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CHAPTER 1
INTRODUCTION
1.1
Background of study
Pumpkin is a vegetable crop from genus Cucurbita belonging to the Cucurbitaceae
family. This family is one of the largest families in plant kingdom comprising of
highest number of edible plant species (Dar & Sofi, 2017). Squash and cucumber
also include in this family which usually grown throughout subtropical and tropical
countries. According to Lee, Chung, & Ezura (2003), Curcurbita pepo, Curcurbita
maxima and C. moschata are three common type of pumpkin worldwide. Pumpkin
mostly found orange in colour when ripe and can be found in various shape and size.
Doymaz (2007) stated that pumpkin usually used as vegetable or as an ingredient in
soups, pies, bread and many other dishes. In addition, since pumpkin is a seasonal
crop and very sensitive to microbial spoilage, the fresh pumpkin must be undergo
processing method to preserve them for a longer shelf life (Guiné, Pinho, & João,
2010).
There are various method to preserve pumpkin. One of the method to
preserve pumpkin are by using drying method. By using drying method, shelf life of
the food product can be extended with lower requirements for packaging and
reducing the weights of shipping. Tonon et al. (2009) stated that spray drying method
is one of the method that is suitable for heat sensitive food product and results in
powder with low water activity, good qualities and facilitate the storage and
transportation.
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The main index to evaluate the drying performance is through the product yield.
However, during drying process, several problems such as stickiness and high
viscosity nature of fruit material can cause low yield product and tend to cause
clogging in the chamber wall of spray drying (Adhikari et al., 2003; Moreira et al.,
2009; Obon et al., 2009; Tan et al., 2011). By using liquefaction of enzyme and
addition of drying agent, these problems can be solved (Chong & Wong, 2015).
According to Mutlu et al. (1999) the separation process can be ease by
degradation of fruit structure into smaller particle through liquefaction process. In
addition, enzyme liquefaction is important as it can help to decrease the viscosity of
the hydrolysates by breaking down the soluble pectin and cell wall of fruits and
vegetables material (Sreenath et al., 1995; Mutlu et al., 1999; Chopda & Barrett,
2001; Rai et al., 2004; Grabowski et al., 2006). This will ease the spray drying
process and prevent from deposition in the wall of spray drying.
Next, one of the important parameter that can affect the enzyme reaction is
incubation time. Increasing the incubation time will increase the enzyme activity.
Similar result showed by Wong & Tan (2017) in which the activity of enzyme
increase with increasing incubation time until the optimum condition was reached
(1.5 hour).
The presence low glass transition temperature 𝑇𝑔 by the low molecular weight
of sugar such as glucose, fructose and sucrose can cause clogging in the chamber
wall of spray dryer (Moreira et al. 2009; Obon et al. 2009; Tan et al. 2011). In order
to prevent from stickiness to occur, drying agent can be used to increase the glass
transition temperature, 𝑇𝑔 of fruit material. In addition, maltodextrin is the most used
drying agent in the food industry as it has neutral taste, colour and low cost
(Shavakhi et al., 2011). It is also mainly used to increase product stability, reduce
stickiness and used in material difficult to dry. Next, one of the important parameter
that must be optimized in spray drying process in order to produce quality powder is
inlet temperature (Liu et al., 2004). Inlet temperature of spray drying can
significantly affect the product yield and the moisture content while no significant
differences in water activity, solubility and hygroscopicity (Wong & Tan, 2017).
Next, the powder form of food material from the spray drying process can be
used in many application. One of the application in this study is the used of pumpkin
powder in the food production that is free from lactose and as a replacement for
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wheat flour. As the lactose intolerance widespread throughout the world, many of the
people avoid milk and dairy product to improve the symptoms (Lomer, Parkes, &
Sanderson, 2008) while gluten that present in the wheat flour can cause several
problems to the people that had celiac diseases. Thus, the further application of this
study can be used to produce food product based on powder ingredient that are free
from lactose and gluten.
1.2
Problem statement
Pumpkin, from genus Curcubita from family Curcubitaceae contained many
nutrient that are beneficial for health such as pectin, vitamins, carotene, mineral salts
and other substances. Various product can be produce from the pumpkin due to its
high nutritional and protective value such as instant noodles, spices, bakery product
and colouring agent in food. Since pumpkin is a fresh seasonal crop, it is very
sensitive to microbial spoilage and have shorter shelf life which cause problem to the
storage. Moreover, according to APO (2006), many tropical fruit like pumpkin tend to
deteriorate and cause wastage during peak harvesting season due to insufficient
storage space and processing facilities (Wong, Pui, & Ng, 2015). Thus, pumpkin
need to be preserved through drying or refrigerated condition to increase its shelf life
and to and maintain its nutritional qualities.
In preservation of pumpkin by using spray drying method, there are several
problems that can affect the product yield in the drying process. The major
degradation of the pumpkin powder produced by spray drying is mainly due to the
stickiness and high viscosity in the nature of fruit material. According to Tonon et al.
(2008) this will lead to the operational problems and low product yield. Thus, the
optimum condition of the enzyme concentration, inlet temperature and incubation
time need to be considered to produce high qualities of powder product. Moreover,
there is also lack of published results on liquefaction of combined enzyme treatment
on spray dried pumpkin powder based on enzyme ratio technique. Thus, this study
aimed to evaluate the effect of enzyme in combination by using enzyme ratio
technique on the viscosity reduction of pumpkin powder.
According to the Makharia et al. (2014), celiac diseases that related with the
ingestion of gluten are rarely occur in Malaysia. Most people in Malaysia tend to
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suffer for non-celiac gluten sensitivity which have similar symptoms as celiac
diseases when they consume food with gluten. Most of the bakery product that are
sold in Malaysia used wheat as one of the ingredient in the product which can cause
problems to people with gluten sensitivity. In addition, the lactose intolerance cases
also quite high in Malaysia. Thus, further research application of pumpkin as
alternatif of wheat ingredient in bakery formulation and as food that are free from
lactose may help to overcome this problems.
1.3
Significance of study
Pumpkin can be found in abundant supply in Malaysia as it is grown all over
Malaysia. The availability of pumpkin has an advantages to be used in this study and
has a potential to be commmercialized in powder form as a food colouring or as a
thickener. Furthermore, since the demand for healthy food is quite high, the presence
of high nutrition in the pumpkin has more significance value in the market.
According to De Escalada Pla et al., (2005) and Yadav et al. (2010) Pumpkin
contained high amount protein (4.0g/100g) and calcium (475 mg/100g) and it is
suitable to be used in the production of healthy food especially in the replacement of
the wheat flour to substitute gluten in the food such as production of pumpkin biscuit
and food for lactose intolerance people.
Since pumpkin contained perishable characteristics and high nutritional
value, spray drying method, one of the well-known method is used to increase shelf
life and for longer storage without degradation to the qualities of pumpkin. In
addition, this study also optimized the condition of enzyme concentration and
incubation time. The enzyme used in this study is Pectinex Ultra SP-L and Celluclast
1.5 L. These enzymes function to degrade the pectin and cellulose in the pumpkin
and useful to ease the process of spray drying and thus can produce high yield of
pumpkin. In this study, enzyme ratio will be used to study the effect of both enzyme
in viscosity reduction of pumpkin puree. Since there is lack of study on the use of
enzyme ratio technique, this study will be helpful to contribute in the further research
about the usage of enzyme liquefaction in the fruit juice. This also can help to save
the cost of enzyme used in the research. This study can be used as a reference for the
further research about optimization of drying agent used for spray drying.
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1.4
Research objective
The main objective of this study is to produce good quality of pumpkin powder while
the specific objective of this study is to :
i.
To determine the effect of the optimize enzyme ratio and incubation time on
viscosity reduction on puree pumpkin
ii.
To evaluate the effect of the inlet temperature on the physicochemical
properties of pumpkin powder.
1.5
Scope of study
In order to achieve the objective of this research, several scope of the study has been
determined. The scope of the study are:
i.
The plant will be use in this study is pumpkin (Curcubita moschata).
ii.
The enzyme concentration will be use is 0.5% v/w for both Pectinex UltraSP-L and Celluclast 1.5 L.
iii.
The enzyme will be treat with different ratio (2.5:7.5, 7.5:2.5, 5.0:5.0, 10.0:0,
0:10.0).
iv.
The incubation time will be use is 30 - 150 min.
v.
The range of inlet temperature in the spray dryer will be use is 150°C - 190
°C.
vi.
The maltodextrin will be 23% w/v.
vii.
The application of a response surface methodology (RSM) will be use as an
experimental design in order to optimize enzyme ratio and incubation time in
spray drying.
viii.
The analytical methods will be use to optimize the optimum condition of
enzyme treatment and incubation time on viscosity of pumpkin puree and
effect of inlet air temperature based on physicochemical properties of
pumpkin powder such as stickiness, moisture content, water activity,
hygroscopicity, solubility, bulk density, process yield.
CHAPTER 2
LITERATURE REVIEW
2.1
Pumpkin
Pumpkin is a vegetable crop belong to the genus Curcubita of the Curcubitaceae
family and widely growth in tropical and subtropical countries. Pumpkin also can
adapt well in hot and humid tropical climate and grown all year round. According to
the Lee, Chung, & Ezura (2003), Cucurbita moschata, Cucurbita maxima, and
Cucurbita pepo are three type of pumpkin that can be found worldwide. In Malaysia,
there are two types of pumpkin present which is Cucurbita moschata and Curcubita
moschata Duchesne. Cucurbita moschata (labu manis) are more commonly grown
throughout Malaysia while Curcubita moschata Duchesne (labu loceng) come solely
from Kedah. According to Norshazila, et al. (2014), the differences between this two
is that Cucurbita moschata (labu manis) have spherical shape while Curcubita
moschata Duchesne (labu loceng) have a bell shape (refer to Figure 2.1).
There are variety of shape, size and colour of pumpkin. For the colour of
pumpkin, it can vary from orange to yellow. Toan et al. (2018) reported that
pumpkin also contain seed and golden-yellow to orange colour pulp in their thick
shell. For the seed of pumpkin, it is non-endospermic, large and usually dark red in
colour. In addition, for the oil in the seed of pumpkin, it contain highly unsaturated
oil with oleic and linoleic acids predominantly present in the oil of the seed.
Furthermore, the low amount of linolenic acid and other unsaturated oil present in
the seed of pumpkin resulting in high oxidative stability and low free radical in
which beneficial to the industrial and human diet (Dar & Sofi, 2017).
There are several active compound present in the pumpkin for example fixed
oils, para-aminobenzoic acid, proteins, polysaccharides, peptides and sterol. Pumpkin
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also rich in protein, polysaccharide, antioxidants, essential amino acids, carotenoids
and minerals (Dar & Sofi, 2017). Furthermore, pumpkin also provide health benefit
to human such as it can help to reduce cholesterol, protect skin, reduce cancer risk,
sharper eyesight, boost immune system and aid weight loss (Klein, 2014).
Pumpkin usually consumed as freshly boiled and steamed or as processed
foods such as soups while in the South-East Asia, the cooked mature fruit of
pumpkin is used for making sweets and dessert by dusted the steam fruit flesh with
cassava flour and fried into chips or steaming the fruit flesh with grated coconut and
sugar (Bhaskarachary et al., 2008). Since pumpkin has perishable characteristics and
cannot be stored for too long without deterioration, it require preservation method
such as drying method to retain its quality and prevent it from spoilage.
One of the drying method to preserve the quality of fresh pumpkin is through
the spray drying process. In the spray drying process, it involved the production of
powder extract. The powder extract of pumpkin can be used as a substitution of fresh
pumpkin and as a pumpkin flour and can be applied in bakery product, instant
noodles, soups, spices and as natural colouring agent in pasta. Furthermore, since
there is high nutrition present in the pumpkin flour, it can be used to substitute the
function of wheat flour or as wheat–pumpkin composite flour blend (Pratyush, Masih
& Sonkar, 2015) and incorporated in the food product such as biscuit or bread as a
gluten free that can help people with celiac diseases.
(a)
(b)
Figure 2.1: (a) Cucurbita moschata (labu manis) (b) Cucurbita moschata Duchesne
(labu loceng) (Norshazila et al., 2012)
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2.1.1
Structure of pumpkin cell wall
Cellulose, pectin, lignin and hemicellulose are the major constituent in the plant cell
wall of the pumpkin. For cellulose, it made up of subunit of glucose from β-1,4 bond
in linear form. Cellulose chain also consists of intermolecular and intramolecular
hydrogen bonds that form rigid and insoluble microfibrils. Moreover, the high tensile
strength of cellulose are important to withstand osmotic pressure and responsible for
plants cell to withstand mechanical stress. For hemicellulose, it consists of complex
polymer carbohydrate with xylan and glucomannan as main components while lignin
contained random polymer with high branched. Lignin usually fill the space between
pectin, cellulose and hemicellulose components. Pectin substance can be found as a
structure of polysaccharide that may be interlined in the middle of lamella and the
primary cell wall of young cells. Furthermore, pectins are made up from
homogalacturonan and rhamnogalacturonan regions which are interrupted by neutral
sugar side chains such as arabinans, galactans or arabinogalactans (Oechslin, Lutz &
Amado, 2003). In fruit juice application, the cellulose and pectin breakdown is
considered as major constituent in decreasing the viscosity of hydrolytes.
2.2
Enzymatic liquefaction
The high ratio of insoluble fiber and solid are responsible for the high viscosity in the
pumpkin. Due to its viscous nature, it would lead to the lower yield of powder in the
spray drying and cause clogging in the chamber wall of spray drying. With the help
of viscosity reducing enzyme in the liquefaction process, the viscosity of pumpkin
could be reduced. In the enzyme liquefaction process, the fruit structure such as
pectin, cellulose and hemicellulose are broke down into small particle by using
enzyme to increase the fluidity of fruit in order to facilitate the separation process
(Chong & Wong, 2015). Without undergo this process, the fruit structure are harder
to clarified especially for the fiber-like molecular structure of pectin.
Next, in the pectin structure, the ripe and unripe fruit can be affected by the
solubility and rigidity of the pectin. For the unripe fruit, the insoluble pectin is
attached to the cellulose microfibrils in the cell wall. This pectin cause the cell wall
to be rigid. However, during ripening process, the presence of the natural enzyme in
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the fruit cause the structure of pectin chain or the side chain which attached to the
unit to be broken down. As a result, the pectin are loosely attached to the cell wall
and become more soluble. Mechanical crushing method of the fruits tissue is not
preferable as it can increase in viscosity and pulp particle due to the soluble pectin
present in the liquid phase, whereas Kashyap et al. (2001) stated that some of other
pectin molecules remain bound to side chain of hemicellulose and thus facilitate
water retention.
The insoluble particles or cloud particles which mostly made up of pectic
substance are highly present in the raw press juice. Pilnik & Voragen (1993) stated
that in this particle, the negative charge pectin molecule will coated the positive
surface charge of protein molecule and causing the molecule between pectin to repel
to each other. With the help of enzyme, this electrostatic repulsion between the cloud
particle will reduce by degrading the pectin structure and exposed the positively
charge protein beneath it. As a result, the aggregation of particle occur which lead to
larger particle. This larger particle eventually precipitate out. This would also cause
the total soluble solid content to increase (Kashyap, Vohra, Chopra, & Tewari,
2001).
Lastly, in this study, two types of enzyme will be used in enzyme liquefaction
process which is Pectinex Ultra-SP and Celluclast 1.5 L enzyme to help to degrade
the structure of pectin and cellulose to decrease the viscosity of pumpkin puree.
2.2.1
Pectinex Ultra-SP
The process of centrifugation or normal hydraulic pressing of tropical fruit juice
usually would lead the juice to be too pulpy and pectinaceous (Aziah, 2011). This
condition will cause problem in spray drying process as it will cause clogging in the
chamber vessel of spray drying machine. Therefore, to reduce the viscosity,
enzymatic treatment by pectinase enzymes is usually done and carried out in order to
degrade the pectins and polysaccharide which is the major contributor to the high
viscosity in the fruit juice. This enzymatic action will cause pectin–protein
complexes to form small clumps which eventually lowering the amount of pectin in
the juice and reducing the viscosity of fruit juice. Jayani, Saxena & Gupta (2005)
stated that pectinases enzyme usually found in bacteria, fungi and plants. Besides
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that, the fungal sources from pectinases usually come from Aspergillus niger species
(Dorota, Agnieszka, & Eugeniusz, 2010). There are several commercial pectinases in
the industry which can be seen in Figure 2.2.
Figure 2.2: Commercial pectinases (Kashyap et al., 2001)
For the mash treatment of fruit, the commercial enzyme Pectinex Ultra SP-L
(Novozymes, Denmark) has potential to produce high juice yield, clear juice and
improve filteration process (Pilnik & Vorange, 1989).
2.2.2
Celluclast 1.5 L enzyme
During the growth of cellulosic material, cellulases can be produced by different type
of microorganisms such as bacteria and fungi (Kubicek,1993; Sang-Mok & Koo,
2001). The structure cellulase from fungal are more simpler compared to the
cellulase from bacteria which known as cellulosomes (Bayer et al., 1998; Percival
Zhang et al., 2006). Other than fungi and bacteria, some of anearobic protozoa and
slime molds able to degrade cellulose material. The enzyme produce by these
microorganisms not only can degrade cellulose but also polysaccharide. Furthermore,
cellulose are more difficult to break down compared to polysaccharide such as
starch.
Celluclast 1.5 L enzyme is one of the commercial cellulase enyzme that are
produced by the selected strain of fungus Trichoderma reesei. Furthermore, T. reesei
secrete three type of enzyme which is cellobiohydrolases, endoglucanases and βglucosidases. For cellobiohydrolases or exo-1,4-b-glucanases, the cellulose chain
11
will degraded starting from the non-reducing end or reducing end in cellulose
amorphous region while for endoglucanases, the reaction by this enzyme will
cleaved the cellulose chain internally in the amorphous region and produced new
terminal end. Although both of the enzyme can hydrolysed amorphous cellulose,
cellobiohydrolases shows the most efficient enzyme to degrade the crystalline
cellulose. Moreover, both of this type of enzyme produced cellobiose molecules. In
order to hydrolysed the cellobiose molecules, the action of β- glucosidases is needed.
As a result, two glucose molecule is released (Perez et al., 2002).
Next, cellulase enzyme contain many benefit and have been widely used in
the food industry. Bamforth (2009) reported that in the wine and brewery industry,
this enzyme help to improve both of the qualities and yields of fermented products.
During process of making wine, enzyme are applied either during primary
fermentation or mashing to hydrolysed glucan, to improve filterability and to reduce
viscosity (Bamforth 2009; Canales et al., 1988). Moreover, cellulase enzyme is used
in maceration process to help decrease the viscosity of the purees and nectars rapidly,
improve cloud stability and texture from tropical fruits such as pineapple juice
(Carvalho et al., 2008).
2.3
Enzyme concentration
Different concentration of enzyme can affect the puree of the fruit such as viscosity
and juice yield of the fruit. A study was done by Aziah (2011) on the effect of
different enzyme treatment (0%, 0.025%, 0.05%, 0.075% and 0.1%) and incubation
time (1-3 hour) on the properties of durian juice such as viscosity and juice yield.
The result showed that the higher the concentration of enzyme used (0.1%) and the
longer the incubation time (3 hour), the greater the yield of juice can be obtained. For
the effect of the enzymatic treatment on viscosity of the fruit, the results showed that
the viscosity reduced significantly at higher concentration of enzyme and incubation
time. However, a study by Chong & Wong (2015) on sapodilla fruits showed that the
viscosity were not significantly (P < 0.05) reduced further with high concentration of
enzyme. Similar findings was also obtained by other researchers on honey jackfruit
(Wong & Tan, 2017).
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Next, different enzyme in combination showed greater effect on viscosity
reduction compared to individual treatment of enzyme. This is due to their
synergistic effect which improve the degradation of both cellulose and pectin (Chong
& Wong, 2015). A study by Chong & Wong (2015) on sapodilla fruits showed that
the used of 0.5% (v/w) of both Pectinex Ultra SP-L and Celluclast 1.5 L reduce the
viscosity about 90.0 ± 2.20%. This result also similar to the study by Wong & Tan
(2017) which showed that the highest viscosity reduction, 94.1 ± 2.1% occurred in
the honey jackfruit puree when treated with the combination of 1.0% (v/w) Pectinex
Ultra SP-L and 0.5% (v/w) Celluclast 1.5 L. However they also stated that individual
enzyme treatment using Pectinex Ultra SP-L showed only 87.3 ± 1.0 viscosity
reduction which is lower compared to the enzyme in combination. Pectin degradation
by enzymatic treatment lead to the loss of wall strength and thus reduced the water
holding capacity. As a result free water is released and the viscosity is reduced (Lee
& Yusof, 2006).
Lastly, the used of cellulase alone in the treatment of fruit material resulting
in poor viscosity reduction compared if using with pectinex (Sreenath, Sudarshana &
Santhanam, 1995). Therefore, using enzyme in combination is more preferable to
reduce the viscosity of fruit material.
2.4
Incubation time
The period of time for the enzyme reaction to occur can affect the viscosity of the
fruit juice. Some of the enzyme are unstable which losing a significant amount of
activity over a period of time. Thus, it will affect the product formed or in the fruit
juice case, it will affect the viscosity of the fruit. Chong and Wong (2015) reported
that the longer the puree treated with enzyme, the greater the viscosity reduction.
However, too long incubation time will lead to the reduction in viscosity of the fruit
juice due to the substrate has been used up during incubation time. Htwe, Bo, & Mya
(2017) stated that the activity of pectinase increase with incubation time up to 3 min
but decrease after that. A similar study by Wong & Tan (2017) on honey jackfruit
showed that at period between 0 to 2.5 hour, the best parameter for viscosity
reduction was obtained at 1.5 hour. The puree treated after 1.5 hour are not
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preferable as it may cause destruction in activity of enzyme. This may due to the cell
may reach the decline phase and displayed low pectinase synthesis.
2.5
Statistical design for optimization process
In optimizing the formulation, design of expert technique provide useful, efficient
and simple tool in optimize the mixture. According to Bezzera et al (2008), in the
past, the optimization technique has been conducted through determine the effect on
the changes of one parameter on a response while others held at constant. However,
this method has disadvantages where there is lack of interaction among the variables
and increase the cost and time due to the increase number of experiment need to be
conducted. In order to solved this problems, response surface methodology (RSM)
is used (Yolmeh & Jafari, 2017).
RSM is a collection of mathematical method and statistical method which
used quantitative data from appropriate experimental designs to determine and
simultaneously solve multivariate equations. It usually uses an experimental design
such as a central composite rotatable design (CCRD) to fit a first- or second-order
polynomial by a least significance technique. An equation is used to describe how
the responses are affected by several test variable, combine the effect of all test
variable and determine test variable relationship (Rashmi, 1999). By using RSM, it
can help to reduce the number of experimental trial to evaluate multiple parameter
and interactions (Lee & Yusof, 2006).
RSM has been applied in the study of spray dried pumpkin powder by
Shavakhi et al. (2011) in which the interaction of cellulase concentration,
maltodextrin and spray dried inlet temperature on the characteristics of pumpkin
powder were investigated by using central composite design. In this study, RSM will
be used to investigate the interaction between the ratio of Pectinex Ultra-SP with
Celluclast 1.5 L and incubation time on the effect of viscosity reduction on pumpkin
puree.
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2.6
Spray drying parameter
Gharasallaoui et al. (2007) stated that spray drying is a unit operation by which a
liquid product is atomized in a hot gas current that used air or inert gas such as
nitrogen gas to produce the powder. The feed is usually in solution, suspension or
emulsion. The heat-sensitive fruit material such as polyphenols that are found
commonly in tropical fruits is suitable to be used as feed material to be preserved
using spray drying machine (Kha et al., 2010; Fang & Bhandari, 2011). Moreover,
carrying agent also used in the spray drying process to be encapsulated or to
entrapped food ingredient in order to protect the heat sensitive component (Caliskan
& Dirim, 2013). The powder formed by this process are easy to transport and storage
and have a longer shelf life than fresh fruit.
In the spray drying process, the liquid system is passed through a spray drying
nozzle in which it come in contact with hot and dried air at highest temperature to
evaporates the solvent and convert the droplets into dry powder and then collected by
using cyclone or drum. Furthermore, the function of the nozzle in spray dryer is to
make small droplets in order to increase the heat transfer and rate of water
evaporation. Since spray drying only involved single step process, it helps to
minimize the process and maximize the profit (Chegini & Ghobadian, 2007;
Murugesan & Orsat, 2011). The process of spray drying is illustrated as in Figure 2.3
and generally involves the following steps (Shahidi & Han, 1993).
i.
Suspension or emulsion preparation
ii.
Homogenization
iii.
Automated feed dispersion into the drying chamber through a fine nozzle
iv.
Dehydration of fine droplets or formation of powder
v.
Collection of accumulated powder
Spray dryer chamber is one of the important component where the drying process
begin with the contacted of spray droplet with hot air. There are several flow of hot
air that will in contact with particle which is co-current, counter-current air and
mixed flow. The co-current flow involved same movement direction of air and
particle while in counter-current flow involved opposite movement of air and
particle. For the mixed flow, the particle are subjected to counter-current and co-
15
current phase. In addition, the inlet temperature, air flow rate, feed flow rate,
atomizer speed, outlet temperature, feed concentration and type of carrier agent are
important factors as it can affect the physicochemical properties of powder (Obon et
al., 2009).
Next, feed material usually in the diluted form for the small scale laboratory
spray dryer in order to prevent clogging in the spray dryer (Chegini & Ghobadian,
2007; Murugesan & Orsat, 2011). If high viscosity of liquid is used to be spray dried,
high inlet temperature are needed to dried the feed and thus increase the power
consumption. Next, feed temperature has a direct effect on the viscosity of the liquid.
High feed temperature will cause the loss of sensitive and volatile compound before
they get microencapsulated. Moreover, high heat temperature also can cause
premature release, cracks on the microspheres and destruction of ingredients
(Zakarian & King 1982).
According to Masters (1979), the liquid droplet size can be directly affected
by viscosity of the liquid at constant atomizer speed. At high viscosity, larger particle
of powder formed due to the formation of larger droplet during atomization of spray
drying. Outlet temperature also can affect the moisture content, sensory properties
and colour of powder (Bielecka & Majkowska, 2000; Koc et al., 2010). Moreover,
the combined simultaneous effect of factors such as agglomeration, nozzle type,
droplet size, interaction between droplets and air, heat and mass exchange cause the
mathematical modelling of spray drying difficult to measure in continuous operation
(Gharsallaoui et al., 2007).
Figure 2.3: Schematic of a spray dryer (Patel & Patel, 2012)
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2.7
Microencapsulation by using various wall of material
Microencapsulation can be defined as the isolation of active substance which is
liquid, solid or gas state to produced products in spherical form and micrometric size.
Since the wall materials is protected from the membrane of surrounding
environment, sensitive ingredient such as antioxidants, flavours, polyunsaturated
oils, drugs and vitamin can be protected from surrounding environment. Moreover, it
can be applied in the food, cosmestic and pharmaceutical industries. The efficiency
of microencapsulation technique is greatly dependence on the microencapsulating
material known as wall material. According to Watson et al., 2017, the wall material
are selected based on the stability of material and ability protect core material from
environment. Moreover, wall material are also chosen based on physical properties
such
as
molecular
weight,
diffusibililty and
solubility (Kandansamy &
Somasundaram, 2012). Raja et al.(1989) also stated that wall material are selected
based on hygroscopicity while Mahdavi et al. (2014) stated that the wall material
needs to be food grade, inexpensive legally allowed and readily available. In this
study, the wall materials used in spray drying will be Maltodextrin.
2.7.1
Maltodextrin
According to Anekella (2011), maltodextrin is a product of hydrolysis of starch
which consisted of D-glucose units linked mainly by α (1→4) glycosidic bond.
Usually maltodextrin can be found in the white granular hygroscopic powder and
soluble in water. The number of dextrose units in maltodextrin is given as Dextrose
Equivalent (DE), which is inversely related to their average molecular weight and its
value is usually between 4 and 20 (Anekella, 2011). Moreover, the maltodextrin that
contained dextrose between 4 to 20 exhibit slightly sweet taste while for the dextrose
syrup or maltodextrin that contained dextrose equivalent (DE) > 20), it is better
perceived by the customers (Descamps, Palzer, Roos & Fitzpatrick, 2013).
In the food industry, maltodextrin have been widely used due to its various
benefit such as dispersing aid, bulking agent, wall material, fat replacer and flavour
carrier. In addition, maltodextrin usually used as ingredient in meat products,
ketchup and sauces, baby foods, confectionery, sport beverages, alcohol-free beers
and dry soups (Descamps et al., 2013). Furthermore, they are useful to reduce the
17
stickiness in the product that are difficult to dry such as flavourings, fruit juice and
sweeteners and thus improve the stability of the product (Bhandari et al., 1993;
Bhandari et al., 1997; Roos & Karel, 1991).
Next, in the spray drying process, the used of maltodextrin as a drying agent
can affect the moisture content and solubility of the powder. Low moisture content
and solubility of powder can be obtained at high maltodextrin concentration but too
high concentration can increase total soluble solid content and viscosity of the puree.
However, Goula, & Adamopoulos (2008) reported that high moisture content of the
tomato powder was obtained by using high concentration of maltodextrin. This
probably due to the chemical structure of maltodextrin that contained high number of
ramifications with hydrophilic groups causing it to easily bind to the water molecules
from the ambient air during powder handling after the spray drying (Phisut, 2012)
The glass transition temperature (Tg), is the temperature at which the polymer
in the amorphous phase is converted between glassy states and rubbery state (Phisut,
2012). Fruit material that have low Tg tend to be sticky in the wall of spray drying
chamber. Thus, with the addition of maltodextrin, it can help to increase the Tg of the
drying material in which is help to overcome the stickiness problem in spray drying
process.
2.9
Inlet air temperature
There are several physical characteristics of powder that can be affected by inlet
temperature such as bulk density, moisture content, particle size and hygroscopicity.
Increasing inlet drying temperature cause the moisture content to be decreased. This
is probably due to the driving force of the moisture content that cause by the larger
temperature gradient between feed droplets and the hot drying air (Quek, Chok &
Swedlund, 2007). Furthermore, increasing inlet temperature will decrease the bulk
density of the powder due to the casehardening on the droplets of the powder
resulting from the rapid formation and dried layer on the droplet surface and particle
size (Chegini & Ghobadian, 2007). This will cause the vapour-impermeable films
formed on the droplet surface which followed by the formation of vapour bubbles
and consequently the droplet expansion.
18
Next, lower moisture content that presence in the powder due to the high inlet
temperature causing the powder to be more hygroscopic. Moisture are usually tend to
absorb easily by powder with low moisture content. Less moist powder usually
contained high water concentration gradient between the surrounding air and the
product thus aids in hygroscopicity of the powder (Phisut, 2012).
2.10
Application of spray dried pumpkin powder
Pumpkin powder have a potential to be commercialized since pumpkin is widely
planted all over Malaysia. Several application of pumpkin powder in the food
production have been done by food industry. For example is the used of pumpkin
powder in the bread production to replace the wheat flour and thus resulting in the
increases of loaf volume and organoleptic of the bread (Pongjanta et al., 2006).
Moreover, the amount of β-carotene present in the pumpkin can increase the
nutritional qualities of the wheat bread in which it can converted into vitamin A and
help to prevent chronic diseases. It also can help to overcome the vitamin A
deficiency where it is common cases for the people living in urban areas
(Chakravarthy, 2000). Pumpkin powder also can be used as additives in snack food
according to Norfezah et al. (2013).
According to Zhan (2003), pumpkin pulp contained good source of essential
amino acid for example lysine (0.508%), leucine (0.700%), valine (0.609%),
phenylalanine (0.483%), isoleucine (0.493%) and theronine (0.381%) In addition,
pumpkin pulp contained high amount of calcium (205.45 µg/g) and potassium
(1840.30 µg/g) but low amount of sodium (Fan & Li, 2005). The present of high
amount of protein in the pumpkin can be commercialized as a substitution of wheat
flour and beneficial for people that have celiac diseases.
Celiac diseases is a genetic diseases that resulting in immune disorder that
triggered by the ingestion of gluten or related rye and barley proteins (Martin, 2012).
The main symptoms that occur by celiac diseases are most commonly diarrhea,
stomach pain, gas and bloating, anemia, weight loss and edema (Holtmeier &
Caspary, 2006). In Malaysia, non-celiac diseases are more common compared to the
celiac diseases in individuals. Non-celiac diseases can be defined as the individual
who have the same intestinal sign, extraintestinal signs or both as the celiac diseases.
19
In order to treat these gluten-related disorder, a strict gluten free diet are needed and
should be followed to avoid the symptoms.
Food ingredient that are possible for inducing celiac diseases and non-celiac
symptoms are wheat, rye, barley and their cross-bred varieties (Cawthorn,
Steinman & Witthuhn, 2010). These food ingredient are widely used in food product
such as bread, cake and biscuit. People that have gluten-related disorder need to
avoid these type of food that contained gluten. However, certain ingredient that have
the same function as gluten can be used in making these type of food. Thus, since
pumpkin contained high amount of protein, it can be used to replace the gluten
function in the wheat to strength and elasticity of the dough production.
Next, the presence of high amount of calcium in the pumpkin make it
applicable to be use in the dairy product that cause lactose intolerance to people.
According to Heyman & Care (2006), lactose intolerance can be defined as incapable
to absorb and digest dietary lactose which can cause gastrointestinal condition or
clinical syndrome for example diarrhea, abdominal pain, nausea, flatulence or
bloating (uncomfortable condition which having gas in the stomach and bowels).
Moreover, the amount of lactose consumed, the form of lactose food substance and
the degree of lactase deficiency can cause different symptoms from individual to
individual (Heyman, 2006). In addition, symptoms for lactose intolerance usually
occur within 30-60 min. The symptoms for lactose intolerance also differ between
infants and older children where older children and adult are less prone to having
diarrhea compared to infants.
The treatment for lactose intolerance usually involve reducing the
consumption of lactose intolerance food but not complete elimination of those food.
By restricted the lactose intake, the duration of gastrointestinal symptoms can be
reduced or shorten. The restriction of lactose consumption to avoid lactose
intolerance rise concern as it reduced the calcium intake compared to those tolerated
lactose. According to Fox et al. (2004) & Doulgeraki et al. (2017), the effect of
avoidance of dairy product in young children can increased fracture risk in later life
due to the nutritional rickets and low mineral density in the body. By replacing the
ingredient mainly from diary product that cause lactose intolerance to the lactose free
ingredient containing the same amount nutrient such as calcium, the nutrient
deficiency can be overcome. Thus, in order to overcome this problem, pumpkin
powder can be used as an alternatif in producing food that are free from lactose.
CHAPTER 3
RESEARCH METHODOLOGY
3.1
Introduction
This chapter covers the reagents, instruments, material and methods used in order in
determining the effect of enzymatic ratio (Pectinex Ultra SP-L and Celluclast 1.5L),
incubation time and spray drier air inlet temperature based on viscosity and
physicochemical properties of pumpkin powder. Figure 3.1 shows the overall
flowchart of the experiment.
Briefly, pumpkin will be treat with different ratio of enzyme (Pectinex Ultra
SP-L and Celluclast 1.5L) and the best reduction of pumpkin puree will be treated
with different incubation time to further reduce the viscosity. After that, different
inlet temperature during spray drying also will be test in order to determine the effect
on the physicochemical properties of the pumpkin powder based on moisture
content, stickiness, water activity, hygroscopicity, solubility, bulk density and
process yield.
21
Material : Pumpkin (Cucurbita
moschata)
Drying agent: Maltodextrin (23% w/v)
Specific
objective
1
Optimization by using Response Surface
Methodology (RSM)
Enzyme liquefaction : 0.5% of Pectinex
Ultra SP-L concentration and 0.5%
Celluclast 1.5 L
Incubation time : (30 min-120 min)
Viscosity reduction (%)
Specific
objective
2
Spray drying inlet temperature (150°C
-190 °C)
Moisture
content
Stickiness
Hygroscopicity
Process yield
Solubility
Water activity
Bulk density
Figure 3.1 : Flow chart design for optimization of spray drying
22
3.2
Materials
Pumpkin fruits (Cucurbita moschata) that will use in this study will be purchase
from the fresh market in area Pagoh and Muar, Johor. Maltodextrin (DE 10–12) will
be obtained from Agrin Chemical Sdn. Bhd. The enzymes, Pectinex Ultra SP-L and
Celluclast 1.5 L (for liquefaction) will be purchase from Novozymes, Denmark and
stored at 4°C.
3.3
Apparatus and instruments
Waring blender (WARING; model 700G), Water bath (Memmert; model WNB 745), viscometer (Brookfield; model DV–II+ Pro), mini spray drying (AGRIDON;
model AG-1-6), moisture analyzer (TOVATECH; model DSC 71P), water activity
meter (DECAGON; model Aqua Lab), texture analyzer (TA.XTPlus100) and
dessicator (LAB; model LAB1354), oven (SHARP; model SHP-R854AS) will be use
at Food Instruments, Food Product Development and Food Analysis Laboratory.
3.4
Experimental Design and Statistical Analysis
The statistical design will be use in this study are response surface methodology to
study the effect for the enzyme ratio and incubation time on physicochemical
properties of pumpkin powder. Table 3.1 shows the value of the response variable
according to the experimental design.
23
Table 3.1: Experimental design of spray drying
Run
Enzyme ratio (0.5% Pectinex Ultra SP: 0.5%
Incubation time (min)
Celluclast 1.5 L)
1
2
3
4
5
6
7
8
9
10
11
12
13
3.5
5.00 : 5.00
5.00 : 5.00
5.00 : 5.00
2.50 : 7.50
5.00 : 5.00
7.50 : 2.50
0.00 : 10.00
5.00 : 5.00
5.00 : 5.00
10.00 : 0.00
7.50 : 2.50
2.50 : 7.50
5.00 : 5.00
90.00
150.00
30.00
120.00
90.00
120.00
90.00
90.00
90.00
90.00
60.00
60.00
90.00
Preparation of pumpkin puree
The pumpkin fruit will be peel, deseed and flesh cut into small pieces which is 2.5 x
0.1x 0.1 cm) (Shavakhi, Boo, & Osman, 2011). Then, by using Waring blender
(WARING; model 700G), the slice pumpkin will be blend for approximately 30-60 s
until a homogenous puree obtain (Chong & Wong, 2015).
3.6
Enzyme liquefaction treatment
The method for enzyme liquefaction was determined by (Wong & Pui, 2015) with
slight modification. In this liquefaction method, 300g of homogenized pumpkin
puree will be treat with different enzyme ratio (2.5:7.5, 7.5:2.5, 5.0:5.0, 10.0:0,
0:10.0) of both Pectinex Ultra-SP and Celluclast 1.5L. Then, the enzyme-added
purees will be incubate at 50°C using water bath (Memmert; model WNB 7-45)
where the viscosity measurement taken at interval 30 min for 2.5 h as shown in
Table 3.1. After that, the puree treat with enzyme will be deactivated at 90ºC. Then,
the lowest viscosity measurement will be choose to prevent the clogging of spray
dryer and maximize the amount of juice yield (Phisut 2012).
24
3.7
Viscosity measurement
According to Chong & Wong (2015), the viscosity of the liquefied pumpkin puree
will be measure by using precalibrated Brookfield viscometer (Brookfield; model
DV–II+ Pro) at 160 rpm with spindle LV-3. The measurement will be carry out at
room temperature (29°C ± 1°C). The viscosity reduction will be expressed in
percentage based on following formula:
Viscosity reduction (%) =
3.8
Viscosity control−viscosity sample
viscosity control
Spray drying process
The spray dry will be performed by using a laboratory mini spray dryer (AGRIDON;
model AG-1-06) and it will be operate concurrently with a 1.4 mm diameter spray
nozzle. The inlet temperature will be between 150°C -190 °C (Shavakhi et al., 2011)
and the outlet temperature will be between 75°C-85°C (Bakar, Ee, & Muhammad,
2012). Then, the solution will be feed into the drying chamber through peristaltic
pump with pressure and flow rate of 6.5 bar and 600 L/h respectively. The air flow
rate will be
30 𝑚3 /h (Shavakhi et al., 2011). During spray drying, the feed
containing maltodextrin will be stirred continuously to ensure its homogeneity
(Bakar et al., 2012) The resulting powder obtained will then immediately remove
and collect in glass collection vessel and store in the amber glass bottle at 4°C before
determining its characteristics (Shavakhi et al., 2011). According to the Kim, Chen
& Pearce (2009), The spray-dryer aspirator rate, pump rate, atomization air
rotameter, and the feed temperature were kept constant at 100%, 10%, 35 mm, and
30±1 °C, respectively. The dryer outlet air temperature could not be controlled
directly, but is a function of the inlet air temperature and the feed rate.
3.9
Analytical method
The analytical methods will be used to determine the inlet temperature based on
physicochemical properties such as moisture content, stickiness, water activity,
hygroscopicity, solubility, bulk density and process yield.
25
3.9.1
Moisture content
The pumpkin’s powder moisture content will be determine by using Moisture
Analyzer (TOVATECH; model DSC 71P).
3.9.2
Stickiness
2.0 g of pumpkin powder will be mix with 3.0 ml of glycerol to formed pumpkin
dough. This study will use a texture analyzer (TA.XTPlus100) to measure the
tension force and provide constant compression force. The probe is a Smc/Chen–
Hosney dough stickiness probe of 25 mm in diameter. It is a special plexiglass probe
which causes easily separation of and probe at their interface. The compression travel
and probe reversing speed are 2 and 10 mm/s, respectively. The probe travel distance
is 10 mm. The dough will then place in the sample chamber of the equipment and the
readings of force (g) for stickiness were made in triplicate (Shavakhi et al., 2011).
3.9.3
Water activity
Measurement of water activity will be use water activity meter (DECAGON; model
Aqua Lab) and the sample prepare in triplicate where the mean will be record.
3.9.4
Hygroscopicity
According to the Cai and Corke (2000), a total of 2 g of pumpkin powder sample will
place in air tight desiccator (LAB; model LAB1354) at 25±1.0 °C containing
saturated Na2 SO4 solution (81% RH). Sample will be weight after 1 week and
hygroscopicity will expressed as grams of moisture per 100 g of dry powder (g/100
g).
26
3.9.5
Solubility
The solubility of pumpkin powder were determined by Cano-Chauca et al. (2005). 1
g of pumpkin powder will be weigh and transfer to the 100 mL of distilled water.
The 25 mL aliquot of supernatant will then transfer to a pre-weighed aluminium and
will be place in an oven (SHARP; model SHP-R854AS) at 105°C until constant
weight. The solubility (%) will be determine by weight difference.
3.9.6
Bulk density
According to Kha, Nguyen & Roach (2010), bulk density can be determine with
slight modifications. By using 50 mL of glass measuring cylinder, 2g of powder will
be freely pour into the glass and tap manually the glass until the level of the powder
become flat. The ratio between the mass (g) of the powder and the volume (mL)
occupied in the cylinder will determines the bulk density (g/mL) value.
3.9.7
Process yield
According to Bhandari et al. (1997b), cyclone and sweep recoveries will be add
together by total up the weight of powder by sweeping of the wall of spray-dryer
glass chamber (sweep recovery) and the weight that will be collect in cyclone
chamber (cyclone recovery). Process yield is expressed as the relationship between
the total solid content in the feed mixture and the total solid content in resulting
powder (Tonon et al., 2008)
CHAPTER 4
EXPECTED RESULT
At the end of this study, experimental design is expected to optimize and evaluate the
formulations in this study, response surface methodology is expected to be
conducted. This method is more suitable and applicable for optimization of
formulation because it gives more reliable data as shown in Table 4.1.
The enzymatic ratio and incubation time will be optimized to determine the
viscosity reduction of pumpkin puree while inlet temperature of spray drying will be
determine based on physicochemical properties of the pumpkin powder such as
moisture content, stickiness, water activity, hygroscopicity, solubility, bulk density
and process yield that will significantly effect on spray drying conditions as shown in
Table 4.1. The optimum condition choose for this study will be 160°C to 180°C for
inlet temperature, 90 minute to 120 minute of incubation time and higher ratio of
pectinex Ultra SP and lower Celluclast concentration. The optimum condition choose
for this study will be low value of viscosity, moisture content, water activity,
stickiness and high value of process yield and bulk density. It also will showed
acceptable qualities of solubility and hygroscopicity.
28
Table 4.1: The effect of enzyme ratio and incubation time on viscosity reduction in
pumpkin puree
Run
Factors
0.5% Pectinex Ultra SP : 0.5
Viscosity reduction
Incubation time (min)
(%)
% Cellulcast 1.5 L
1
5.00 : 5.00
90.00
2
5.00 : 5.00
150.00
3
5.00 : 5.00
30.00
4
2.50 : 7.50
120.00
5
5.00 : 5.00
90.00
6
7.50 : 2.50
120.00
7
0.00 : 10.00
90.00
8
5.00 : 5.00
90.00
9
5.00 : 5.00
90.00
10
10.00 : 0.00
90.00
11
7.50 : 2.50
60.00
12
2.50 : 7.50
60.00
13
5.00 : 5.00
90.00
Table 4.1: Yield and physicochemical analysis of pumpkin powder spray-dried under
different inlet temperatures (150–180°C)
Inlet temperature (°C)
Physicochemical
characteristics
150
Moisture content (%)
Stickiness
Water activity (𝐴𝑤 )
Solubility (g)
Hygroscopicity (%)
Bulk Density (g/ml)
Process yield (%)
160
170
180
190
CHAPTER 5
PLANNING
The figure 5.1 shows the flow chart of the overall of this studies from the day which
task given until the submission of the corrected final report and the technical paper.
This flow cover the Bachelor Degree Project 1 and 2 workloads. This studies has to
be done accordingly within the planned period which shown in Table 5.1 and Table
5.2 which represented in the format of the Gantt Chart. This is to ensure this studies
will be completed in the required duration of time.
Table 5.2: Gantt chart for Bachelor Degree Project (BDP) I and the milestones
Activities
BDP I (weekly)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Bachelor Degree Project briefing
Meeting with supervisor
Literature review proposal assessment
Proposal feedback from supervisor
Submission of final proposal
Preparation of slide presentation
Proposal presentation
Submission of corrected proposal
milestone
30
Table 5.2: Gantt chart for Bachelor Degree Project (BDP) II and the milestones
1
2
3
4
5
BDP II (weekly)
6
7
8
9
10
11
12
13
14
Discussion with supervisor
Thesis writing
Experimental design
Sample preparation
Physicochemical properties determination
Analyse and finalize
Optimization and validation of data
Final report submission
BDP II presentation
Corrected final report submission
Submission of thesis
milestone
31
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