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Effects of Calcium Addition during Enzymatic Hydrolysis and Different Pretreatments on Drying Duration of Brown and White Sago Sugars

Nurazureen binti Matnin

(43375)

Bachelor of Science with Honours (Resource Biotechnology)

2016

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Effects of Calcium Addition during Enzymatic Hydrolysis and Different Pre-

treatments on Drying Duration of Brown and White Sago Sugars

Nurazureen binti Matnin (43375)

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This project is submitted in partial fulfillment of the requirements for the Degree of

Bachelor Science with Honours

(Resource Biotechnology)

Supervisor: Professor Dr Kopli Bujang

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Faculty ofResource Science and Technology Universiti Malaysia Sarawak

2016

ACKNOWLEDGEMENT

First of all, I would like to express my deepest gratitude to God the highest for

granting me the strength to accomplish my final year project. A special thanks and sincere

appreciation dedicated to my supervisor, Professor Dr. Kopli Bujang for his guidance and

support throughout this project. The insight, motivation and experience were certainly

appreciated.

Million thanks also dedicated to all postgraduate students of the Biochemistry

Laboratory, Faculty of Resource Science and Technology especially to my advisor, Miss

Nadia Dayana Sikem for her insight, guidance and willingness to offer suggestions and

commentary for improvements during my thesis topic research. Additionally, I am

indebted to Miss Sharifah binti Mohammad, Mr Muhammad Norhelmi bin Ahmad, Miss

Aimi Nadia binti Saharuddin and Miss Daisy Dinsi for their support and advice throughout

this proj ect.

Next, I am grateful to all the members of my lab and friends for their

companionship and support while perfonning this project. Last but not least, I would like

to thank my family for their understanding and encouragement in many ways throughout

this project.

I

DECLARATION

I hereby declare that no portion of the work has been submitted in the support of an

application for another degree qualification of this or any other university or institution of

higher learning .

........ ... ... ~.... ....... ....... .. . .

(NURAZUREEN BINTI MATNIN)

Resource Biotechnology Programme

Department of Molecular Biology

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

II

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T ABLE OF CONTENTS

ACKNOWLEDGEMENT I

DECLARATION II

TABLE OF CONTENTS III

LIST OF ABBREVIATIONS VI

LIST OF TABLES VII

LIST OF FIGURES

ABSTRACT 1

VIII

1.0 INTRODUCTION 2

1.1 Overview and Problem Statement 2

1.2 Objectives 4

2.0 LITERATURE REVIEW 5

2.1 Sugar Industry 5

2.1.1 World Market and Demand 5

2.1.2 Sources of Sugar 5

2.1.3 Uses of Sugar 6

2.2 Sago Industry 7

2.2.1 The Sago Palm 7

2.2.2 Production of Sago Starch 8

2.2.3 Utilization and Bioconversion of Sago 8

2.3 Types of Sago Sugars 10

2.3.1 Brown Sago Sugars 11

2.3.2 White Sago Sugars 11

III

,....

123.0 MATERIALS AND METHODS

3.1 Materials

3.1.1

3.1.2

3.1.3

3.1.4

3.2 Methods

3.2.1

3.2.2

3.2.3

3.2.4

Sago Starch 12

Hydrolytic Enzymes 12

Calcium 12

Powdered Activated Charcoal 12

13

Standard Hydrolysis of Sago Starch into Sugar 14

Purification of Sago Sugar 14

Drying and Crystallization of Sago Sugar 14

Analysis of Sago Sugar 15

3.2.4.1 Reducing Sugar using DNS Method 15

3.2.4.2 Total Phenolic Content 16

3.2.4.3 Total Flavonoid Content 17

3.3 Analytical Methods 17

3.3.1 Effects of Calcium during Hydrolysis of Sago Starch 17

3.3.2 Effects of Different Pre-Treatments on Brown Sago Sugars (with

and without addition of Calcium) 18

3.3.2.1 Sedimentation 18

3.3.2.2 Centrifugation 19

I' 3.3.3 Effects of Calcium on White Sago Sugar (with and without

Calcium) 20

3.3.4 Comparison of Drying Time and Crystallization between

Different Pre-Treatments in Brown Sago Sugars (with and

IV

J

20 without Calcium)

,....

3.3.5 Comparison of Drying Time and Crystallization on White Sago

Sugars (with and without Calcium) 20

4.0 RESULTS AND DISCUSSIONS 21

4.1 Effects of Different Pre-Treatments on Brown Sago Sugars (with and

without Calcium) 21

4.2 Effects of Addition of Calcium on White Sago Sugars 24

4.3 Comparison of Drying Time and Crystallization between Different Pre-

Treatments on Brown Sago Sugars (with and without Calcium) 27

4.4 Comparison of Drying Time and Crystallization on White Sago Sugars

(with and without Calcium) 31

5.0 CONCLUSIONS 34

6.0 REFERENCES 35

APPENDICES 38

v

II

ANOVA

AMG

BSS

°C

DE

DNS

DSS

GA

GC

HSS

mL

nm

PAC

QE

rpm

t

TFC

TPC

WSS

LIST OF ABBREVIATIONS

Analysis of Variance

Amyloglucosidase

Brown Sago Sugar

Degree Celsius

Dextrose Equivalent

Dinitrosalicylic Acid

Dried Sago Sugar

Gallic Acid

Glucose Concentration

Hydrolyzed Sago Sugar

millilitre

Nanometre

Powdered Activated Charcoal

Quercetin Equivalent

Revolutions per minute

Ton

Total Flavonoid Content

Total Phenolic Content

White Sago Sugar

VI

II

,.....

Table

Table 1

Table 2

Table 3

Table 4

Table 5

Table 6

Table 7

Table 8

Table 9

Table 10

Table 11

Table 12

Table 13

Table 14

Table 15

•

LIST OF TABLES

Page

Development of food products from sago 9

Development of energy products from sago 10

Reducing sugar, TPC and TFC of different pre-treatments on brown 21

sago sugars

Reducing sugar, TPC and TFC of white sago sugars 24

Drying time between different pre-treatments on brown sago sugars 27

Mass of sugar, glucose recovery and glucose yield between different

pre-treatments on brown sago sugars 28

Drying time of white sago sugars (with and without calcium) 31

Mass of sugar, glucose recovery and glucose yield of white sago

sugars 31

Drying time and production of brown and white sago sugar 38

One-way ANOY A of reducing sugar, TPC and TFC analyses between

different pre-treatments on brown sago sugars (without calcium) 39

One-way ANOY A of reducing sugar, TPC and TFC analyses between

different pre- treatments on brown sago sugars (with calcium) 39

One-way ANOY A of reducing sugar, TPC and TFC analyses of white

sago sugars (with and without calcium) 40

One-way ANOY A of final mass, glucose recovery and glucose yield

between different pre-treatments on brown sago sugars (without 40

calcium)


between different pre-treatments on brown sago sugars (with calcium) 41


of white sago sugars (with and without calcium) 41

YII

1

LIST OF FIGURES

Figure Page

Figure 1 Flowchart of method involved during production and analysis of brown

and white sago sugars 13

Figure 2 Drying process of; (a) brown sago sugars and (b) white sago sugars. 15

Figure 3 Different pre-treatments used on hydrolyzed brown sago sugars; (a)

refrigerator, and (b) sedimentation in the oven 19

Figure 4 The clear liquid BSS was collected by using centrifugation pre-treatments

Figure 5 Reducing sugar between different pre-treatments on brown sago sugars

Figure 6 Total phenolic and flavonoid content between different pre-treatments on

on brown sago sugars 19

(with and without calcium) 22

brown sago sugars (with and without calcium) 24

Figure 7 Reducing sugar of white sago sugars (with and without calcium) 25

Figure 8 Total phenolic and flavonoid content of white sago sugars (with and

without calcium) 26

Figure 9 Final mass, glucose recovery and glucose yield between different pre

Figure 10 Crystallization of brown sago sugars (without calcium) between different

Figure 11 Crystallization of brown sago sugars (with calcium) between different

Figure 12 Final mass, glucose recovery and glucose yield of white sago sugars (with

Figure 13 Crystallization of white sago sugars; (a) without calcium and (b)with

Figure 14 Graph of concentration and absorbance of stock standard solution of

treatments on brown sago sugars (with and without calcium) 29

pre-treatments 30

pre-treatments 30

and without calcium) 32

addition of calcium 33

glucose concentration 42

Figure 15 Graph of concentration and absorbance of GA stock standard solution 42

Figure 16 Graph of concentration and absorbance of stock standard solution of

Quercetin 43

VIII

,.....

Figure 17 Drying process of brown sago sugar without the addition of calcium

(refrigerator) 44



Figure 20 Drying process of brown sago sugar with the addition of calcium

(oven) 44

(centrifuge) 45

(refrigerator) 45

Figure 21 Drying process of brown sago sugar with the addition of calcium (oven) 46

Figure 22 Drying process of brown sago sugar with the addition of calcium

(centrifuge) 46

Figure 23 Drying process of white sago sugar without the addition of calcium 47

Figure 24 Drying process of white sago sugar with the addition of calcium 47


pre-treatments; sedimentation in the (a) refrigerator and (b) oven 48






pre-treatments; sedimentation in the (a) refrigerator and (b) oven 48

pre-treatments; (a) sedimentation in the refrigerator and (b) centrifuge 49

pre-treatments; (a) sedimentation in the refrigerator and (b) centrifuge 49

pre-treatments; (a) centrifuge and (b) sedimentation in the oven 50

pre-treatments; (a) centrifuge and (b) sedimentation in the oven 50

Figure 31 Crystallization ofwhite sago sugars; (a) (b) (c) without calcium 51

Figure 32 Crystallization of white sago sugars; (a) (b) (c) with calcium 51

IX

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Effects of Calcium Addition during Enzymatic Hydrolysis and Different PreTreatments on Drying Duration of Brown and White Sago Sugars

Nurazureen binti Matnin

Resource Biotechnology Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

Abstracts

Melroxylon sagu is the starch-producing crop that can produces sago sugar via an enzymatic hydrolysis process. This project has been aiming to identify which pre-treatments (sedimentation and centrifugation) and types of sago sugars can be an alternative to give rise to various qualities of sago sugars. In this project, analyses of white and brown sago sugars were compared by observing the result of drying time, reducing sugar, total phenolic and flavonoid contents, glucose recovery and yield . By the end of this project, sedimentation have been shown to be an excellent pre-treatments of brown sago sugars as it recorded the highest amount of reducing sugar, total phenolic content and total flavonoid content with the shortest drying time (6 days) whereas 221.32 ± 15.54 gIL, 0.099 ± 0.019 mglmL, 0.0097 ± 0.0007 mglmL (without calcium/oven) and 175.44 ± 15.54 giL, 0.091 ± 0.002 mglmL and 0.0119 ± 0.0006 mglmL (with calcium/refrigerator) respectively. Centrifugation is proven to be the most effective pre-treatments as it recorded the highest production of brown sago sugars, which is 298.5 ± 30.41 g (without calcium) and 346.5 ± 6.36 g (with calcium) and yield the finest, powder-like and lighter color of brown sago sugars (with and without calcium). Besides, white sago sugar (without calcium) have the best reducing sugars (175.15 ± 11.l2), TPC (0.00049 ± 0.00046) and TFC (0.00014 ± 0.00012). It is also revealed that white sago sugars (without calcium) have the best results for drying time (6 days), production of white sago sugars (283 ± 10.82 g), glucose recovery (97.30 ± 6.18 %), glucose yield (80.99 ± 5.82 %) and yield the finest, powder-like and whiter color of white sago sugars. However, it does not give different results despite adding calcium in white sago sugars as ANOV A indicated that p = 0.674 (final mass), p = 0.956 (glucose recovery) and p = 0.780 (glucose yield) at (l = 0.05.

Keywords: Melroxylon sagu, brown sago sugar, white sago sugar, calcium, pre-treatment

Abslrak

Melroxylon sagu adalah hasil kanji yang boleh menghasilkan gula sagu melalui proses hidrolisi enzimalik. Projek ini berlujuan unluk mengkaji kaedah (pemendapan dall pengemparall)dan jenis gula sagll unluk dijadikan a/lernalif yang boleh meningkalkan kualili gula sagu. Dalam projek in i, pembandingan anlara analisis gula sagu perang dan pulih lelah dijalankall dengan membandingkan masa pengeringan, kepekalan glukosa, TPC, TFC, pemulihan dan penghasilan glukosa .. Hasilnya, kaedah pemendapan merupakan kaedah lerbaik dalam gula sagu perang kerana menghasilkan kepekatan gula, TPC dan TFC yang tinggi iaitu 221.32 ± 15.54 giL, 0.099 ± 0.019 mglmL, 0.0097 ± 0.0007 mg/mL (tanpa kalsium/oven) dan 175.44 ± 15.54 giL, 0.091 ± 0.002 mglmL, 0.0119 ± 0.0006 mg/mL (tambahan kalsium/peti sejuk) dengan masa pengerillgan yang singkat (6 hari).Kaedah pengemparan merupakan kaedah yang efektif apabila menghasilkan jumlah gula saguperang yang tinggi iaitu 298.5 ± 30.41 g (tanpa kalsium) dan 346.5 ± 6.36 g (tambahan kalsium) serla menghasilkan gula sagu perang yang paling halus (tanpa dan tambahan kalsium). Selain ilu, gllia sagu pUlih (Ianpa kalsium)mengandungi kepekalan glukosa (175.15 ± 11.12), TPC (0.00049 ± 0.00046) dan TFC (0.00014 ± 0.00012), masa pengeringan (6 hari), berat gula (283 ± 10.82 g), pemulihan glukosa (97.30 ± 6.18 %), penghasilan glukosa (80.99 ± 5.82 %) yang lerbaik serla menghasilkan gula sagu pUlih yang paling halus dan putih. Namun begitu, lambahan kalsium tidak memberi kesan kepada gllia sagu pulili kerana ANOVA menunjukkan p = 0.674 (beral gula), p = 0.956 (pemulihan glukosa) dan p = 0.780 (penghasilan glukosa) pada a = 0.05.

Kata kunci:Melroxylon sagu, gllia sagu perang, gula sagll puti", kalsium, kaedah

1

1.0 INTRODUCTION

1.1 Overview and Problem Statement

Brown and white sago sugars are made from Metroxylon sagu that has been transformed

into sago starch via an extraction process. Metroxylon sagu grows in the hot humid tropics

of South-East Asia and Oceania and contains a valuable starch in its trunk (Flach, 1997).

This pinnate-leaved palm is able to grow naturally in swampy areas or peat soils without

the need of pesticide and also herbicide (Pei-Lang et al., 2006). The sago palm is also

resistance to floods, drought and strong winds because it is an extremely hardy plant.

Bujang (2014) pointed out that the sago palm is the only commodity that able to grow in

peat soil which occupies around 75% of coastal plains and lowland river basins in the state

ofSarawak in East Malaysia. This major advantage makes sago palm better than other cash

crops.

Bujang (2010) stated that recently the global consumption of sago starch had accounted for

about 3% of the total world market which initially were dominated by com, potato and

tapioca starches and achieved between 200,000 to 300,000 tons per annum. The

productivity of sago palm was measured to be four times than of paddy rice because it

contains a large amount of starch. Bujang (2010) stated that one ton of sago starch can be

converted into one ton of liquid sugar by using Ishizaki process and then produce 640 litres

of ethanol. Sugar derived from sago starch has the highest concentration at 205 giL with

recovery ofmore than 100% dextrose equivalent (DE) compared to com and tapioca starch

(Bujang, 2011). Besides, sago sugar contains mostly glucose (94%), with maltose and

other impurities, both at 3% each (Bujang, 2011). Nowadays, the consumption of sugar

derived from sugarcane and sugar beet contribute to certain health problem especially

diabetes. Thus, high glucose content in sago sugar may act as alternative for diabetics to

2

control and maintain the sugar level in their body. Furthermore, the presence of flavonoids

has increases the potential of sago sugar to be utilized in pharmaceutical industry (Bujang,

2015).

This study has been almmg to use sago sugar as the ongm to study the effects of

sedimentation, centrifugation and addition of calcium during solid liquid separation on its

quality and drying duration. Drying time of brown and white sago sugars is the interest of

the study because different methods of sugar processing can give rise to various quality of

sago sugar. It could shot up the production of brown and white sago sugars in compromise

with quality performance. The production of white sago sugar does not undergo different

pre-treatments because it will be purified by filtration using powdered activated charcoal

(PAC). Moreover, there are several analyses such as glucose concentration determination;

total phenolic and flavonoid content estimation are conducted on brown and white sago

sugar. Consequently, there are several problems occurred in conversion of sago sugar.

Different pre-treatments (sedimentation and centrifugation) before drying of brown sago

sugars may show different productivity of sugars production. After hydrolysis process,

there is sediment left in the hydrolysate which may not only contain the remaining of

valuable sugar, but also impurities that may contribute to bitter taste in sugar production.

Moreover, sedimentation of brown sago sugar is time consuming meanwhile centrifugation

technique is costly and may lead to fmancial constraint.

3

1.2 Objectives

The objectives of this research are:

I. To identify the effects of calcium addition as a stabilizer on the production and

drying time of brown and white sago sugars.

2. To determine the effects of glucose concentrations, total phenolic compounds and

flavonoid content with different pre-treatments, sedimentation and centrifugation

on brown and white sago sugars.

3. To study the effects of sedimentation and centrifugation on drying time of brown

and white sago sugars.

4

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2.0 LITERATURE REVIEW

2.1 Sugar Industry

Today, global production of sugar is increasing from developed to developing countries.

Up to now, sugar consumption and demand have increased rapidly based on the increase of

population indeveloping countries.

2.1.1 World Market and Demand

In crop year 2013114, the scale of global sugar production is about 174.8 million tonnes

and achieves average growth rate of 2 %/year (Pham et al., 2014). According to Pham et

al. (2014), the largest producing countries are Brazil (22% of total production), India

(15%), China (8%) and Thailand (6%) with sugar trading volume on the world market

around 55-60 million tonnes. The largest sugar producer and exporter in the world, Brazil

is also one ofthe largest ethanol manufacturers and consumers (Martinelli et al., 2011). A

huge impact on sugar supply and price in the world will happened due to the decision to

expand ethanol production in this country (Pham et al., 2014).

2.1.2 Sources of Sugar

Sugar can be processed from sugar cane (75-80% of the global supply), sugar beet and

Metroxylon sagll. Approximately 75% and 25% of the world's sugar production are

produced from sugar cane and sugar beets, respectively (Potts et al., 2014). About 66% of

the sugar consumption in Malaysia is for domestic uses. According to F AO (2009), the

production of sugarcane in Malaysia is concentrated in the northwest of Peninsular

Malaysia in Perlis and Kedah and its cultivation is relatively small. The sugar recovery is

rather low at only 7%/kg fresh weight even though the annual production of sugarcane is

5

relatively high at 1.3 to 1.6 million tonnes (Bujang, 2010). The higher price of this

commodity is caused by the lack of raw materials and then, increase in industrial

application ofsugarcane (Bujang, 2014). Sugar made from sugarcane is profitable and easy

plant to grow compare to Metroxyion sagu that takes up to 10 years for the plant to be

harvestable (Flach. 1997). However, Bujang (2014) stated that sago starch produced the

highest glucose recovery among other starches with (99% DE), followed by corn starch

(84% DE), then tapioca starch (76% DE) and sweet potato (72%). Sago starch has the

potential to replace sugarcane in the sugar industry because it involves enzymatic

hydrolysis of the starch (HSS) process and purified using PAC (Bujang et ai., 2006). On

the other hand, sugar beet has sugar content of 14-18% and grown primarily in temperate

regions (FAO. 2009). Although the harvest time of sugar beet is shorter than of sugarcane

(5 - 6 months), it is more costly and less competitive compare to sago sugar and sugarcane

(FAO, 2009).

2.1.3 Uses of Sugar

Many types of sugars which include glucose, fructose, sucrose and lactose are found in the

diet on a daily basis. Sugar is usually referring to sucrose or known as table sugar. Sugar

cane and sugar beets contain large quantities of sucrose (Potts et ai., 2014), meanwhile

sago starch contain glucose in large quantities (Bujang et ai., 2011). As an example of

carbohydrates, sugar provides our body with the energy that required by organs and

muscles to function. Moreover, sugar is known as food additive used efficiently in drinks

and foodstuffs. They also have important biological, physical and chemical properties as

well as bringing sweetness. For instance, sugar can be used in the canning, cooking and

freezing of foods to improve flavor and texture, and to preserve natural color and shape

(Potts et ai., 2014). During freezing, sugar protects the surfaces of frozen fresh fruit from

6

contact with air that can produce enzymatic browning (due to oxidation). However,

excessive consumption of sugars, combined with insufficient energy expenditure increased

risk ofoverweight, obesity and serious diseases (Potts et al., 2014).

2.2 Sago Industry

Currently, interest in sago industry has increased rapidly. Sago also is one of the important

commodities which contribute to the economic value in the countries. As a carbohydrate,

sago is required to support human life by preventing a human food crisis due to large world

population.

2.2.1 The Sago Palm

Metroxylon sagu is derived from Javanese word and contains starch in its trunk. According

to Ubi and Dransfield (1987), this sago palm is belongs to the family Palmae Jussieu and

genus Metroxylon Rottboell. There are many uses ofsago-palm derived products. The sago

leaves are used for roof thatch and wall sliding and can be woven into bags, baskets, cages

and rope (Adeni et al., 2010). Spoons and food wrappers also can be made from sago

leaves. On the other side, the rachis of fronds is used for walls or house building. The

cortex of trunk is used as flooring and frring in factory. Plank that is used for building or

making things also can be made. Other parts of the sago palm can be used in traditional

medicines and many different things (Adeni et al., 2010). Moreover, ground pith is rasped

and dried as an animal feed for pigs, horses and chicken. In order to extract good quality

and quantity of sago starch, the pith of sago palm that contains starch need to be separated

from the cellulosic materials, then has to undergo several processes (Bujang and Ahmad,

2000). The starch obtained can be converted into many uses, for instance, dextrose

7

glucose, human food products, animal feed, paper industry, textile industry, foundry

molds, single cell protein production and as a biodegradable filler in plastics.

2.2.2 Production of Sago Starch

Several processes have to be taken for production of sago starch. According to Bujang and

Yusop (2006), steps taken in sago starch production include debarking, pulping, extraction,

dewatering, drying and packing. Sago starch was extracted from the pith after removing

the cortex, rachis and leaflets from the pith (Singhal et al., 2008). The liquefied starch was

poured and precipitated in a settling container. Then, the starch settled on the bottom of the

container was washed several times until white starch was obtained (Kamal et al., 2007).

Sago is grown commercially for the production of sago starch and conversion to animal

food or to ethanol in Malaysia, Indonesia and Papua New Guinea (Singhal et al., 2008).

One of the main potential of sago starch is that it has multifunctional uses and can be

utilized for energy and food products.

2.2.3 Utilization and Bioconversion of Sago

Sago can be utilized and converted into many uses. Nowadays, the products made from

sago are marketable and provide a regular cash flow to the sago farmers. Thus, the

utilization and bioconversion of sago has been developed. Table 1 shows the development

of food products by using several enzymes and substrate. Sugars, single cell protein, lactic

acid, kojic acid and cyclodextrin are some of food products that can be produced from

sago.

8

Table 1: Development offood products from sago

DescriptionFood Product Enzyme! References

Substrate

The starch slurries were enzymatically (Bujang,Sugars • Termamyl-120L

hydrolysed for 4-6 hours at the optimum 2014)

concentration and then, purified using powdered

activated charcoal (PAC) by filtration . Sugar

also can be obtained from sago hampas because

hampas is the fibrous wastes that are discarded

with sago effluent into the river.

• Amyloglucosidase

Spirulina platensis Spirulina platensis has very high protein (Bujang,

content. The cyanobaterium cultivated in sago 2014)

Single

Cell Protein effluent is treated for production of single cell

protein.

L-Iactic acid, the only form of lactate that is (Bujang,

absorbed entirely by the human body is 2014)

Lactic Acid Lactococcus

lactis 10-1 produced from fermentation of starch by

Lactococcus lactis 10-1. It is importance in the

food and cosmetic industries.

I In order to produce high yields of kojic acid by (Ros farizan I Aspergillus flavus, gelatinized sago starch has

Kojic Acid Aspergillus sp.

et al., 2002)been chosen as a carbon source in different

fermentation modes In an 8-L stirred tank (Satoko

fermentor. It is importance In the field of et ai., 2006)agriculture, food science, medicine and

agriculture and recently, there are reports on the

genotoxicity of kojic acid.

Cyclodextrin Cyclodextrin Cyclodextrin glycosyltransferase produced by (Charoenlap

(CD) from glysyltranferase various microorganisms, such as Bacillus sp. et aI., 2004)

sago starch produced synthesizes the CD enzymatically by converting

particularlyby Bacillus starch into CD. CD IS useful in food ,

sp. pharmaceutical, cosmetic and agricultural

applications.

9

On the other side Table 2 shows the development of energy products by using several

enzymes and substrate. Starch to ethanol, fiber to ethanol and effluent to biodiesel are

some ofenergy products that can be produced from sago.

Table 2: Development of energy products from sago

Energy

Product

Enzyme/Substrate Description References

Starch • Nitrogen sources

(mielU or com steep

The sago starch is used as substrate together

with nitrogen sources (mieki or com steep

(Bujang,

2014) to Ethanol

liquor)

• Saccharomyces

cereviceae CSI-l (for

continuous ethanol

fermentation)

liquor) as the alternative to yeast extract for

production of ethanol for biofuel.

Fiber

to Ethanol

• Cellulose

• p-gLucosidase

--Enzymatic hydrolysis of steam-treated sago

fiber production of sugars from cellulose can

be used as substrate for production ofbiofuel.

(Bujang,

2014)

Eftluent Scenedesmus dimorphus Scenedesmus dimorphus is cultured in l6L of

modified sago effluent with NaHC03.

(Bujang,

2014) to

Scenedesmus is the preferred species for oil Biodiesel

yield in the production ofbiodiesel. A process

used in conversion lipid to biodiesel, trans

esterification is the most economical with 98%

conversion yield.

2.3 Types of Sago Sugars

There are two types of sago sugars studied; hydrolyzed sago sugars (brown sago sugars)

and purified liquid sago sugars (white sago sugars).

10

2.3.1 Brown Sago Sugars

Consumption of brown sago sugar is beneficial to health due to the presence of

antioxidant, which analyzed based on total phenolic content (TPC) and total flavonoid

content (fFC). Flavonoids are based on a C6-C3-C6 backbone and belong to polyphenol

family. As the secondary metabolite, it plays vital function in plant such as growth

regulators, gene expression modulators, intracellular signaling and also stress responses

(Kamtekar et al., 2014). The most important is that they can acts as anticancer, antioxidant

and anti-inflammatory properties on activated cell lines of animal and possible role in

several chronic diseases prevention involving oxidative stress that acts as their protective

effect against low-density lipoprotein (LDL) oxidation (Sudha et at. , 2011). According to

Bujang (2015), brown sago sugar has the highest total phenolic and total flavonoid

contents at 20% (w/v). Furthermore, types of flavonoid that has been discovered in brown

sago sugar are Gallic acid, Quercetin and Kaempferol (Bujang, 2015).

2.3.2 White Sago Sugars

White sago sugar contains mostly glucose (94%), maltose (3%) and other impurities (3%)

(Bujang et al., 2011). Several types of mono- or oligo-saccharides as impurities (3%) are

produced when the hydrolytic enzymes used contain a-amylase and attacks gelatinized

starch randomly (Bujang, 2015). PAC is used to purify brown sago sugars into white sago

sugars (Bujang et al., 2012). This process is known as purification of BSS. Different

amount of PAC gives different recovery of sugars (Bujang et al., 2012). Based on the

research, the yield of white sago sugars was lower when higher amount of PAC was used

during purification (Bujang, 2011). According to Ang et al. (2006), the absorption between

PAC is higher towards protein and color but lower towards glucose and lactate.

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3.0 MATERIALS AND METHODS

3.1 Materials

3.1.1 Sago Starch

Sago starch was obtained from Herdsen Sago Industries in Pusa, Sarawak.

3.1.2 Hydrolytic Enzymes

Termamyl-SC (alpha-amylase from Bacillus licheniform is 120KNU-S/g)

Amyloglucosidase (AMG) from Aspergillus niger, ~ 300 U/mL purchased

Novozyme Biomass Kitt were used for sago starch hydrolysis (Bujang, 2012).

and

from

3.1.3 Calcium

Calcium powder obtamed from calcium granule was added into another set of enzymatic

hydrolysis of sago sugar in both brown and white sago sugars.

3.1.4 Powdered Activated Charcoal (PAC)

AC was used in purification ofHSS (with and without calcium) aided by a vacuum pump

produce Purified Sago Sugar (PSS). Sterilized PAC which was washed with 0.2 M HCI

deionized water was used for all samples (Bujang et al., 2012). Then, cellulose acetate

filter paper was used to filter hydrolyzed sago sugar.

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3.2 Method

Flowchart presented in Figure 1 shows the summary of experimental method involved

during production and analysis of brown and white sago sugars. Detail methods for each

step are explained in the next proceeding pages.

Standard Enzymatic Hydrolysis of Sago Starch into Sugar (With and Without Calcium)

- Enzymatic hydrolysis:

- Liquefaction (Tennamyl-SC)

- Saccharification (Amyloglucosidase)

- Effect ofdifferent pre-treatments on brown sago sugar:

- Sedimentation in the Refrigerator

- Sedimentation in the Oven

- Centrifugation

Purification of Sago Sugar

Drying and Crystallization of Sago Sugar

Analysis of Sago Sugar:

- Reducing Sugar using DNS method

- Total Phenolic Contents using Follin-Ciocalteu method

- Total Flavonoid Contents using colorimetric method

Statistical Analysis

Jlpre 1: Flowchart ofmethod involved during production and analysis of brown and wh ite sago sugars

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