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PILOT SCALE PRODUCTION OF SUGARS FROM SAGO STARCH

Hafizah Binti Booty

Master of Science 2011

Pusat Khidmat Maklumat Akademik

IJNIVER. SITI MALAYSIA SARAWAK

P. KHIDMAT MAKLUMAT AKADEMIK UNIMAS

1111111111111111111111 11111 1000246247 PILOT SCALE PRODUCTION OF SUGARS FROM

SAGO STARCH

HAFIZAH BINTI BOOTY

A thesis submitted in fulfillment of the requirement for the Degree of

Master of Science (Biotechnology)

Faculty of Resource Science and Technology UNIVERSITI MALAYSIA SARAWAK

2011

DECLARATION

I hereby declare that no portion of the work referred to in this thesis has been submitted in

support of an application for another degree or qualification to this or any other university

or institution of higher learning.

HAFIZAH BINTI BOOTY

07021255

August 2011

i

ACKNOWLEDEMENTS

First of all, I would like to convey my deepest gratitude to Allah S. W. T for His blessings

throughout the project. Also, I would like to express my sincere appreciation and

heartfelt gratitude to my supervisor, Prof. Dr. Kopli Bujang for his advice, guidance,

encouragement and comment during this project and thesis preparation.

Special thanks to all my seniors who had given useful technical advice, patience and help

in the laboratory work. I would also like to thank my lab mates and friends namely,

Merlina, Lenny, Ugam, and Nur Hafizah, for their continuous encouragement,

friendship and advice while working together in the laboratory. I am also thankful to

Lab assistants Mr. Ajis and Mr. Amin for their assistance.

Lastly, I thank my parents Mr. Booty Osman and Mdm. Halimah Bujang for the

blessings, financial support, encouragement and patience throughout this project. Thank

you so much.

ii

ABSTRACT

(Production of sugars was performed at lab scale (1L) from hydrolysis of various types of

starch (sago, corn, tapioca and sweet potato flour). The starch slurries was enzymatically

hydrolysed for four hours at the starch concentration of 20% DS (200g of starch powder

suspended in 1L water). Filtration of sugar syrup with powdered activated charcoal

(PAC) was made and the measurement of glucose was based on the yield referred as

dextrose equivalent (DE). Upon filtration, the highest sugar (mainly glucose) recovery

was produced by sago starch at 99% DE, followed by corn starch (84% DE), tapioca

starch (76% DE) and sweet potato starch (72% DE). The effectsl of different starch

concentrations in hydrolysis of sago starch (HSS) were then studied) Evidently, 50% DS

generated the highest amount of total reducing sugars (TRS) compared to 40%, 30% and

20% DS at 413 g/L, 377 g/L, 298 g/L, and 205 g/L, respectively. However, the amount of

glucose produced from filtered HSS (20% DS) gave the highest recovery (99% DE), a

much higher concentration of glucose produced compared to 30% DS (89% DE) and 40%

DS (81% DE). Furthermore, the concentration of 50% DS produced the lowest sugar yield

at only 63% DE. Enzymatic hydrolysis of sago starch was performed thereafter at a

larger scale using 20% DS of sago starch at 5L and 50L working volumes. It was

observed that 1,000g of sago starch (suspended in 5L water) yields 66% DE after PAC

compared to 62% DE produced from hydrolysed of 10,000g of sago starch. Consequently,

scaling up the process from 200g to 1,000g reduced the sugar yield by 33% (99% to 66%),

but scaling up further 1,000g to 10,000g reduced the sugars yield by only 4% (66% to

62% DE). These results confirmed that the process could be further scaled up without

significant loss in sugar yield. In addition, 60°C was proved to be the best

111

temperature conditions for sugar syrup or hydrolysed sago starch (HSS). Sago

starch seems to be the most promising as an alternative raw material for the sugar

industry of Malaysia.

Key words: Sago starch, enzymatic hydrolysis of starch, powdered activated charcoal

(PAC), dextrose equivalent (DE)

iv

PENGHASILAN GULA DARIPADA KANJI SAGUDAI. AM SKALA INDUSTRI

ABSTRAK

Penghasilan gula melalui proses hidrolisis kanji berenzim telah dijalankan pada skala

IL menggunakan empat jenis tepung kanji (sagu, jagung, ubi kayu dan keledek). Cairan

kanji telah berjaya dihidrolisiskan oleh enzim dalam tempoh empat jam pada kepekatan

kanji sago 20% DS (200g tepung sago dilarutkan dalam IL air). Larutan gula pula

kemudiannya dijerap warnanya menggunakan serbuk arang teraktif (PAC) di mana

paras gula diukur dengan merujuk kepada nilai setara dektros (DE). Terbukti bahawa

hidrolisis kanji sagu menghasilkan paras glukosa yang tertinggi iaitu 99% DE, diikuti

hidrolisis kanji jagung (84% DE), kanji ubi kayu (76% DE) dan kanji keledek (72% DE).

Kesan terhadap perbezaan kepekatan substrat (kanji sagu) ke atas proses hidrolisis

kanji sagu (HSS) turut dikaji. Terbukti bahawa HSS dengan kepekatan 50% DS

menghasilkan paras gula penurun yang tertinggi berbanding kepekatan 40% 30% dan

20% DS, maisng-masing pada 413 g/L, 377g/L, 298 gIL dan 205 g/L. Tetapi larutan gula

HSS (20% DS) yang telah dijerap warnanya oleh serbuk arang teraktif telah berjaya

menghasilkan paras glukosa tertinggi iaitu sebanyak 99% DE, diikuti kepekatan 30%

(89% DE) dan kepekatan 40% (81% DE). Kepekatan substrat 50% DS kanji pula

sebaliknya hanya mampu menghasilkan glukosa sebanyak 63% DE. Kajian hidrolisis

enzim ke atas kanji sagu (20% DS) diteruskan lagi dengan mengekalkan kepekatan

substrat 20% DS pada skala 5L dan 50L air. Keputusan menunjukkan kanji sago yang

diampaikan dalam 5L air) menghasilkan 66% DE selepas proses penyahwarnaan

dengan karbon teraktif berbanding dengan 10, OOOg kanji sagu diampaikan dalam 50L

air yang menghasilkan 62% DE sahaja. Manakala, apabila kuantiti kanji sagu

ditingkatkan daripada 200g kepada 1,000g, telah berlaku pengurangan paras glukosa

V

sebanyak 33% (99% kepada 66%). Apabila, kuantiti kanji sagu ditingkatkan daripada

1,000g kepada 10,000g pula, hanya 4% penurunan yang dicerap. Berdasarkan nilai

glukosa yang terhasil, kajian lanjut ke atas proses hidrolisis kanji sago pada skala yang

lebih besar masih boleh diteruskan. Tambahan pula, suhu 60°C merupakan suhu

optimum untuk menyimpan larutan gula sagu. Sebagai kesimpulan, kanji sagu boleh

dijadikan bahan alternatifdalam industri gula di Malaysia.

Kata kunci: Kanji sagu, hidrolisis kanji sagu berenzim, serbuk karbon teraktif, nilai

dektros

V1

Pusat Khidmat Makiumat Akademik UNIVERSITI MALAYSIA SARAWAK

TABLE OF CONTENTS

Declaration Acknowledgements Abstract Abstrak Table of contents List of Tables List of Figures List of Abbreviations

CHAPTER 1 INTRODUCTION

1.0 Introduction

1.1 Objectives

CHAPTER 2 LITERATURE REVIEW

2.1 Sugars

2.1.1 Sources of sugars

2.1.2 Sugar Productions

2.1.3 Types of sugars

2.1.4 Application of sugars

2.1.5 Sugar industries in Malaysia

2.2 Starch

2.2.1 Sago Palm (Metroxylon sagu)

2.2.2 Sago starch and its properties

2.2.3 Extraction of sago starch

2.2.4 Applications of sago starch

2.2.5 Sago starch industries in Malaysia

2.2.6 Other starch sources

Pages

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4

5

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9

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21

23

29

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vii

2.3 Conversion of starch to sugars 34

2.3.1 Enzymatic hydrolysis of starch 36

2.3.2 Large scale enzymatic hydrolysis of starch 38

2.4 Purification of sugars 39

2.4.1 Sugar de-colorization using activated carbon 41

2.5 Storage and handling sugar products 42

CHAPTER 3 MATERIALS AND METHODS 44

3.1 Materials 44

3.1.1 Starch 44

3.1.2 Enzymes 44

3.1.3 Powdered Activated Charcoal (PAC) 44 3.1.3.1 Pretreatment of PAC 45

3.1.4 Pilot scale (50L) stainless steel hydrolyser 45

3.2 Methods 47

3.2.1 Enzymatic hydrolysis of starch 47

3.2.2 Lab scale production of sugar from enzymatic 48 hydrolysis of starch 3.2.2.1 Effects on various types of starch 48 3.2.2.2 Effects on different sago starch 48

concentrations

viii

3.2.3 Pilot scale enzymatic hydrolysis of 20% DS

sago starch 3.2.3.1 Hydrolysis of 1Kg sago starch

suspended in 5L water 3.2.3.2 Hydrolysis of 10Kg sago starch

suspended in 50L water

3.2.4 Purification of sugars using Powdered Activated Charcoal (PAC)

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49

51

54

3.2.5 Effects of storage at different temperatures 56

3.3 Analytical methods

3.3.1 Reducing sugars

3.3.2. Starch

3.3.3 Colour

3.3.4 Protein

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57

58

59

60

CHAPTER 4 RESULTS 61

4.1 Lab scale enzymatic hydrolysis of starch 61

4.1.1 Effects on various types of starch 61

4.1.2. Effects on different starch concentrations 64

4.2 Purification of sugars using Powdered Activated 67 Charcoal (PAC)

4.2.2 Effects on different sago starch concentrations

4.2.1 Effects on different types of starch 67

73

4.3 Pilot scale enzymatic hydrolysis of 20% DS sago 78 starch

4.4 Effects of storage at different temperature 82 conditions on PAC purified sago syrup

ix

CHAPTER 5 DISCUSSION

CHAPTER 6 SUMMARY

REFERENCES

APPENDIX A

APPENDIX B

APPENDIX C

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87

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102

104

107

X

List of Tables

Table 1 Physicochemical properties of sago starch (Ahmad et al., 1999). 22

Table 2 Sugar recovery obtained from enzymatic hydrolysis of various starch, 62 sago, corn, tapioca and sweet potato.

Table 3 Sugar production from enzymatic hydrolysis of sago starch at 65 different starch concentrations (20%, 30%, 40% and 50%).

Table 4 Percentage of protein and colour removal before and after PAC

69 treatment on filtered sugar syrups.

Table 5 Sugar recovery obtained from hydrolysed starch (sago, corn, tapioca 71 and sweet potato) before and after PAC treatment (from 5 replicates).

Sugar recovery obtained from HSS before and after PAC treatment at Table 6 different sago starch concentrations (from 5 replicates). 76

Sugars recovery obtained from pilot scale hydrolysis of sago starch Table 7 (20% DS) at 5L and 50L, (from 5 replicates). 79

Table 8 Effects of storage temperature on TRS concentration of PAC purified 83 sago syrup.

Table 9 Dried matter, moisture and starch content based on (1%, w/v) of 107 sago, corn, tapioca and sweet potato flour.

Table 10 Amount of protein loss after purified with PAC. 107

Table 11 Percentage of color removal in after PAC treatment from hydrolysed;

107 sago, corn, tapioca and sweet potato starch.

Percentage of color removal after PAC treatment from HSS at various Table 12 concentrations; 20%, 30%, 40% and 50% DS. 108

Percentage of color removal after PAC treatment from pilot scale Table 13 enzymatic hydrolysis of sago starch (20% DS) at 5L and 50L working 108

volume. Amount of total reducing sugar obtained from enzymatic hydrolysis of Table 14 sago, corn, tapioca and sweet potato starch.

109

Amount of TRS obtained from HSS at various concentrations; 20%, Table 15 30%, 40% and 50% DS before and after PAC treatment. 109

Table 16 Amount of TRS obtained from pilot scale of HSS at 5L and 50L working volume before and after PAC treatment.

Table 17 Amount of TRS obtained from HSS when stored at various temperatures; 60°C, 4°C and room temperature (RT).

109

110

XI

List of Figures

Figure 1 Flow diagram of operation in a raw sugar mill (Andreis et al., 1990). 9

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

Figure 16

Figure 17

Figure 18

Sago palm (Metroxylon sagu) in Sarawak.

Typical sago estate in Sarawak.

19

20

Harvested sago logs awaiting collection. 24

Sago logs are transferred using (a) lorry or as (b) sago rafts to sago mills.

Sago logs are debarked using either an auto debarking machine or manually with a machete.

Debarked sago logs are rasped and mixed with water for starch extraction.

Schematic plan of a typical sago mill in Sarawak, (Bujang pers. comm., 2009).

25

26

26

27

Separation of starch slurry from the waste water. 27

Bagged sago starch ready for export. 28

(a) Schematic design and (b) 50L vessel for Pilot-scale enzymatic hydrolysis of starch.

Lab scale enzymatic hydrolysis of sago starch using a stainless steel vessel (5L).

46

50

Process of saccharification using the pilot scale (50L) hydrolyser. 51

Harvesting process of the hydrolysed sago starch from the prototype 50L system.

52

Hydrolysed sago starch (HSS) with reddish brown colour. 53

Removal of colour and impurities from purified HSS using PAC columns.

55

Effects of starch concentrations on HSS after 6 hours. 64

(a) Sugar syrups from enzymatically hydrolysed starch upon filtration on Whatman (0.45 µm); From left: sago starch (MA); tapioca starch (MB); corn starch (MC); sweet potato starch (MD)

(b) Sugar syrups upon filtration and purification on PAC; From left: sago starch, tapioca starch, corn starch and sweet potato starch

68

xii

Figure 19 Comparison of sugar recovery (DE) before and after treatment 70 with PAC.

(a) Filtered sugar syrups using Whatman 0.45µm cellulose nitrate membrane filters.

Figure 20 From left: HSS 20%, 30%, 40% and 50% DS (b) Purified sugars syrup on PAC.

From left: HSS 20%, 30%, 40%, and 50% DS

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Comparison of sugar recovery (DE) from HSS at different starch Figure 21 concentrations before and after PAC treatment. 75

Comparison of sugar recovery (DE) obtained from pilot-scale Figure 22 enzymatic hydrolysis of sago starch before and after treatment 78

with PAC.

Removal of colour and proteins from HSS (a) before and (b) after Figure 23 PAC filtration. 80

Figure 24 Stability of PAC purified sago sugar syrup under storage at 82 different temperature after 21 says (from 5 replicates).

Figure 25 Formation of melanoids (brown colouration) in PAC purified sago 84 syrup after 21 days during storage at 4°C.

Figure 26 Starch standard calibration curve from Iodine method at 590nm. 104

Figure 27 Glucose standard calibration curve from DNS method at 575nm. 105

Figure 28 Protein standard calibration curve from DC Protein Assay Kit

106 using Bovine Serum Albumin (BSA) as standard at 750nm.

X111

List of Abbreviations

% Percentage

%/kg Percent per kilogram

cm Centimeter

DE Dextrose equivalent

DS Dry substrate

g Gram

g/L Gram per liter

HCl Hydrochloric acid

hr Hour

hrs Hours

H2SO4 Sulfuric acid

HSS Hydrolysed sago starch

HPLC High performance liquid chromatography

kg Kilogram

L Liter

M Molarity

mg/L Milligram per liter

min Minute

mL Milliliter

mug Milliliter per gram

nm nanometer

NaCl Sodium chloride

NaoH Sodium hydroxide

OD Optical Density

xiv

PAC Powdered activated charcoal

RM Ringgit Malaysia

R2 Correlation coefficient

RT Room temperature

t Tones

tons/ha Tones per hector

TRS Total reducing sugar

USD US dollar

v/v Volume per volume

w/v Weight per volume

w/w Weight per weight

pLJg Microliter per gram

µm Micrometer

µL Microliter

xv

CHAPTER 1

INTRODUCTION

1.0 Introduction

Sugar industries in Malaysia can be categorized as well developed as reflected by the

rapid increase in direct domestic consumption which is amplified by an equally fast

growing food processing industry (FOMCA, 2006). Commercial sugar that we consumed

these days is derived from sugar cane. Sugar cane is a very easy and profitable plant to

grow but rather ineffective in reproducing naturally (Braun, 1997). Up till now, sugar

processing industries in Malaysia still depend on imports for about 90% of its raw

materials which has reached a record of 1.0 million tones, compared to export at 101,000

tones. Owing to lack of raw materials and increases in industrial application of cane

sugar naturally lead to higher price of this commodity.

Starch is one of the essential energy source of the living world. Nevertheless, only some

plant species can actively accumulate and store starch (Chulavatnatol, 2001). Sago, corn,

potato, cassava and rice are among the known plants with high starch content which is

a natural raw material alongside other starch-producing plants such as tapioca, rice and

wheat. Sago starch is extracted from the sago palm (Metroxylon spp. ), also known as

"rumbia" by local people (Ahmad et al., 1999). This crop is found abundantly in the state

of Sarawak mainly Mukah, Igan and Oya and well-known as one of the great starch

producer in Malaysia. More than 90% of all sago-planting areas are found in the state of

Sarawak in East Malaysia. The largest (75%) sago planting area is in Mukah where over

1

50% of the sago starch is produced (Bujang and Ahmad, 1999). A fully cultivated sago

estate has about 138 palm/ha/year, and at about 185 kg starch/palm, a total of 25.53 tons

starch/ha/year can be expected.

Sago starch is utilized in the form of sago flour or sago pearl. Other than foodstuffs, sago

starch can also be used to produce adhesives for paper or even as a stabilizer in

pharmaceuticals (Aziz, 2002). Sago starch is highly recommended in the production of

sugar for fermentation products, pharmaceutical application and cosmetics. About

100,000 tones of sago starch are used annually in Malaysia for various applications

including the food industries, household, and glue manufacture. With about 90% of all

sago planting areas in the country, sago sugar industry has a remarkable potential to be

commercialized in Sarawak. A study done by Bujang (2004) has discovered that

bioconversion of sago starch into glucose is a more sensible alternative since glucose

(US$0.50/kg) fetches a higher price then sago starch (US$0.20/kg).

2

Our previous study has shown that sago starch is highly recommended as the starchy-

substrate for sugar production to be used in the production of ethanol (Adeni and

Bujang, 1998) and lactic acid (Bujang et al., 2000). This study highlights the importance

and potentials of sago starch as an alternative source to sugarcane for the production of

commercial sugars. The aim of the study is to determine the highest recovery of sugar

(mainly glucose) produced from sago starch, for the production of commercial sugars

using sago starch, a locally available and cheaper substrates.

3

1.1 Objectives

The principle aim of this research is to maximize glucose production, purification

recovery and consequently to enhance the value of sago starch in Malaysia. In order to

achieve this aim, the objectives of the research project are to:

i) compare the amount of sugars produced from enzymatic hydrolyzed of different

starch sources

ii) develop the separation and purification procedures of sago sugars

iii) study the effects of different starch concentrations during hydrolysis in order to

maximize glucose recovery

iv) study the recovery of sugars from pilot scale enzymatic hydrolysis of sago starch

(20% DS) at 5L and 50L

v) develop the optimum conditions for storage of liquid sago sugars

4

Pusat Khidmat fNaklumat Akademik UNIVERSITI MALAYSIA SARAWAJi

CHAPTER 2

LITERATURE REVIEW

2.1 Sugar

Sugar is a class of edible substance, mainly sucrose. It is a broad term applied to a large

number of carbohydrates present in many plants and characterized by a more or less

sweet taste. In non-scientific use, the term sugar refers to sucrose or "table sugar", a

white crystalline solid disaccharide (Anonymous, 2010; Wikipedia, 2009a). Scientifically,

sugar refers to any monosaccharide (simple sugar) or disaccharide. It is composed of

carbon, hydrogen and oxygen belonging to a class of carbohydrates. It can be categorized

into three main groups; monosaccharide, disaccharides and polysaccharides. Glucose is

the simplest sugars in the monosaccharide family. The disaccharides are formed by the

union of two monosaccharides with loss of one molecule of water, which includes lactose,

maltose and sucrose. Polysaccharides are polymers that contain many monosaccharide

residues; one of the common example is starch.

Sugar has a central position in human consumption and serves as a major foodstuff for

animals. The sugar we normally used nowadays is made of sucrose obtained from

sugarcane; therefore, the industrial production of sugars today is mostly based on cane

sugar and sugar beet processing. Sucrose is a common table sugar that is used to alter

flavor and properties such as preservation, mouth feel and texture in foods and

5

beverages. Sugar may dissolve in water to form syrup. Generically known as "syrup",

they also have specific name such as "honey" or "molasses". Manufacturing and

preparing foods may involve other sugars such as palm sugar and fructose, obtained

from corn (maize).

According to Toth and Rizzuto (1990), back in the 15th century, sugar was economically

important to all European. European sugar was mainly refined in Venice. Later

sugarcane was planted in large plantations in other regions in the world including India,

Indonesia, Philippines and the Pacific. Toth and Rizzuto (1990) revealed that over

110,000,000 tons of sugar per year was used in manufactures and consumed worldwide.

One of the early applications of sugar, it was a crude pharmaceutical ingredients, as it is

still used today to masked the bitter or unpleasant taste of medicine.

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2.1.1 Sources of Sugars

Sugar primarily comes from sugar cane and from sugar beet (Andreis et al., 1990). It also

appears in fruits, honey, sorghum, maple sugar and in many other sources. Sugar is

normally synthesized in plant leaves and as a source of energy for growth and at the

same time will be sent to the stalks for storage. The sweet sap in the stalk source gives

rise to sugar.

Sugarcane cultivation requires a tropical or subtropical climate (Andreis, 1990; Toth and

Rizzuto, 1990), with a minimum of 600mm annual rainfall. It is one of the most efficient

photosynthesizer that can convert as much 20% of incident solar into biomass. One thing

about sugarcane is that it usually propagates from cutting with at least one bud, rather

than from seed. Once planted, a stand of cane can be harvested several times. Usually,

each successive harvest gives a smaller yield, and most eventually the declining yields

justifies replanting. Average yields is about 100 tons of sugarcane per hectare producing

10 tons of cane sugar.

Sugar beet is a member of the Chenopediaceae subfamily under the family of

Amerenthaceae, a plant whose root contains high concentration of sucrose (Food-Info,

2009). It is a temperate climate biennial root crop, producing sugar during the first year

of growth in order to see it over the winter and for the flowers and seeds in the second

year. It is therefore sown in spring and harvested in the first autumn or early winter.

The sucrose is stored in the bulbous root, which bears a strong resemblance to a fat

parsnip (Food-Info, 2009). Typical sugar content for mature beets is 17% by weight but

the value depends on variety and location, and it does vary from year to year. Up untill

7