STUDY OF FABRICATION AND ANALYSIS OF NANOCELLULOSE

HYDROGEL FROM OIL PALM EMPTY FRUIT BUNCH

SITI HAMIZAH BINTI ASPIN

MN2011006T

FAKULTI SAINS MAKANAN DAN PEMAKANAN

UNIVERSITI MALAYSIA SABAH

2020
 CHAPTER 1

INTRODUCTION

1.1RESEARCH BACKGROUND
Nanocellulose hydrogel has flexible uses in diverse fields such as livestock, fruit, oil, textiles,
biomedical, and bio-composites. It has its benefits as opposed to hydrogels made from non-
degradable and harmful products in agricultural fields. Besides, nanocellulose hydrogels are
helpful to soil pH as they will not substantially alter soil pH (Azeem, KuShaari, Guy, Basit &
Thanh, 2014). While hydrogel in different fields has already been studied, minimal studies have
been recorded on hydrogels produced from OPEFB nanocellulose. However, future OPEFB
nanocellulose hydrogels' future applications may be expected based on studies published on
cellulose-based hydrogels. In conclusion, the usage of OPEFB nanocellulose hydrogels in the
food industry will create a different phenomenon and render our food industry more
competitive with other developing countries.

Malaysia is one of the leading producers of agricultural commodities in the South East
Asian zone. Agricultural waste is thus plentiful and readily accessible (Ozturk et al., 2017). The
key agro-based waste in the country is oil palm biomass in fronds, trunks, and fibers, paddy
straw, rice husk, banana residues, and sugar cane bagasse, coconut husk, and pineapple waste.
Oil palm biomass is the primary source of biomass in Malaysia. Apart from its abundance,
OPEFB has a high degree of cellulose, rendering it a very promising feedstock to extract
nanocellulose and manufacture various cellulose-based goods. Regenerated cellulose hydrogel,
in particular, was formed by dissolving OPEFB cellulose and sodium carboxymethylcellulose
(NaCMC) in the sodium hydroxide/urea method (Salleh et al., 2019).

1.2 Problem statement

1.2.1 Abundance of biomass product
Oil palm biomass is the primary biomass resource in Malaysia. Malaysia is one of the leading
agricultural commodity producers in the Southeast Asian region. Therefore, agricultural wastes

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are abundant and readily available (Ozturk, Saba, Altay, Iqbal, Hakeem, Jawaid & Ibrahim,
2017). OPEFB has high potential lignocellulosic material suitable for the production of
bioalcohol, solid fuel, pulp, and much other value-added product. However, the usage of the
hydrogel in various fields has already been explored, minimal studies on hydrogels made OPEFB
nanocellulose has been reported (Farah, Lee, Zuriyati, Lee, and Luqman, 2019).

1.2.2 Humidity control

Recently, bio-based hydrogels have been applied to food packaging. However, the use of
superabsorbent polymers as bio-based hydrogels in the food industry remains limited. There are
increasing interest to develop bio-based hydrogels and innovative technologies that reduce the
use of fossil fuel related to conventional non-biodegradable petroleum-based materials (Santana
et al., 2017). Besides, hydrogels are polymeric matrixes with the ability to absorb water or any
water compatible fluids up to 100% of their weight. Moreover, superabsorbent hydrogels are a
kind of hydrogel which are constituted by polymers that can absorb over 100% and up to
thousands of times their dry weight in water (Feng, Ma, Wu, Wang, & Lei, 2014).

The main role of hydrogel in food packaging is humidity control inside of a packaging.
Their potential application in the food preservation field include to remove the moisture or
exudates generated inside a package because of vegetables transpiration and water loss caused
by physicochemical changes in packaged foods or by the permeation of water vapor due to
environmental conditions, especially in the case of fresh foods (Ahmed, 2015). Furthermore, the
use of hydrogel can decrease water activity, slowing down the growth of mold, yeast, and
spoilage bacteria on foods such as ready-to-eat meals (e.g., freshly cut vegetables) and
hygroscopic products (e.g., powder food) or to decrease the softening of dry crispy products
(e.g., fried potatoes, biscuits). To increase hydrogel applications, diverse materials and
methodologies have been studied.

Most studies focused on improving specific features of the polymers used to prepare
hydrogels, as well as decreasing their environmental impact by finding biodegradable
alternatives (Thombare, Jha, Mishra, & Siddiqui, 2016). The use of hydrogel comprises an
interesting strategy for food packaging systems, especially for the control of water content,

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which greatly limits food durability. In this regard, the development of hydrogels based on
superabsorbent polymers allows the creation of alternative packaging systems.

Although studies on food shelf-life extension using hydrogels from natural polymers are
limited, this approach is promising, since the features of these materials may add considerable
value to packaged products due to their biocompatibility and low toxicity (Caló & Khutoryanskiy,
2015). Because of that, the integration of hydrogels into packaging systems constitutes an
evolving and eco-friendly alternative technology.

1.3 Significant of Study

This study highlight the usage of low cost and plentiful food waste with effective nanocellulose

hydrogel which waste from oil palm empty fruit bunch can be alternative sources for

development of nanocellulose hydrogel. This research will able to reduce air pollution and

greenhouse effect.

1.4 Research Objectives

The main objectives of this research are to develop nanocellulose hydrogel from oil palm empty
fruit bunch:
1. To characterize the physiochemical of oil palm empty fruit bunch (OPEFB) CNFs
hydrogel.
2. To optimize condition of CNFs hydrogel from oil palm empty fruit bunch (OPEFB).
3. To analyze the humidity of spinach (Spinacia oleracea) using CNFs hydrogel.

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 CHAPTER 2

LITERATURE REVIEW

2.1 Oil palm fruit empty bunch (OPFEB)

Malaysia has endowed with a considerable amount of biomass resources from Gan et al. (2015)
crops such as sugarcane, cassava, and corn; Tayeb et al. (2018) agricultural residues such as
rice straw, cassava, rhizome, and corncobs; Anuar et al. (2019) woody biomass such as fast-
growing trees, wood waste from wood mill, and sawdust; (Chiew & Shimada, 2013) agro-
industrial wastes such as rice husks from rice mills, molasses, and bagasse from sugar refineries
and residues from palm oil mills; Kong et al. (2014) municipal solid waste; and Chiew at el.
( 2011) animal manure and poultry litter (Malaysia, 2013). Malaysia is one of the leading
agricultural commodity producers in the Southeast Asian region. Therefore, agricultural wastes
are abundant and readily available Ozturk et al. (2017).
The primary agro-based wastes in the country are oil palm biomass (fronds, trunks, and
fibers), paddy straw, rice husk, banana residues, sugarcane bagasse, coconut husk, and
pineapple waste Goh et al. (2010). The primary agricultural wastes from the oil palm biomass
accounted for 46,000 kilotons in fronds and 11,000 kilotons in the form of trunks. In 2007,
approximately 880 kilotons of paddy straw and 484 kilotons of rice husk were produced from
the replanting of paddy Goh et al. (2010). Banana residues, sugarcane bagasse, and coconut
husks accounted for 530, 234, and 171 kilotons. In pineapple farms, 48 kilotons of pineapple
wastes are generated after the fruits are extracted, Goh et al. (2010).

2.2 Nanocellulose Hydrogel

Nanocellulose is considered a sustainable nanomaterial due to its availability, biodegradability,
and biocompatibility. It exists in several forms, including nanofibrillated cellulose (NFC),
nanocrystalline cellulose (NCC) or cellulose nanocrystals (CNCs), and bacterial cellulose (BC).
Nanocellulose is usually used in the food, biological, cosmetics agricultural sector, paper, and
film industry. The hydrogel is the one of application nanocellulose: highly hydrated porous

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cellulosic soft materials with good mechanical properties. These cellulose-based gels can be
produced from bacterial or plant cellulose nanofibrils, which are hydrophilic, renewable,
biodegradable, and biocompatible.
The hydrogel can absorb a large amount of water, physiological, or saline solution due
to its hydrophobicity and permeability, good compatibility, biodegradability, and suitable applied
in many areas such as food and food packaging (Luo and Zhang, 2013). Hydrogels are
designed to react with changes in the surrounding environment, such as pH, temperature, and
specific mixture (Im, Choi, Subramanyam, Huh & Park, (2017). The hydrogel worked as a
carrier for pH-responsive dyes (bromothymol blue/methyl red), in response to external CO2
stimuli that changed color according to the food’s freshness. Freshness indicators detect food
spoilage by analyzing metabolites of micro-organisms in food packaging (Wu et al., 2020).

2.3 Hydrogel
Hydrogel has ability to absorb a large amount of water, physiological, or saline solution
due to its hydrophilicity and permeability, good compatibility, biodegradability and suitable
applied in many areas such as food and food packaging (Luo and Zhang, 2013). Hydrogels are
also designed to react with the changes in the surrounding environment such as pH,
temperature and certain mixture (Im, Choi, Subramanyam, Huh & Park, (2017).
In addition, hydrogel worked as a carrier for pH-responsive dyes (bromothymol
blue/methyl red), in response to external CO2 stimuli which changed colour according to the
freshness of the food. Freshness indicators detect food spoilage by analysing metabolites of
micro-organisms food packaging (Wu et al., 2020).

2.4 Application of nanocellulose to the food sector

Nanocelluloses are nano-sized materials derived from cellulosic sources utilising chemical ,
physical or biological methods (Zhuo, Liu, Pan, Dong, & Li, 2017). Most articles reporting on the
production of nanocellulose-based packaging composites present findings on their barrier and
mechanical properties, with certain testing of their antimicrobial properties, as well as the
quality of nanocellulose-based food items. This paper explores their possible uses in the food
industry.

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2.4.1 Green food packaging
Few researches have measured the enhancement of the consistency and/or shelf life of food
packed with nanocellulose-based composites. Azeredo et al . ( 2012) have improved edible films
(based on alginate-acerola purée) with cellulose whiskers or MMTs. The application of these
coatings to fresh acerolas minimised the weight loss of fruit, the frequency of rot, the rate of
ripening and the preservation of ascorbic acid. However, nanocomposite coating was the most
effective in preserving red colour and visual acceptance, as well as in minimising weight loss.
In their study, Dehnad et al. (2014) added a biocomposite of chitosan-nanocellullose to prolong
the shelf life of ground meat, finding a decrease in the population of lactic acid bacteria relative
to nylon packed samples at 3 and 25 C after 6 days of storage. Furthermore, the coating of
blueberries with aqueous slurry of cellulose nanofibrils and calcium carbonate nanoparticles has
shown that the leakage of anthocyanin pigments from blueberries during thermal treatment has
been practically removed (Zhao et al., 2014).

In addition, Dong et al. (2015) was able to increase the shelf life and consistency of
strawberries by adding a mixture of 1% chitosan and 5% nanocellulose. In reality, the weight
loss and decay rate of the coated strawberries decreased due to a decline in senescence and
metabolism. The coating also enabled the maintenance of total phenols and total anthocyanins
and improved visual appearance. Bionanomaterials are a promising alternative for use in food
packaging applications, replacing traditional plastics, particularly those manufactured from oil.
However, the low mechanical and water vapour barrier properties of these products restrict
their widespread usage in the food packaging industry. Techniques intended to enhance the
properties of these biodegradable products include the manipulation of biopolymers and their
usage in nanocomposites.

Bio-based product packaging

Food packaging must have adequate cover to maintain the quality and safety of food. The use
of packaging products is also of vital significance on the food industry. In reaction to the
increasing demands of our community, work on nanotechnology applications in packaging has
drawn considerable interest in recent years (Huang, Li & Zhou, 2015). Cellulose fabrics such as
paper or paperboard are increasingly used as food packing materials. However, as their plastic

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polymer equivalent, bio-based packaging must have the same roles as defence, consistency
preservation and food safety (Lavoine, Desloges, Manship & Bras, 2015).
A great deal of focus has lately been given to the usage of durable biopolymer coatings on
paperboards for food packaging. This approach enables the consistency and freshness of the
commodity to be preserved over time, as needed for marketing before use (Stewart, Tompkin &
Cole, 2002).
Ideally, this coating may also be used as a buffer against organic solvents (increase in
hydrophobicity) or to reduce the rate of transmission of water vapour and oxygen permeability
of paper and paperboard (Koivula et al . , 2016). The use of biopolymers as dispersion coatings
is also growing, in particular the use of cellulosic fibres (nanocellulose or microfibres) that are
biodegradable and recyclable (Lavoine, Desloges, Bras, 2014).
In addition, cellulose nanofibres have shown strong tolerance to oxygen in addition to their
outstanding mechanical properties leading to the formation of a close grid (Lavoine, Desloges,
Bras, 2012). However, the strong water affinity of cellulose is a big annoyance for certain
wrapping. Blends of biopolymers or synthetic polymers may solve the issue. Polypyrrole (PPy)
associated with cellulosic content has shown an improvement in hydrophobic character and a
decrease in the water vapour transmission rate (WVTR) (Bideau, Bras, Adoui, Daneault &
Loranger,2017).

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 CHAPTER 3

METHODOLOGY

3.1 Material
Oil palm empty fruit bunch will be used as raw material to make nanocellulose. 2,2,6,6-
tetramethyl piperidine-1- oxyl (TEMPO), sodium bromide (NaBr), sodium hypochlorite (NaClO),
bromothymol blue (BB), methyl red (MR), phosphotungstic acid, sodium hydroxide,
hydrochloride (HCl), zinc chloride (ZnCl2), and sodium hydrogen carbonate (NaHCO3) will be of
chemical grade from Aladdin (Aladdin®, Shanghai, China) without any purification. Miller LB
agar and phosphate-buffered saline (PBS) will be purchased from Sigma Aldrich.

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 3.2 Experimental Design
Figure 1 below shows that the experimental design for the research to achieve the
propose objectives

1. Extraction of
Nanocellulose from
oil palm fruit empty
bunch

2. Fabrication of
nanocellulose CNFs 5. Statistical analysis
Hydrogel.

3. Physicochemical 4. Analyze the

characterization humidity of spinach

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 3.3 Extraction of nanocellulose hydrogel
The extraction of nanocelullose from oil palm empty fruit bunch (OPEFB) will be prepared based
on the method from Lu et al. (2020). OPEFB (2.0% w/w) will be a grind, going through 11
passes to get an OPEFB water suspension with a mass content of 2.48% (w/w). TEMPO-
oxidised OPEFB will be prepared according to a reported method (Isogai, Saito, & Fukuzumi,
2011). Typically, the mixture of OPEFB water suspension (1.0% w/w, 100g), TEMPO (0.016 g),
and NaBr (0.103 g) will be prepared first, and then NaClO (8.0 mmol/g cellulose) will be added
slowly with constant stirring. The reaction will be performed at 25 °C, with the mixture
maintained at a pH of 10. The reaction will be quenched by ethanol (10 mL) once the mixture's
pH stopped decreasing, and then the final product will be washed thoroughly with distilled
water.

3.4 Crosslinking of nanocellulose hydrogel

CNFs hydrogels will be produced by adding a zinc chloride solution to the top of TEMPO-
oxidized CNFs aqueous dispersions without stirring. The TEMPO-oxidized CNFs dispersion will be
first homogenized by a high shear mixer at 3000 rpm for 5 min, followed by degassing and
transferring to a cylinder-shaped Teflon mold. A designed amount of the ZnCl2 solution (20 wt.
%) will be added dropwise along the container's wall into the CNF dispersion without stirring.
After standing for overnight, the liquid TEMPO-oxidized CNFs dispersion will be transferred to a
solid CNFs hydrogel. The resulting hydrogel will be rinsed with water four times to remove
unbounded metal ions and stored at 4 ◦C for analysis after that.

3.5 Characterization of hydrogel

3.4.1 Scanning Electron Microscopy (SEM), Transmission electron microscopy
(TEM)
The as-prepared hydrogels' morphology will be assessed by scanning electron microscopy
(SEM) on PhenomPro (Phenom-World, Eindhoven, The Netherlands) at 10 kV. The specimens
will be first rapidly frozen using liquid nitrogen and then freeze-dried for 24 h before SEM
observation.

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 3.4.2 X-ray diffraction spectra (XRD) and Fourier transform infrared (FT-IR)
spectroscopy.
The X-ray diffraction (XRD) analysis of nanocellulose hydrogel will be performed using a smart
lab x-ray diffractometer with Cu Kα (0.154 nm) radiation generated at 40 kV and 40 mA. The
scan parameters will be set at 2 s per step with a 0.05° step resolution in the range of 2θ.
Subtraction of background intensity from the measured intensity of nanocellulose samples will
be performed. The Fourier-transform infrared spectroscopy of nanocellulose hydrogel will be
measured using an FTIR spectrometer to identify the functional group. The spectra will be
recorded from 3600 to 500 cm−1 at 4 cm−1 resolution and 64 scans per spectrum in the
transmission mode.

3.4.3 Thermogravimetric Analysis (TGA)

The thermal degradation behavior of nanocellulose hydrogel will be determined by the
Thermogravimetric analyzer based on the method from Castilhos et al. (2017), the dry
hydrogel, and its components, i.e., pure alginate and pure zein, will be analyzed concerning
theirs. A simultaneous thermal analyzer, fabricated by Netzsch, model STA 449 F3 Jupiter, will
employ N2 as the purge gas, at a flow rate of 50 ml min−1. The experiments will be conducted
from 25 to 900 °C at a heating rate of 10 K min−1. Approximately 75 mg of the sample will be
put into an alumina crucible. An empty crucible will be used as a reference.

3.4.4 Swelling index

The swelling behavior of CNFs hydrogels will be evaluated by determination of the swelling ratio
temperature for 24 h. The samples will be taken out from the water at selected time intervals
and dried gently with blotting paper to remove excess surface water and weighed (mt). The
swelling ratio will be calculated by water uptake of the hydrogel (mt − m0) divided by the dry
weight (m0). Each value will be averaged from three parallel measurements. Freeze-dried CNFs
hydrogels will be weighted (m0) and then immersed in deionized water at room. The following
equation will calculate the swelling power:
Swelling (%)= Wt-Wo/Wo x 100% (1)

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3.4.5 Water absorption capacity
The water absorption capacity of the CNFs/ZnO was measured by drying the in an oven at 50°C
for 24 hours. After 24 hours, the CNFs/ZnO will be immediately weighted. The conditioned
CNFs/ZnO will be fully immersed in distilled water for 5 minutes. Subsequently, the remaining
water on the surface of CNFs/ZnO will be removed by using filter paper and immediately
weighted. All measurements will be performed for five replicates and averaged. The water
absorption capacity was calculated using the following:

W A −W I
Water absorption capacity : X 100 %
WI

Where w Ais the weight of film after being immersed in distilled water and W I is the initial dry
weight of the film.

3.4.6 Compression and thickness

The hydrogels will be subjected to compression tests using a universal testing machine. All
hydrogels will be cylinder shape with a diameter of 12 mm and a height of 20 mm. The
compression tests will be carried out at room temperature at a velocity of 1 mm/min. All
engineering stress will be determined to a compressive strain level of 90% and presented as an
average of three individual trials. The thickness of hydrogel will be measure by micrometer
and taking 5 spots differently to calculate the average.

3.5 Optimization condition of CNFs

The optimization was based on the gel content and degree of swelling of the hydrogel. In this
research, four parameters were studied in order to optimize the formation of CNFs hydrogel.
The parameters will be; pH, concentration, reaction time and reaction temperature. The pH will
be 1, 5, 10 and the temperature 4ºC, 25ºC and 37ºC. For the concentration will be 1%-5%
and the reaction time will be recorded when the CNFs can control the humidity of spinach.

3.6 HUMIDTY CONTROL OF SPINACH

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To monitor the humidity level of spinach, the sample will be placed inside the beaker that
contains CNFs hydrogel. The beaker will be sealed with an adhesive tape. The whole
experiment will be conducted at room temperature for 7 days.
3.7 Statistical Analysis
The results are expressed as the means ± standard deviation (SD) of three duplicate
measurements to ensure the data's accuracy. The experimental data will be submitted to the
analysis of variance (ANOVA). Significant differences between the average amount will be
established at p≤0.05.

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 Expected result

Fig. 2: The mechanism of CNFs hydrogel formation

From the figure 2 show that the formation of CNFs hydrogel mechanism. When the CNFs react
with zink ion, the zink will cause of the cross link between cellulose component. Hence,
nanocellulose hydrogel from oil palm empty fruit bunch can be produce.

CONCLUSION
To conclude, the CNFs hydrogel will be prepared from an empty fruit bunch and can absorb the

water during the dehydration of spinach. The porous morphological structure of CNFs hydrogel

will be expected to provide the pathway to diffuse this moisture from spinach easily. Thus, the

CNFs hydrogel will be an ideal indicator for humidity control of vegetables.

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 MILESTONES
Flow chart of research activities october 2020 to July 2021 (1 years).
Milestones Timeline
Research proposal and slide preparation October 2020

Preparation and extraction of nanocellulose

November-January 2021
hydrogel from oil palm empty fruit bunch.

Fabrication of nanocellulose hydrogel throught

January 2021
crosslinking of Zn2+
Characterization of nanocellulose hydrogel:
 Morphology
 Physical January 2021-June 2021
 Chemical
 Mechanical
 Optimization condition of CNFs
hydrogel. July 2021- November 2021
 Analyze the humidity of spinach
Statiscal analysis January 2021- April 2021
Complete of the final project, poster,
June 2021
presentation and publications.

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