : microorganism isolated from caecal Wistar rats

SCFA comparison above showed similar pattern of SCFA production during in vitro fermentation where acetic acid and butyric acid were the most dominant fatty acid. Propionic acid formation followed similar pattern of butyric acid but the concentration was lower. RS concentration and fermentation time were important to influence profile of SCFA. The difference of concentration and fermentation time resulted difference profile of SCFA.

22 In this research, 2% RS of jago sweet potato fermentation resulted in lower SCFA level than 2% RS of salosa sweet potato. Fermentation of 2% RS of salosa sweet potato resistant starch during 36 h and 48 h resulted in very high acetic acid level (939.12 mM and 591.84 mM) but the butyric acid level was very low at 48 h fermentation and it was not detected. Fermentation 2% RS of sukuh sweet potato did not result acetic acid and resulted in lower SCFA level than 2% RS of jago sweet potato. Lower concentration of jago sweet potato RS (1%) resulted in higher SCFA level than rice, sago, and corn resistant starch, especially in the acetic acid and the butyric acid. Molar ratio resulted from fermentation of 1% jago sweet potato RS during 48 h was 453 mM : 248 mM : 225 mM (acetate:propionate:butyrate) and it was the highest among the substrates. Rice, sago, and corn resistant starch showed similar molar ratio of SCFA but the difference was in the propionic level. Propionic acid level in the rice and the sago resistant starch were very low and lower than the butyric acid level whereas in the corn resistant starch, the propionic acid level was higher than the butyric acid. Fermentation of 0.5% rice and sago resistant starch resulted in higher molar ratio of SCFA than 1% RS. This result indicated that lower concentration of RS resulted in higher formation of SCFA, especially the acetic acid and the butyric acid.

Longer fermentation time also resulted that increasing level of SCFA. Thus data above confirmed that longer fermentation time until 48 h resulted in higher level of SCFA, especially the acetic acid and the butyric acid. Fermentation of sweet potato resistant starch during 48 h resulted in the highest molar ratio of SCFA 453 mM : 248 mM : 225 mM (acetate:propionate:butyrate). Similar result was also achieved at 48 h fermentation of rice, sago, and corn resistant starch. Fermentation of salosa sweet potato resistant starch during 36 h resulted in higher molar ratio of SCFA 939 mM : 0 mM : 14 mM (acetate:propionate:butyrate) than during 48 h fermentation which resulted in molar ratio of SCFA 591 mM : 0 mM : 0 mM (acetate:propionate:butyrate). It indicated that Clostridium butyricum need longer time to produce higher level of the acetic acid and the butyric acid. Longer fermentation time has relationship with growth cycle of microorganisms where after 48 h fermentation indicated microorganisms have entered death phase which was pointed by decreasing medium turbidity than in the early fermentation.

Besides RS concentration and fermentation time, glucose content in the medium also influenced the profile of SCFA produced by Clostridium butyricum. Data from Table. 7 showed that fermentation of all the substrates (except corn resistant starch) with higher content of glucose (0.5 %) resulted higher level of SCFA. Higher content of glucose in the medium means that bacteria will have more carbon sources to grow so it can be utilized by the Clostridium butyricum

to duplicate itself at the growth phase. Rapid growth of Clostridium butyricum at the beginning of the fermentation will reduce amount of the carbon source. The consequence, Clostridium butyricum will begin to hydrolize the resistant starch to glucose and ferment the undigest carbohydrate to form short chain fatty acid. Higher total bacteria in the medium will give more chances for the Clostridium butyricum to form short chain fatty acid.

The differences in physicochemical properties of substrates such as the crystalline structure of starch and hydrogen bonds may affect the accessibility of microbial enzymes and the colonic fermentation, modifying the quantity of SCFA produced and the rate of fermentation (Goni et al. 2000). Simple crystalline structure of starch enable the microbe’s enzyme to convert glucose to pyruvate as the intermediate compound in further catabolism. More hydrogen bonds indicate more complex structure which means microorganisms need more energy (ATP) to convert carbohydrate to glucose. If energy required at higher level, it will reduce energy source for microorganisms to convert glucose to pyruvate which limit the production of SCFA. The result may imply that jago sweet potato resistant starch have simpler crystalline structure and less of hydrogen bonds than

23 other substrates such as rice resistant starch, sago resistant starch, and corn resistant, and it can be better utilized by Clostridium butyricum to produce higher level of acetate and butyrate.

High level of butyric acid has relationship with the acetic acid level. Increasing level of butyric acid was possitively correlated with increasing level of acetic acid. Relationship between acetic acid and butyric acid was observed in all substrates fermentation. In this research, increasing the acetate and the butyrate level occured after 48 h fermentation. This result has similar pattern with rice resistant starch, sago resistant starch, and corn resistant starch. This result indicated that after 48 h fermentation, SCFA production tend to shift to the acetate. The lack of nutrient in the medium could be one of the factors which cause why Clostridium butyricum would rather produce acetic acid because more ATP is required to form the butyric acid.

Clostridium is an acidogenic bacterium, produce acetate and butyrate as the main fermentation products. The result of fermentation also showed that acetate and butyrate were the main fermentation product. Sharp and Macfarlane (2000) reported that saccharolytic Clostridia is well adapted to grow faster in high substrate concentration and resistant starch granules are advantageous for their growth. Figure 8 shows the metabolic pathways of SCFA production.

Figure 8. The metabolic pathways of SCFA production. 1a) Butyrate kinase; 1b) Butyryl CoA-Acetate Transferase (Zhang et al. 2009)

Glucose as the carbon source is metabolized into pyruvate through glycolisis pathway. Pyruvate is the main substrate for further enzymatic metabolism. In the acetate pathway, one mole of glucose is metabolized into acetyl-CoA with pyruvate-feredoxin oxidoreductase enzyme which release CO2 and 2 ATP, then acetyl-CoA is metabolized into acetate with acetate kinase releasing 2 ATP, so 4 ATP are formed during conversion of the glucose to two acetic acids. In the butyric pathway, CoA is metabolized into butyryl CoA then metabolized into butyrate by acetyl-CoA transferase releasing 1 ATP, so 3 ATP are formed during conversion of glucose to butyric. More energy are released from conversion of the glucose to acetic acid. In this case bacteria prefer

24 to produce acetic acid than butyric acid in the exponential phase (Zhang et al. 2009). Beside through butyrate kinase, glucose can be metabolized to butyric acid with Co-A transferase enzyme which convert butyryl-CoA and acetate into butyric acid and acetyl-CoA.

1a pathway is present generally within Clostridium species, such as Clostridium acetobutylicum, Clostridium tetani, Clostridium perferingens, and Clostridium difficile. In this pathway, butyrate kinase expressing gene is found in Clostridium sp which is isolated from soil and water while in the 1b pathway, it is found from bacteria which is isolated from human gut so this pathway is dominant in colon. Louis et al (2004) reported that CoA transferase enzyme activity is the most dominant pathway in the human colon.

Chen and Blaschek (1999) reported that activity of CoA transferase, acetate kinase, and butyrate kinase influence acid metabolism during fermentation. Some research reported that enzyme activity in human colon is influenced by substrates, pH, and end product level. In this research, SCFA profile in Stage I showed that butyric acid was the most dominant. This result showed that Co-A transferase activity to form butyric acid was higher than acetate kinase whereas in Stage II, acetic acid level was higher than the butyric acid. This result indicated that acetate kinase activity was higher than the Co-A transferase. The difference resistant starch which was used proved that substrate influenced enzyme activity, where fermentation of 1% RS resulted presumably in higher acetate kinase activity and fermentation of 2% RS resulted presumably in higher butyrate kinase activity. The pH also influenced the enzyme activity, lower pH (4.69) increased acetate kinase activity and decreased CoA transferase activity.

Aman et al (2001) reported that the butyric acid formation by Clostridium butyricum is affected by high level of butyril CoA : CoA ratio. Increasing the acetate level from 226 mM to 453 mM indicated that butyril CoA : CoA ratio was low such that the metabolism aimed to produce acetic acid. Louis et al (2007) reported that limited carbon source in the medium will stimulate bacteria to produce acetic acid because they will produce more ATP than butyric acid production. This statement explained why the butyrate level was lower than the acetate level in the end of fermentation.

Many factors are able to influence the metabolics pathway of the microorganism during fermentation. In the case of butyrate-producing Clostridia, the concentrations of glucose, pH, H2 partial pressure, acetate, and butyrate impact the growth rate, the final product concentration and the distribution of products (Kong et al. 2006). Excess the carbon source often affects osmotic dehydration of the microorganisms in the fermentation process. The significant increase of the butyrate-acetate ratio was observed in the glucose-limited culture without sparging nitrogen into fermenter in the butyrate fermentation with Clostridium butyricum as the working microorganism (Saint-Amans, 1995).

This research result is expected to give information about short chain fatty acid (SCFA) profile produced by Clostridium butyricum BCC B2571 during fermentation of jago sweet potato resistant starch. This resistant starch has potency to develop as functional food because result high butyric acid level during fermentation which can be utilized to prevent colon cancer.

25

V. CONCLUSION AND RECOMMENDATION

A. CONCLUSION

Based on the research, RS type 3 (RS3) could be successfully developed from jago sweet potato through enzyme hydrolisis with pullulanase enzyme (34,779 U/ mg protein) at 24 hours at 50oC. Jago sweet potato starch contains water 13.73%, protein 0.44%, ash 0.23%, fat 0.56%, and carbohydrate 85.04%. Amylose content of jago sweet potato starch is 25.83% and RS content of jago sweet potato starch treated with pullulanase is 28.15%. In vitro fermentation resulted in short chain fatty acid (SCFA) at different levels depending on the RS and the concentration of glucose in the medium. Fermentation of 20 g/l (RS3) and 1 g/l (glucose) resulted in SCFA with molar ratio of acetate, propionate, butyrate 50 mM : 37 mM : 68 mM (1.3:1:1.8) at 12 h fermentation whereas fermentation of 10 g/l (RS3) and 5 g/l (glucose) resulted in SCFA with molar ratio of acetate, propionat, butyrate 453 mM : 248 mM : 225 mM (2:1.1:1) at 48 h fermentation. This result indicated that longer fermentation time and higher glucose concentration could increase butyric acid level but there was metabolic shift to higher formation of acetic acid

B. RECOMMENDATION

1. Increase yield of resistant starch could be conducted by homogenizing size of retrogradation starch to optimize drying with spray drier.

2. In vitro fermentation could be pursued with mixture microorganisms from human intestine to find out the real profile of SCFA in intestine.

3. Optimize further fermentation condition to increase the butyric acid level could be obtained by modifying pH medium and sodium acetate level in the medium composition.

26

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30

 

Appendix 1. Amylose Content Analysis

 

Amylose Standard Curve

 

Calculation example for amylose content:

 Sample 1 Absorbance = 0.322 Y = 23.37X + 0.010 0.322 = 23.37X + 0.010 X = 0.01 (mg/ml)

Amylose Content (%) =

Amylose Content (%) = 0.01 (mg/ml) x 100 ml x 20 x 100 %

` 102.40 mg x 1000

= 25.98%

y = 23,37x + 0,010 R² = 0,998

0 0,2 0,4 0,6 0,8 1 1,2

0 0,01 0,02 0,03 0,04 0,05

Ab

so

rba

n

ce

31

 

Appendix 2. RS Content Analysis

Glucose Standard Curve

Sample Abs Sample weight (mg) Concentration (mg) Glucose content (mg) RS content (%) Average (%)

Jago 1 0,088 100,00 0,01 0,67 16,72

28.19

Jago 2 0,168 101,30 0,04 1,59 39,67

Calculation example for glucose concentration:  Sample Jago 2

Absorbance = 0.168 Y = 3.835X + 0.031 0.168 = 3.835X + 0.031 X = 0.04 (mg/ml)

Glucose Content (%) = . %

Glucose Content (%) = 0.41 (mg/ml) x 0.9 x 50 x 100%

101.30 mg

= 1.59%

RS content (%) = Glucose content x Volume = 1.59 x 25

= 39.67%

y = 3,835x + 0,031 R² = 0,984

0 0,1 0,2 0,3 0,4 0,5

0 0,02 0,04 0,06 0,08 0,1 0,12

Ab

sor

b

an

ce

32

 

Appendix 3. Absorbance and pH value during fermentation

 Change of absorbance during fermentation

Time (hour) Absorbance

2% RS 1% RS

6 0.632 NM

12 0.644 0.757

24 0.646 NM

36 0.649 NM

48 0.647 0.700

*NM : Not Measured

 Change of pH value during fermentation

Time (hour) pH

2% RS 1% RS

0 6.81 6.80

6 5.00 NM

12 4.80 5.09

24 4.75 NM

36 4.74 NM

48 4.74 4.69

33

 

Appendix 4. Acetic, propionic, and butyric acid standard curve for fermentation of 2% RS

y = 37,05x - 3,833 R² = 1

0 5 10 15 20 25 30

0 0,2 0,4 0,6 0,8 1

Are

a

Concentration (M) Acetic Acid

y = 63,05x - 1,985 R² = 0,996

0 5 10 15 20 25 30 35 40 45

0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8

Are

a

Concentration (M) Propionic Acid

y = 78,85x + 0,715 R² = 0,999

0 5 10 15 20 25 30 35 40 45 50

0 0,1 0,2 0,3 0,4 0,5 0,6

Ar

ea

Concentration (M) Butyric Acid

46  

48  

50