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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
1
11
111
V
Vii
xi
X11
xiv
1
1
4
5
5
7
9
12
13
14
16
17
21
23
29
30
32
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)
49
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
57
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
85
87
88
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
74
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.
6
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