Synthesis and Characterization of Starch Adipate Nanoparticles from Native Sago Starch

Synthesis and Characterization of Starch Adipate Nanoparticles from Native Sago Starch

Donny Goh Wah Ern (41045)

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Synthesis and Characterisation of Starch Adipate Nanoparticles from Native Sago Starch

Donny Goh Wah Ern (41045)

This project is submitted in partial fulfilment of the requirements for the Degree of Bachelor of Science with Honours

Supervisor: Assoc. Prof. Dr. Chin Suk Fun Co-supervisor: Dr. Samuel Lihan

Resource Chemistry Department of Chemistry

Faculty of Resource Science and Technology University Malaysia Sarawak

2016

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Acknowledgement

This final year project would not been accomplished without the support of several people. It is my pleasure to acknowledge these individuals who were instrumental for completion of my project. I would like to express my appreciation and gratitude to my dedicated and hardworking supervisor, Associate Prof Dr. Chin Suk Fun for her guidance and encouragement in helping me to conduct my research and thesis writing. I sincerely enjoyed working in a research environment that stimulates my way of thinking and using knowledge that I have learnt in application.

I also wish to sincerely thanks and express appreciation to Mr. Shafri for assisting me in scanning electron microscopy analysis. Not to forget Mr. Wahad for the knowledge in using fourier transform infrared spectroscopy. I would like to acknowledge the valuable input of Miss Lim and Mr. Tan for willing to share opinions, knowledge and experience with me.

Besides, this work would not have been possible without fully support from my family.

My deepest appreciation belongs to them for their patience and understanding.

Last but not least, thanks for numerous friend who endured for this long process with me, offering support and love during my work in research and analysis. Without your help, this project would not be successfully done.

UNIVERSITI MALAYSIA SARAWAK

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Table of Contents

Acknowledgement. . . ... . . . .. I

Declaration ... .... II

Table of Contents ... .... III

List of Abbreviations ... V

List of Figures . . . ... . . ... VI

List of Tables. ... VIII

Abstract ... .

1.0 Introduction ... ... 2

2.0 Literature Review... ... 4

2.1 Sago Starch ... ... .... 4

2.2 Starch Nanoparticles ... 5

2.3 Adipic Acid... ... 6

2.4 Modification of Starch ... 7

2.5 Esterification of Starch. ... ... ... ... 8

3.0 Materials and Method... ... .... . ... 9

3.1 Materials... ... 9

3.2 Synthesis of Starch Adipate Nanopaticles ... ... 9

3.3 Characterization... 12

3.3.1 Fourier Transform Infrared Spectroscopy (FTIR) ... 12

3.3.2 Scanning Electron Microscope (SEM)... ... 12

III

3.3.3 Degree of Substitution ... ... 12

3.3.4 Solubility. .... ... .... ... 13

3.3.5 Moisture Absorbency... ... ... ... 14

3.3.6 Antibacterial Study... ... 14

3.3.6.1 Diffusion Assay... ... ... 14

4.0 Results and Discussion... 15

4.1 Fourier Transform Infrared Spectroscopy (FTIR) ... ... 15

4.2 Scanning Electron Micrscope (SEM) ... ... 16

4.3 Effect of Sodium Hydroxide Molarity on Degree of Substitution (DS) ... 18

4.4 Effect of Adipic Acid Molarity on Degree of Substitution (DS) ... 23

4.5 Effect of Reaction Temperature... ... .... 29

4.6 Effect of Precipitating Media... 30

4.7Solubility... .. ... 31

4.8 Moisture Absorbency... ... 32

4.9 Antibacterial Study... ... 33

4.9.1 Diffusion Assay... ... ... 33

5.0 Conclusion... .... ... ... 34

6.0 References... ... ... 35

Appendixes.. ... ... ... ... ... 43

AGU DS FfIR HCI IUPAC KBr MHA NaOH SANPs SEM

List of Abbreviations

Anhydrous Glucose Unit Degree of Substitution Fourier Transform Infrared Hydrochloric Acid

International Union of Pure and Applied Chemistry Potassium Bromide

Muller-Hinton Agar Sodium Hydroxide

Starch Adipate Nanoparticles Scanning Electron Microscope

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Figure 1

Figure 2 Figure 3 Figure 4 I

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

List of Figures

Molecular structure of starch;

amylose

Molecular structure of adipic acid

(A) amylopectin and; (B) 4

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Esterification processes of SANPs 10

Esterification reaction of native sago starch with adipic acid 11 FTIR spectrums of native sago starch and starch adipate nanoparticles (A) native sago starch and; (B) SANPs with molar ratio 1: 1: 4 (AGU: NaOH: Adipic Acid)

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SEM micrographs of, (A) native sago starch; (B) regenerated sago starch and; (C) SANPs (DS=0366)

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FTIR spectra for native sago starch and SANPs at different molarity ofNaOH

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Degree of substitution of samples with different molar ratio of (NaOH)/ AGU)

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SEM micrographs of native sago starch and SANPs synthesized with different molar ratio of AGU: NaOH: adipic acid where, sample (C) 1: 1: 4 and sample (D) 1: 1.5: 4

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FTIR spectra of native sago starch and SANPs sample

synthesise~y different molarity of adipic acid; (A) 1: 1: 1; (B) 1: 1: 2; (C) 1: 1: 3 and (D) 1: 1: 4

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Degree of substitution of SANPs with different molar ratio of adipic acid! AGU

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SEM micrographs of SANPs synthesized by different molar ratio (AGU: NaOH: adipic acid) where, (A) 1:1: 1; (B) 1: 1: 2;

(C) 1: I: 3; and (D) I: I: 4

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Size of particles (nm) synthesized by different molar ratio of adipic acid / AGU

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Figure 14

Figure 15

Figure 16 Figure 17

Figure 18

FTIR spectra of sago starch and SANPs synthesized in various reaction temperatures

29 FTIR spectra of SANPs (DS=0.366) precipitated in different

media (A) ethanol, (B) propanol, and (C) butanol

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Solubility ofSANPs with different DS value 31 The percentage moisture absorbency of SANPs with different DS value

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Agar diffusion assay which incubated with E. coli and paper disks contained 5 and 20 III of starch adipate nanoparticles (DS=0.366) solution and paper disk with water as control, (A) before incubation, (B) after 18 hours incubation

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VII

List of Tables

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Table 1 Initial screening process of different molarity of N aOH used in 18 starch gelatinization

Table 2 SANPs samples with different molarity of adipic acid 23

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Synthesis and Characterization of Starch Adipate Nanoparticles from Native Sago Starch

Donny Goh Wah Ern

Resource Chemistry

Faculty Resource Science and Technology University Malaysia Sarawak

ABSTRACT

This research aimed to synthesize and characterize physicochemical properties of starch adipate nanoparticles (SANPs). SANPs was synthesized by esterification reaction of native sago starch and adipic acid. The physical and chemical properties of SANPs were characterized. Throughout the screening of the esterification, with the condition temperature of 100°C and ethanol as precipitating media, gave the carbonyl group between starch and adipic acid. Different molar ratio of sodium hydroxide and adipic acid were used in synthesizing SANPs. The substitution of adipic acid onto starch chain was confinned by FTIR spectroscopy and the degree of substitution (DS) as determined by the back-titration method was within range of 0.004 - 0.366.

Key Words: Starch adipate nanoparticles, acid modified starch, esterfication ABSTRAK

Objektif kajian ini adalah mensintesis dan menentukan sifat jizio-kimia nanopartikel adipik kanji sagu. Nanopartikel adipik kanji disintesis dengan cara pengesleran kanji sagu tempatan dengan asid adipik. Sifat jizikal dan kimia nanopartikel adipik kanji telah dikaji. Berdasa~n pengimbasan pengesteran, kondisi suhu 100°C dan etanol sebagai media mendakan adalah pemangkin pemberian kumpulan karbonU antara sagu dengan asid adipik. Perubahan dalam nisbah molar asid adipik dan natrium hidroksida lelah digunakan untuk mensintesis adipik kanji. Penggantian asid adipik dalam rantai sagu dikenal pasti dengan menggunakan FTIR spektroskopi dan darjah penggantian menggunakan cara pentitratan batik adalah dalam tinkungan 0.004 - 0.366.

Kalil Kunci: Nanopartikel adipik kanji, kanji modifikasi asid, pengesteran

1.0 Introduction

Starch is a major food component of living orgamsm and it is a complex biodegradable carbohydrate made up of thousands of glucose units (Zhou et al., 2009).

Starch consists of linear and branched chains glucose molecules, named amylose and amylopectin. Starch is the main energy storage for plant growth and this has provided living organism the main source of energy. Plant such as maize, grain, sago, wheat, tapioca, rice and potato contain high amount of starch and they are being commercialize widely.

Starch derivatives such as acetylated starch are commonly used in food and beverage products as gelling agents, thickeners and encapsulating agents (Abas et al., 2010). Besides that, in papermaking industry, starch derivatives act as wet-end additives for dry strength and surface improvement aid. Different starch products can help in controlling loss of fluid in subterranean drilling, workover and completion fluids (Chiu &

Daniel, 2009). Neveen and Salaman, 2011 reported that due to environmental protection, some starch is assimilated into plastics component to enhance the process of environmental fragmentation and degradation. Recently, researchers implemented starch as precursor material in synthesizing starch-b'1l~d nanopartic1es in biomedical and pharmaceutical industry such as plastic fillers and drug delivery carriers (Kaur et al., 2007).

Starch is one of the most frequently used natural occurring polysaccharides in production of biopolymers products because of its properties, nontoxicity, biodegradability, biocompatibility, low cost, renewable and easily to get in this nature (Kim et al., 2012).

However, natural starch without any modification physically and chemically is ._IU<O.I,uentally incompatible for most applications. Therefore, modifying starch is useful

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Due to numerous industry demands for functionality of different starch products, starch is commonly modified by chemical, physical, genetic or enzymatic process.

(Richardson & Gorton, 2003). Singh and Chawla (2009) stated that chemical modification involves in substituting free hydroxyl group in the starch polymer with functional groups, yielding different types of starch derivatives. Physical modification implies pre­

gelatinization, and heat-treatment of starch (Miyazaki et at., 2006). Factors such as degree of substitution, solubility and types of substituent are the determinant of the properties of modified starch. (Kavitha & BeMiller, 1998; Ruan et at., 2009).

There is various chemical modification of starch being investigated and studied.

Esterification of starch is one of the common chemical modification (Singh et at., 2007).

Starch undergoes esterification by substitution or cross-linking because dicarboxylic acids contain two carboxylic groups and each of the carboxylic group can react with free hydroxyl group from different starch chains. Modification of potato starch with mixture of adipic acid and acetic anhydride is commonly applied reaction of starch cross-linking (Singh et a!., 2007; Mali & Grossman 2001). In addition, distarch glutarates (Kim et al., 2008), starch succinates (LawaI et al., 2008), maleates (Biswas et a!., 2006), and starch­

dicarboxylic acid complexes (John & Raja, 1999) have been prepared. Nonetheless, less research is carried out on

esterifica1f~n

of sago starch with adipic acid.

In this study, the objectives are to synthesis and characterize adipic acid modified

starch nanoparticles (SANPs). The starch adipate nanoparticles produced were more hydrophilic than native sago starch. Native sago starch has been esterified by using different molarity of adipic acid. The chemical composition, degree of substitution, solubilty, moisture and surface morphology of SANPs were investigated by using fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM).

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2.0 Literature Review

2.1 Sago Starch

,-~~"

_-HO~

HOC~ 0 o I

~v\. C~

--OHO~~

OH 0

HO OH o· "

(A) HOC~ 0

--O~

^{HOCH2 0}

HO OH O~v\. ^, HO~.'

OH 0 (B)

Figure l. Molecular structure of starch; (A) amylopectin and; (B) amylose

Sago starch or Metroxylan sagu is a source of carbohydrates that has been utilized for long time ago (Gwaiseuk, 2001). Sago starch is rich in carbohydrates whereby its content exceeds most other type of carbohydrates such as wheat, com, sweet potato and rice. Sago starch is composed of27% amylose and 73% amylopectin (Wiyono & Silitonga, 1989). Sago palm is found in tropical lowland forest or freshwater swamps. These areas include Thailand, Malaysia and Indonesia to Micronesia, Fiji and Samoa (lshizuka et al., 1996).

Sago starch is a fine and white powder. This characteristic makes sago starch as a major ingredient in cosmetic products such as perfumed body powders, cool body powders and mosquito repellent powders (Boonme et al., 2009). Besides that, it can also be modified to become drug delivery assistant, plastics, instant food and many more. Sago starch is biodegradable, cheap and easy to get locally. However, few studies have been reported in using sago starch in starch modification compared to potato, cassava and com

2.2 Starch Nanoparticles

Starch is usually used in production of biopolymers and biomedical industries because of its properties, nontoxicity, biocompatibility, biodegradability, renewable low cost, and easily to get in this nature (Kim et al., 2012). It is widely used in the cosmetics, food, plastics, textiles, paper, and pharmaceutical industries.

Distinctive synthetic methods for synthesis of starch nanoparticles have been discovered such as high-pressure homogenization, mini-emulsion cross-linking and nanoprecipitation. High-pressure homogenization is a simple technique which is useful for diluted and concentrated samples (Shi et al., 2011). However, a bulk of homogenization cycles is required and contamination of product may occur (Chingunpituk, 2007).

Nanoprecipitation method is more prioritized as it is very familiar and uncomplicated. Starch nanoparticles that have size range between 300nm and 400nm were synthesized by a simple nanoprecipitation method from native sago starch. Starch nanoparticles usually formed by simple nanoprecipitation through drop-wise addition of dissolved native starch solution to excess absolute ethanol. Different solvent used for precipitation of starch nanoparticles may alter the structure and chemical properties of the nanoparticles (Pang et al., 201". The shape and size of starch nanoparticles were restrained by changing the process parameters and using of specific surfactant (Chin et al., 2007). Starch nanoparticles with an average diameter of 150 nm were acquired in the presence of surfactants during precipitation. Both solvent and non-solvent systems used when synthesizing starch nanoparticles are mainly aqueous-based and the practice is easy to perform.

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2.3 Adipic Acid

Figure 2. Molecular structure of adipic acid

Adipic acid with IUPAC name hexanedioic acid and molecular formula, C6HlO04 is

a white crystalline solid which is soluble in water and organic solvent (Gaivoronskii &

Granzhan, 2005). It has no substituted groups and nonhygroscopic properties. The molecular weight of adipic acid is 146.14 glmol. In room temperature, adipic acid exists in solid state and has a slight odour.

Adipic acid has a wide variety of uses and over 4.4 billion pounds of it has been produced worldwide. It is the biggest amount of product mainly used in the production of polymers, especially as a monomer for the synthesis of nylon 6-6 (Derek et 01., 2016).

Adipic acid is also mainly used in food industry as additives (Abbas et 01., 2010). Besides that, adipic acid is a main ingredlPm used in the pharmaceutical, chemical and perfume industry (Danly & Campbell, 1978). It is used also in a wide range of applications, for example in coatings, plasticizers and detergents (Mahalakshmi et 01., 2016).

Adipic acid are eco-friendly chemical because based on its physico-chemical properties, adipic acid is a readily biodegradable, low potential for volatility, not persistent and not potentially bio accumulate (Mahalakshmi et 01., 2016). It is expected to partition Jftdominantly into the aquatic compartment and not to adsorb on soil or sediment particles

2.4 Modification of Starch

Modified starch can be classified as food additive which is prepared by treating starch granules which degrade the starch partially (Mahalakshmi et a/., 2016). Most modified starches are white and odourless powders. Modification of starch are carried out to improve its properties in certain application such as increasing in water holding capacity, reinforce its binding minimized synthesis of starch, heat resistant behaviour, and thickening improvement (Adzahan, 2001; Miyazaki et a/., 2006).

Consideration on market and production are needed in selection of modified starch for particular application. The market-related properties are product properties such as the organoleptic consideration, shelf durability and aesthetics (Sajilata & Singhal, 2004); while the production-related properties such as viscosity, pH and temperature. There are varieties of modified starch being used in food and beverage industry. Modified starches usually contain very low level of substituent group (Mahalakshmi et a/., 2016).

Enzymatic modification of starch is the formation of low molecular weight starch by hydrolization by amylolytic enzymes to become (Miyazaki et a/., 2006). These modified enzyme usually used in pharmaceutical and food industry. Chemical modification is the majority modification methof~owadays. Many approaches in chemical modification of starches have been improvised in food, pharmaceutical and textile industries. Physical modification includes heat-treatment and pre-gelatinization of starch. Heat-treatment modification involves annealing treatments and heat-moisture whereby this modification occurred without any damage to granular integrity and loss of birefringence (Miyazaki et a/., 2006; Mahalakshmi et a/., 2016).

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Physiochemical properties of modified starch are detennined by using different reaction conditions such as type of substituents, molar substitution and distribution of substituents (Ruan et al., 2009).

2.5 Esterification of Starch

Starch esterification reaction is one of the most popular method of starch modification. The properties of modified starch were affected by various factors, such as sources of starch, the degree of substitution, reaction temperature and types of solvent system (Chen et al., 2004). The most common starch esters that have being researched are starch maleate (Lehmann & Volkert, 2011). Starch esters are being used in applications such as substitutions for sealing adhesives, petroleum-based plastic materials, and biodegradable packing materials (Mano et al., 2003).

Various chemicals have been used to synthesis starch esters. The esterification of starch is nonnally carried out by reacting starch with fatty acid chlorides (Jorge et al.,

1999), acetic anhydrides (Volkert et al., 2010), succinic anhydrides (Yoshimura et al., 2006), sodium selenite (Staroszczyk et al., 2007) , or dicarboxylic acid (John & Raja, 1999) in various types solvents and under basic conditions. Nonetheless, less research is investigated by using adipic a~ Common parameters that will affect esterification process are presence of catalyst, reaction temperature and type of substituent used.

3.0 Materials and Methods

3.1 Materials

Native sago starch was purchased from a local grocery store (Kuching, Malaysia).

Sodium hydroxide (NaOH), adipic acid, ethanol, propanol and butanol were used. E. coli, Miller's Luria broth and Muller-Hinton Agar (MHA) were used for antibacterial studies.

Ultrapure water (18.2 MQ/cm, 25°C) was obtained from a Miiipore Mili Q-system and used in all synthesis. All other chemicals were of reagent grade and were used without further purification.

3.2 Synthesis of Starch Adipate Nanoparticles (SANPs)

SANPs were synthesized modified from the methods reported by (Pang et a/., 2012). 2 g (0.0125 mol of AGU) of native sago starch was dissolved in 40 ml of ultrapure water. An amount of NaOH is added to the solution. The solution is heated to 60°C with stirring until gelatinization occurred. Next, adipic acid was added to the mixture. The resulting mixture was heated at (60°C, 80°C or 100 °C) for 4 hours with stirring. The mixture was then added drop wise into (ethanol, propanol or butanol) for precipitation.

Subsequently, precipitate formed was vacuum filtered, rinsed with excess ethanol and dried at 60°C overnight. Approximate 0.15 g of dried precipitate was further purified by dissolved in 50 ml of ultrapure water and re-precipitated in excess absolute ethanol.

Collected SANPs was dried at 60°C overnight.

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Sago Starch

• Ultrapure water, NaOH ( -60 °C)

Gelatinization

• Adipic acid (100 °C) for 4 hours Esterification

• Ethanol ( drop wise) Precipitation

•

Rinsed with absolute ethanol Filtration

• 60 °C overnight Impure SANPs

•

Ultrapure water Repurification

(Precipitation and filtration)

• Oven dry (60 °C) for 24 hours SANPs

o

+ _HO~OH

(.)

o

"

o~OH o

NaOH

)

o

Figure 4. Esterification reaction of native sago starch with adipic acid

o

o

"

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3.3 Characterization

3.3.1 Fourier Transform Infrared Spectroscopy (FTIR)

FTIR spectroscopy was used to study the esterification of starch with adipic acid.

All samples were dried at 55°C for 24 h. Then, the sample were grounded with potassium bromide (KBr) in the ratio of 1: 100 and then pressed under high pressure to obtain 1-2mm thick pellet. Spectra of samples from KBr/sample pellets within the wavenumber range of 400 - 4000 cm-^I were obtained using a FTIR spectrometer (SHIMADZU Model FTIR­

8201 PC)

3.3.2 Scanning Electron Microscopy (SEM)

SEM was used to study the particle sizes of SANPs and surface morphology of SANPs. Dried sample was mounted onto double-baked adhesive carbon tab stub to circular aluminum stub. Then, the stub was placed in the SEM chamber and sputter-coated with gold and observed under SEM (lEOL Model JSM 6390LA). The size of samples was being measured by using image of the sample in Smite View software.

3.3.3 Degree of Substitution (DS)

OS of starch adipate was determined using titration method referring to the method from Ogawa el al., (1998) with modifications. 0.05 g of dried SANPs was dissolved in 10 ml of 0.1 M NaOH and stirred continuously for 30 minutes. A few drops of phenolphthalein indicator were added into the mixture. Then, the mixture was titrated with 0.1 M hydrochloric acid (HCl) solution until pink colour of the mixture decolorized. DS was calculated using the formula (equation 1) below.

M ^{NaOH X} V^{HC) X} Mw Percentage of Substitution, Ad % = W ST

162 x Ad%

DS 12914.12 - (128.14

x

Ad%) - - - (equation 1)

Wbere,

M NaOH = Molarity of NaOH V HCl

=

Volume (ml) ofHCI used W ST = Weight of SANPs

Mw

=

Molecular Weight of Adipic Acid Substituents [COOH(CH2)3CO-]

= 129.14

3.3.4 Solubility

Firstly, 0.5 g of sample was weighed, transferred and mixed with 0.5 g of ultrapure water. The mixture was sonicated for 30 minutes. Subsequently, the mixture was centrifuged at 4000 rpm for 5 minutes and the supernatant was removed. The precipitate was dried at 60°C for 24 hours. Dried sample was cooled in desiccator and weighed.

Solubility test was repeated for

an~her

^{2 times.}

Solubility in 1 g of solution =

Initial Weight of Sample - Weight of Dried Sample 1

x~---

Initial Weight of Sample O.Sg of solution

- - -(equation 2)

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