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EFFECTS OF STARCH CONCENTRATION ON HYDROLYSIS OF SAGO STARCH

Jason Chan Tze Sien

Bachelor of Science with Honours (Resource Biotechnology)

2015

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EFFECTS OF STARCH CONCENTRATION ON HYDROLYSIS OF SAGO STARCH

Jason Chan Tze Sien (36324)

This dissertation is submitted in fulfilment of the requirements for the Degree of Bachelor of Science with Honors in Resource Biotechnology.

Supervisor: Prof. Dr. Kopli bin Bujang

Resource Biotechnology Department of Molecular Biology

Faculty of Resource Science and Technology Universiti Malaysia Sarawak

11/6/2015

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Acknowledgement

I would like to express my gratitude towards the provision of facilities, equipment and materials by Department of Molecular Biology, Universiti Malaysia Sarawak for the accomplishment of my final year project.

Furthermore, I would like to express my deep sense of gratitude and appreciation to my supervisor, Prof. Dr. Kopli bin Bujang for his professional guidance, valuable knowledge and advices, and for spending his precious time to assist me and helped to clear my doubts. I am truly thankful for Prof's dedication and encouragement all along the way towards completing my final year project.

I would also not forget to thank the master students and postgraduates for their advices especially Miss Nadia who guides me a lot in my experiments. Lastly, I would also like to thank my lab mates for sharing their knowledge with me, as well as my parents for their supports and encouragement.

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Declaration

I hereby declare that the content of this report is original and 100% of efforts of own in completion of this FYP report. There is no portion of work referred in this project has been submitted in support of an application for another degree qualification of this or any other university or institution of higher learning.

e

(Jason Chan Tze Sien) Resource Biotechnology

Department of Molecular Biology

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

11 JUNE 2015

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Table of Contents

Acknowledgement Declaration Table of Contents List of Abbreviations List of Tables List of Figures Abstract

1.0 Introduction

2.0 Literature Review

2.1 Sago (Metroxylon sagu) Starch

2.2 Enzymatic Hydrolysis of Sago Starch

2.3 Different Factors Affecting Sago Starch Hydrolysis 2.3.1 Concentrations of Starch

2.3.2 pH of the Mixture 2.4 Types of Starch Hydrolysis 3.0 Materials and Methods

3.1 Materials

3.1.1 Sago Starch

3.1.2 Hydrolytic Enzymes 3.2 Methods

3.2.1 Analysis of Sago Starch 3.2.1.1 Moisture Content

II

III IV VI VII

VIII

IX

1

3

4

5

6

8

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3.2.2 Preparation of Sago Starch Slurries 3.2.3 Liquefaction

3.2.4 Saccharification

8 9 10

3.2.5 Analytical Methods 11

3.2.5.1 High Performance Liquid Chromatography (HPLC)

Determination 11

3.3 Experimental Methods 12

3.3.1 Effects of Starch Concentration on Hydrolysis of Sago Starch 12

4.0 Results and Discussion 14

4.1 Determination of Glucose Content in Each Sample by High-Performance

Liquid Chromatography (HPLC) 14

4.2 Effects of Starch Concentration on Hydrolysis of Sago Starch and

Glucose Recovery 19

5.0 Conclusion 23

References Appendix Appendix A Appendix B

24

25

26

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List of Abbreviations

Abs Absorbance

AmG Amyloglucosidase

DE Dextrose equivalent

DNS Dinitrosalicylic acid

DS Dry weight basis

HCl Hydrochloric acid NaOH Sodium hydroxide

OD Optical density

°C Degree Celsius

PI micro litre

ml millilitre

L litre

g gram

kg kilogram

nm nanometer

% percent

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List of Tables

Table Title Page

1 Glucose recovery of samples with different sago starch concentrations

(10%, 15%, 20%, 25%, 30%, 35% and 40%) 19

2 Weight of crucibles, and weight of crucibles and starch samples

before and after oven drying 25

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List of Figures

Figure Title Page

1 Sago starch kept in clean polypropylene woven bag 6

2 Termamyl SC 7

3 Amyloglucosidase (AmG) SC 7

4 Electronic balance used for weighing sago starch 9

5 1 L volumetric flask 9

6 Heating of mixture of starch and liquefaction enzyme in water bath

at 90°C for 2 hours 10

7 Orbital incubator shaker used for saccharification process 11 8 High Performance Liquid Chromatography (HPLC) with RID-20A

detector column used 12

9 Sago starch (100 g) was weighed using electronic balance 13

10 Duran bottle labeled 10% for 10% starch concentration to produce

sago sugars 13

11 HPLC chromatogram generated by sample of 10% sago starch 15 12 HPLC chromatogram generated by sample of 15% sago starch 15 13 HPLC chromatogram generated by sample of 20% sago starch 16 14 HPLC chromatogram generated by sample of 25% sago starch 16 15 HPLC chromatogram generated by sample of 30% sago starch 17 16 HPLC chromatogram generated by sample of 35% sago starch 17 17 HPLC chromatogram generated by sample of 40% sago starch 18 18 Glucose recovery of samples with different sago starch concentrations 20 19 Difficulty of attempting to mix voluminous amount (400 g) of

starch into 1 L of water at one go 21

20 The products of hydrolysed sago starch (10% to 40% accordingly) 22

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Effects of Starch Concentration on Hydrolysis of Sago Starch Jason Chan Tze Sien

Resource Biotechnology

Faculty of Resource Science and Technology Universiti Malaysia Sarawak

ABSTRACT

High dependence on imports of raw sugar and application of cane sugar by sugar processing industries has been a problem as the price of sugar is rising. Because of this, production of sugar from enzymatic hydrolysis of sago starch must be maximized. Enzymatic hydrolysis of sago starch producing raw sugar is affected by the concentration of starch. A study was carried out to determine the optimum starch concentration which yields highest amount of sugar and study the effects of various starch concentrations on sago starch hydrolysis. Different concentrations (w/v) of starch (10%, 15%, 20%, 25%, 30%, 35% and 40%) were prepared to study the effects of starch concentration on sago starch hydrolysis, with other variables (pH, temperature and concentration of enzymes) kept constant. Glucose produced was studied and analysed using High Performance Liquid Chromatography (HPLC). Glucose recovery (%) was used to express starch conversion yield into glucose. The optimum starch concentration which yields highest amount of glucose in sago starch hydrolysis obtained from this research is 35% (350 g/L). The yield of glucose increases as the starch concentration increases but it drops beyond starch concentration of 35%, probably due to the insufficient amount of enzyme molecules to digest the starch. High amount of starch (400 g or above) also poses problems during mixing with distilled water as the medium for enzymatic hydrolysis.

Key words: Enzymatic hydrolysis, sago starch, starch concentrations, High Performance Liquid Chromatography (HPLC), glucose recovery

ABSTRAK

Pergantungan yang terlalu melampau pada import gula mentah dan penggunaan gula tebu oleh industri memproses gula telah menjadi masalah kerana harga gula semakin meningkat. Oleh itu, penghasilan gula dari hidrolisis enzimatik kanji sagu mesti dimaksimumkan. Hidrolisis enzimatik kanji sagu yang menghasilkan gula mentah dipengaruhi oleh kepekatan kanji. Satu kajian telah dijalankan untuk mengetahui kepekatan optimum kanji yang menghasilkan amaun gula yang paling tinggi dan mempelajari kesan-kesan kepekatan kanji terhadap hidrolisis kanji sagu. Beberapa kepekatan (w/v) kanji (10%, 15% 20% 25%, 30%, 35% dan 40%) telah disediakan untuk tujuan tersebut, dengan faktor faktor lain (pH, suhu dan kepekatan enzim) dimalarkan. Glukosa yang dihasilkan telah dikaji dan dianalisis dengan menggunakan Kromatografi Cecair Prestasi Tinggi (HPLC). Pemulihan glukosa (%) telah digunakan untuk menyampaikan hasil penukaran kanji kepada glukosa. Kepekatan optimum kanji yang menghasilkan amaun glukosa yang paling tinggi diperolehi daripada kajian ini ialah 35% (350 g/L). Penghasilan glukosa meningkat dengan bertambahnya kepekatan kanji tetapi menurun selepas kepekatan kanji sebanyak 35%, mungkin disebabkan kekurangan molekul enzim untuk mencerna kanji. Amaun kanji yang tinggi (400 g atau lebih) juga menyebabkan masalah apabila pencampuran dengan air tulen sebagai medium untuk hidrolisis enzimatik.

Kata kunci: Hidrolisis enzimatik, kanji sagu, kepekatan kanji, Kromatografi Cecair Prestasi Tinggi (HPLC), pemulihan glukosa

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1.0 Introduction

The highest production of starch (about 25 tons per hectare) has been contributed by sago every year. The production is approximately four times higher than that of wheat, rice and corn (lshizaki, 1997). It is fortunate that Malaysia has outsized sago planting areas and is suitable for the growth of sago plantation. Sarawak, the state which contributes the highest production of starch, has the most abundant sago planting areas of about 30000 to 50000 hectares. This has made Sarawak the world largest sago starch exporter in 2013 with a record of approximately 47,946 tonnes of sago starch in total export (DOS, 2013). The abilities of sago such as surviving in peat soils and growing without much care have made it to become one of the major sources of energy or biofuel and bioplastic. Basically, one hectare of sago plantation can give rise to 12.5 tons of biofuel as about 500 kg of ethanol can be produced from one ton of glucose through fermentation (Bujang et al., 2000).

However, too much dependence on imports of raw sugar and application of cane sugar by sugar processing industries has been a problem as the price of sugar is rising (Bujang, 2011). Besides, another two problems faced by the society are the discarding of non-degradable plastics and deficiency of fuel. With the availability of sago starch, our country is able to utilize its advantages fully to solve these problems by generating biofuel and bioplastics. Therefore, to fully utilize sago starch for lactate and ethanol fermentation, enzymatic hydrolysis of sago starch to sugar must be maximized. In this, concentration of sago starch used is an important factor in affecting the efficiency of enzymatic hydrolysis.

It has been reported by Bujang et al. (2000), Booty and Bujang (2009) that the best starch concentration to produce sago sugars is 20% (200 g/L). Hence, this research study is

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carried out to study the effects of different starch concentrations during enzymatic hydrolysis and to observe its possibility to increase the starch concentration beyond 20%.

The aims of this project are to:

1. Produce sugar from sago starch.

2. Determine the optimum starch concentration which yields highest amount of glucose in sago starch hydrolysis.

3. Determine the effects of various starch concentrations (10%, 15%, 20%, 25%, 30%, 35% and 40%) on sago starch hydrolysis.

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2.0 Literature Review

2.1 Sago (Metroxylon sagu) Starch

Sago palm is one of the most abundant palms found distributed in South East Asia region.

The regular temperature and humidity (25 °C and 70% respectively) in these tropical areas are suitable for the growth of the palm (Bujang, 2011). Sago palm does not depend on herbicide and pesticide to survive in the peat soils or wet areas (Pei-Lang et al., 2006).

Singhal et al. (2008) found that the utmost growth of sago palm required light contact (more than 800 K/cm2) and soil with low salt concentration. Sarawak has always been one of the contributors of highest starch production. According to Ishizaki (1997), sago starch production was reported to be around 15 to 25 tons/ha in Sarawak. The demand for starch has enlarged yearly for many countries because of numerous products can be derived from sago palm. There were two huge plantations of total 30000 ha commenced by Sarawak state government at Mukah and Dalat districts to meet the growing requirement (Singhal et al., 2008). The acknowledgment of world largest sago starch exporter was entitled to Sarawak in 2013 with a record of approximately 47,946 tonnes of sago starch in total export (DOS, 2013).

One of the main factors why glucose production from sago starch has to be maximized is that the country still depends too much on imports of raw materials from other countries. According to Bujang (2011), approximately 1 million tons of raw materials have been imported from countries such as Thailand, Australia and Brazil. Besides, sugar production from sugar cane is insufficient to meet the demand as sugar cane is not so effective in natural reproduction (Braun, 1999). Consequently, the price of this commodity rises with the escalating utilization of cane sugar in sugar processing industry.

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2.2 Enzymatic Hydrolysis of Sago Starch

Researches on the hydrolysis of sago starch have been carried out to study the effects of different variables such as concentration of starch or enzymes, pH, and types of starch.

These researches were done by UNIMAS researchers at lab-scale stage between years 1998 to 2010 (Bujang, 2011). Later, improvements were made by using 50 L prototype starch hydrolyser to increase the volume. This was followed by 1000 L pilot-scale levels by early 2012 (Bujang, 2011). These previous researches have shown that the problem of insufficiency in sugar production from sugar cane could be solved as the production of sago sugars is an on-going progression with much enhancement. Termamyl-120L was used in the liquefaction process whereas Dextrozyme was used in saccharification (Bujang &

Jobli, 2002). The incubation time for saccharification was longer if the volumes of hydrolysis were larger (Bujang & Law, 2006).

2.3 Different Factors Affecting Sago Starch Hydrolysis 2.3.1 Concentrations of Starch

According to Booty and Bujang (2009), the optimum concentration of starch for enzymatic sago starch hydrolysis generating more than 100% recovery of sago sugars was 20%. This concentration was used in enzymatic hydrolysis to test for its effects in scaling up process (1 L, 5 L and 50 L). The production yield of sago sugars for 200 g starch (in 1 L water), 1000 g starch (in 5 L water) and 10000 g starch (in 50 L water) were 99%, 66%, and 62%

respectively (Bujang, 2011). The yield difference between 1000 g starch (66%) and 10000 g starch (62%) is only 4% whereas yield difference between 200 g starch (99%) and 1000 g starch (66%) is 33%. This shows that the recovery of sago sugars reduced less

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significantly when the process was scaled up from 1000 g starch to 10000 g starch, and

also possibly the same when higher scaling-up is carried out (Booty & Bujang, 2009).

2.3.2 pH of the Mixture

The optimum pH for sago starch hydrolysis has not been established although enzyme supplier Novo has validated that the optimum pH for liquefaction and saccharification in general starch hydrolysis were pH 6.5 and pH 4.5 respectively (Bujang et al., 2000).

2.4 Types of Starch Hydrolysis

There were other types of starch that have been tested for enzymatic hydrolysis before, for example sweet potato, tapioca and corn (Booty & Bujang, 2009). When compared to the other starch hydrolysis, sago starch gave rise to highest recovery of sugars (over 100% DE), with 205 g/L of sugars converted from 200 g (20% w/v) of sago starch (Bujang, 2011).

Results showed that the dextrose equivalent (DE) of sweet potato, tapioca and corn were

86% DE, 93% DE, and 99% DE respectively (Bujang, 2011). This showed that sweet

potato starch contributed to the lowest recovery of sugars among them.

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3.0 Materials and Methods

3.1 Materials

3.1.1 Sago Starch

Food grade sago starch was obtained from Herdsen Sago Mill, Pusa. The sago starch was kept in clean polypropylene woven bag with thick plastic lining until further use as shown in Figure 1.

Figure 1: Sago starch kept in clean polypropylene woven bag

3.1.2 Hydrolytic Enzymes

Enzyme for liquefaction (Termamyl SC) and enzyme for saccharification (Amyloglucosidase SC) were supplied by Novo Nordisk in Denmark. Termamyl SC (Figure 2) was thermostable a-amylase extracted from Bacillus licheniformis (120 KNU/g) whereas Amyloglucosidase (AmG) SC (Figure 3) was glucoamylase

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isolated from Aspergillus niger. They were kept in the refrigerator until further use.

0.5 pl Termamyl SC was added to starch slurry per gram for liquefaction whereas 0.6 pl Amyloglucosidase SC per gram of starch was added to the liquefied suspension for saccharification.

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Figure 2: Termamyl SC

Figure 3: Amyloglucosidase (AmG) SC

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3.2 Methods

3.2.1 Analysis of Sago Starch

3.2.1.1 Moisture Content

The moisture content of sago starch was determined using the equation below.

wl - w2

Moisture content (%) = w1-w

X 100%

where, w= weight of crucible (g)

w1= weight of crucible and starch sample before oven drying (g)

w2= weight of crucible and starch sample after oven drying at 60 °C for 24 hours (g) Average of moisture content of three trials was taken.

3.2.2 Preparation of Sago Starch Slurries

The concentration of starch was adjusted and prepared as 10%, 15%, 20%, 25%, 30%, 35%

and 40% on a dry weight basis (DS). 10% (DS) of starch slurry was prepared by dissolving 100 g of sago starch in I L of water. This was followed by dissolving each 150 g, 200 g, 250 g, 300 g, 350 g and 400 g of sago starch in six separate beakers (1 L of water each) to prepare 15%, 20%, 25%, 30%, 35% and 40% (DS) of starch slurry respectively. The amount of sago starch needed was weighed and measured by using an electronic balance as shown in Figure 4. Each of the samples was stirred well and poured into a I L volumetric flask and then topped up till 1 L as shown in Figure 5. Each of the starch slurries was then poured into a beaker.

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Figure 4: Electronic balance used for weighing sago starch

Figure 5: 1 L volumetric flask

3.2.3 Liquefaction

The pH of starch suspension in liquefaction was kept constant at pH 6.5 throughout the whole process, adjusted using I M of sodium hydroxide (Bujang et al., 2000). 0.5 µl Termamyl SC was added to starch slurry per gram using pipette during liquefaction. Then,

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the heating of mixture of starch and liquefaction enzyme was carried out in water bath at 90 °C for 2 hours, as shown in Figure 6. The mixture was stirred constantly.

Figure 6: Heating of mixture of starch and liquefaction enzyme in water bath at 90 °C for 2 hours

3.2.4 Saccharification

After liquefaction, it was cooled down and the temperature was allowed to drop till around 60 °C. The pH of starch suspension in saccharification was kept constant at pH 4.5 using I M of hydrochloric acid (HCl) throughout the whole process (Bujang et al., 2000). 0.6 µl Amyloglucosidase SC per gram of starch was added to the liquefied suspension that had been treated with Termamyl SC. Then, the mixture was incubated at 60 °C for another 24 hours using orbital incubator shaker (Bujang & Jobli, 2002) as shown in Figure 7.

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Figure 7: Orbital incubator shaker used for saccharification process

3.2.5 Analytical Methods

3.2.5.1 High Performance Liquid Chromatography (HPLC) Determination

Determination of reducing sugars present in HSS was done using HPLC, on a Refractive Index (RID-20A) detector with a column of prominence CTO-20A and 0.005 M H2SO4 as the mobile phase as shown in Figure 8. The column temperature and flow rate used was 60 °C and 0.8 ml/min respectively. The analysis for each sample was done for 5 minutes.

Sugar standard was used to determine the amount of glucose in HSS. The samples were diluted 10 times prior to analysis and 20 pl of each diluted sample was injected.

Cihicncv RPCnvPrv= Concentration of Glucose

X 100%

vI-v--- ".,.,., Total Amount of Starch Sample per Litre X 1.11

* 1.1 1= constant, total conversion of starch into glucose

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Figure 8: High Performance Liquid Chromatography (HPLC) with RID-20A detector column used

3.3 Experimental Methods

3.3.1 Effects of Starch Concentration on Hydrolysis of Sago Starch

10% of starch slurry was prepared by dissolving 100 g of sago starch and poured into a I L volumetric flask and then topped up till I L. The amount of sago starch needed (100 g) was weighed and measured by using an electronic balance as shown in Figure 9. The starch slurry was then poured into a Duran bottle labeled 10% as shown in Figure 10. After adding 50 µl Termamyl SC to starch slurry, the mixture was incubated in water bath at 90 °C for 2 hours at pH 6.5. After 2 hours, 60 µl Amyloglucosidase SC was added to the liquefied suspension and the mixture was incubated at 60 °C for another 24 hours at pH 4.5.

The concentration of glucose present was analysed using High Performance Liquid Chromatography (HPLC). Glucose recovery was calculated for the conversion yield of starch into glucose.

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Figure 9: Sago starch (100 g) was weighed using electronic balance

Figure 10: Duran bottle labeled 10% for 10% starch concentration to produce sago sugars

This process was repeated for starch concentration of 15% (150 g/L), 20% (200 g/L), 25%

(250 g/L), 30% (300 g/L), 35% (350 g/L) and 40% (400 g/L).

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4.0 Results and Discussion

4.1 Determination of Glucose Content in Each Sample by High-Performance Liquid Chromatography (HPLC)

High-Performance Liquid Chromatography (HPLC) is an effective method for separation and quantification of sugar components. The analysis of HPLC includes both qualitative and quantitative analysis. Qualitative analysis could be done by identifying the peaks formed in the chromatogram. A peak represents a signal which is produced when a chemical is detected by the instrument. Retention time is assigned to every peak in the chromatogram. Retention time is the time taken for a compound to exit from HPLC column.

For quantitative analysis, response generated by the detector is dependent on the amount of analyte and matrix. There are also some requirements to be met before the analysis, otherwise less accurate results will be obtained. There are various analytical approaches and mathematical models available for quantitation of analytes.

In this research, the column temperature and flow rate used was 60 °C and 0.8 mu min respectively with injection volume of 20 µl. Figures 11, 12, 13, 14, 15, 16, and 17 show the HPLC chromatograms generated by samples of 10%, 15%, 20%, 25%, 30%, 35%, and 40% sago starch respectively.

When 10% (100 g/L) starch was used, the highest glucose concentration was shown in Figure 11. The actual glucose yield obtained is shown in Table 1.

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The highest glucose concentration when 15% (150 g/L) starch was used was shown in Figure 12. The actual glucose yield obtained is shown in Table 1.

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When 20% (200 g/L) starch was used, the highest glucose concentration was shown in Figure 13. The actual glucose yield obtained for this starch concentration is shown in Table 1.

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There are basically five peaks for all my samples except for 40% sample. The peak for glucose with retention time of about 3.9 minutes is shown clearly and is the highest peak in every sample. Other peaks include the first two small peaks which represent the

"noise" and peaks for ethanol. This means that the HPLC results of all my samples are generally good except for 40% sample which has too many "noise" peaks as shown in Figure 17. This is probably due to the contamination of sample during filtering or dilution process. The amount of glucose in each sample was calculated using the standard method.

With this, the glucose content in each sample was determined and the glucose recovery for every sample was calculated.

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4.2 Effects of Starch Concentration on Hydrolysis of Sago Starch and Glucose Recovery

Glucose recovery or conversion yield of sago starch into glucose by enzymes is the amount of glucose that has been converted from sago starch in percentage. Table 1 shows the glucose recovery of samples with different sago starch concentrations (10%, 15%, 20%, 25%, 30%, 35% and 40%).

Table 1: Glucose recovery of samples with different sago starch concentrations (10%, 15%, 20%, 25%, 30%, 35% and 40%)

Samples (%) Glucose Content (gIL) Glucose Recovery (%)

10 115.8 104.32

15 169.6 101.86

20 232.7 104.82

25 297.1 107.06

30 357.1 107.24

35 429.1 110.45

40 261.9 58.99

Based on the table, the glucose recovery of 10% sample is 104.32%, then it drops a little bit to 101.86% in 15% sample, after that it continues to increase gradually to the highest, 110.45% in 35% sample. However, the glucose recovery declines tremendously after 35% sample, which is 58.99% only in 40% sample. This means that the highest glucose recovery is 110.45%, contributed by sample with 35% of starch. Glucose content recorded for 35% starch sample is 429.1 g/L. On the other hand, sample with 40% of starch

19

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contributes to the lowest glucose recovery (58.99%), with glucose content of 261.9 g/L only.

The results of the research show that sugar has been produced from sago starch by enzymatic hydrolysis. All of the samples (up to 35%) show positive results for the detection of glucose with glucose recovery over 100%. A variety of starch hydrolysis including tapioca, corn and sweet potato has been carried out to produce sugars in previous studies. The sugar recovery produced by corn starch, tapioca starch and sweet potato were 99% DE, 93% DE and 86% DE respectively (Bujang, 2011). When compared to the other starch hydrolysis, sago starch in this research gave rise to highest recovery of glucose (110.45%), with 429.1 g/L of glucose converted from 35% (350 g/L) of sago starch. This means that sago starch is better in the production of sugars when compared to other starches. The glucose recovery of samples with different sago starch concentrations (10%, 15%, 20%, 25%, 30%, 35% and 40%) is also illustrated in the bar chart as shown in Figure

18.

Glucose Recovery of Sago Starch (%)

120 100

80 60 40 20

0

104.32

10

101.86 104.82 107.06 107.24 110.45

I'll

15 20 25 30

Samples (%)

35

58.99

i

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Figure 18: Glucose recovery of samples with different sago starch concentrations

Glucose Recovery of Sago Starch (%)

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The optimum starch concentration which yielded highest recovery of glucose (110.45%) in hydrolysis is 35% (350 g/L), as shown clearly in the bar chart. This means that enzymes Termamyl and Amyloglucosidase work best in hydrolysis processes when the starch concentration is 35%. Termamyl converts the gelatinized starch into liquefied starch most effectively and decreases viscosity of starch slurries whereas Amyloglucosidase works to produce highest amount of glucose at this starch concentration. As for other starch concentrations, 10% to 30% samples generally yielded glucose recovery in the range of 101% to 107%, in an increasing manner. After the optimum concentration of 35%, there is a tremendous decline of glucose recovery in 40% sample, which is only 58.99%. This means that the enzymes work fairly well in 10% to 30% starch samples, but not as good as in 35% sample. In contrast, the great decline of glucose recovery in 40% sample is most probably due to the inhibiting action of reducing sugars produced on enzymes (Kolusheva

& Marinova, 2007). This was also due to the difficulty of attempting to mix the voluminous amount (400 g) of starch into 1 L of water at one go, as shown in Figure 19.

Figure 19: Difficulty of attempting to mix voluminous amount (400 g) of starch into I L of water at one go

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The products of hydrolysed sago starch (10% to 40% accordingly) or sago sugar is shown in Figure 20. The figure shows the amount of suspended solids in HSS product increased with increasing substrate concentration up to 40%. 10% HSS product has the lowest amount of undissolved or suspended solids, whereas at 40%, product has the highest amount of suspended solids on the bottom. Clearly, increasing the concentration of sago starch caused higher amount of suspended solids which needs to be removed by centrifugation for production of brown sago sugar.

As in the 40% HSS product, it contained too much undissolved suspension which gave it cloudy colour. This is most probably because of the failure of enzymes to work on the high concentration of starch as mentioned earlier, causing the hydrolysis process to be incomplete.

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5.0 Conclusion

The results of the research show that sugar has been produced from sago starch by enzymatic hydrolysis. All of the samples (up to 35%) show positive results for the detection of glucose with glucose recovery over 100%. According to Monib (2015), sago sugar is 94% glucose, 3% maltose and the remaining 3% are impurities. As such, the high yield of `glucose' at starch concentration more than 20% probably are reducing sugars and not glucose (Bujang et al., 2000).

The optimum starch concentration which yielded highest recovery of glucose (110.45%) in hydrolysis is 35% (350 g/L), with 429.1 g/L of glucose converted from 350 g/L of sago starch. However, in an actual hydrolysis process, 20% (200 g/L) starch concentration is recommended as there is only an increment of 6.3% in the yield for 15%

increase in starch concentration. If the objective is just to produce reducing sugar for lactate or ethanol fermentation, then it is quite acceptable to use more than 30% starch in the mixture. But if pure glucose is needed, the enzyme concentration will need to be increased to ensure glucose (not just reducing sugars) is produced. In this case, starch concentration can be increased up to a maximum of 35%.

As for other starch concentrations, 10% (100 g/L) to 30% (300 g/L) samples generally yielded glucose recovery in the range of 101% to 107%, in an increasing manner.

In contrast, the great decline of glucose recovery (58.99%) in 40% (400 g/L) sample is most probably due to the difficulty of attempting to mix voluminous amount (400 g) of starch into I L of water at one go. Therefore, it is not advisable to utilize 40% or above of starch concentration in hydrolysis for high glucose yield purposes as it will be quite impossible.

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References

Booty, H. B., & Bujang, K. B. (2009, Oct). Maximising production of sugars from enzymatic hydrolysis of various starch sources, compared to sago starch. Paper presented at the I"ASEAN Sago Symposium, Kuching, Sarawak, Malaysia.

Braun, T. (1999). Sugarcane. Retrieved from http: //www. ethnoleaflets. com

Bujang, K. B. (2010, July). Production and processing of sago: A food and fuel alternative.

Paper presented at the International Seminar on Sago and Spices for Food Security, Ambon, Indonesia.

Bujang, K. B. (2011, Oct). Potential of sago for commercial production of sugars. Paper presented at the 10`h International Sago Symposium, Bogor, Indonesia.

Bujang, K. B., Awang-Adeni, D. S., & Jolhiri, P. (2000). Effects of starch concentration and pH on enzymatic hydrolysis of sago starch. ICBiotech. Osaka Univ. Japan, (14), 32-35.

Bujang, K. B., & Jobli, S. (2002). Effects of glucose feed concentrations on continuous lactate production from sago starch. Inter Symp. Tropical Natural Bio-Resources and Green Chemistry Strategy. ICBiotech. Osaka Univ. Japan, 358 - 363.

Bujang, K. B., & Law, P. L. (2006, Dec). Pilot-scale production of sugars from sago starch. Paper presented at Bio-Malaysia 2006, Kuala Lumpur Convention Centre.

DOS. (2013). Export of sago starch by country of destination 2009-2013, pp 31. Sarawak:

Department of Statistic Malaysia.

Ishizaki, A. (1997). Concluding remarks for the 6th International Sago Symposium, Riau, Indonesia. Sago Comm. July, 8(2), 22-24.

Kolusheva, T., & Marinova, A. (2007). A study of the optimal conditions for starch hydrolysis through thermostable a-amylase. Journal of the University of Chemical Technology and Metallurgy, 42(1), 93-96.

Monib, N. J. (2015). Development on Sago Sugars Recovery and Purification by Filtration on Powdered Activated Carbon (PAC). Master Thesis. Dept. of Molecular Biology.

Faculty of Resource Science and Technology. Universiti Malaysia Sarawak. 94300 Kota Samarahan, Sarawak, Malaysia.

Pei-Lang, A. T., Mohamed, A. M. D., & Karim, A. A. (2006). Sago starch and composition of associated components in palms of different growth stages. Carbohydrate Polymers, 63, 283-286.

Singhal, R. S., Kennedy, J. F., Gopalakrishnan, S. M., Kaczmarek, A., Knil, C. J., &

Akmar, P. F. (2008). Industrial production, processing, and utilisation of sago palm-derived products. Carbohydrate Polymers, 72, 1-20.

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Appendix

Appendix A

Moisture Content

Table 2: Weight of crucibles, and weight of crucibles and starch samples before and after oven drying

Crucible Weight Weight of Weight of crucible and starch sample

of crucible and after oven drying at 60°C for 24 hours,

crucible starch sample w2 (g) wl-w2 wl-w

, w (g) before oven

Day 1 Day 2 Day 3 Day 4 drying, wl (g)

A 94.094 114.094 112.381 111.759 111.752 111.685 2.409 20

B 91.263 111.263 109.312 108.752 108.735 108.725 2.538 20

C 79.244 99.244 97.577 97.183 97.151 97.132 2.445 20

Calculations

Moisture content of sample A

Moisture content of sample B

2.409

= 2 0 X 100%

= 12.05%

2.538 20

= 12.69%

X 100%

2.445 Moisture content of sample C =

20 X 100%

= 12.23%

Average moisture content of sago starch =

12.05+12.69+12.23 , 3

= 12.32%

Therefore, the average moisture content of sago starch is 12.32%.

U/0

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Appendix B

Glucose Recovery

Concentration of glucose in 10% sample = 11.58 g/L X 10

Glucose recovery of 10% sample 115.8

g/L X 100%

100X 1.11

= 104.32%

Glucose recovery of 15% sample _

169.6

g/L X 100%

150X 1.11

= 101.86%

Concentration of glucose in 20% sample = 23.27 g/L X 10

200X 1.11 g/L X 100%

= 104.82%

Glucose recovery of 25% sample

= 250X 1.11^297.1 gJL X 100%

= 107.06%

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g/LX100%

300X 1.11

= 107.24%

Glucose recovery of 35% sample

429.1 _ýý,

ý ,, g/LX100%

350X 1.11

= 110.45%

Concentration of glucose in 40% sample = 26.19 g/L X 10 261.9

Glucose recovery of 40% sample = g/L X 100%

400X 1.11

= 58.99%