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. 2 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 3 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 4 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. 5 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 7 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) 8 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 9 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 10 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). 12 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 13 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. 14 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) 16 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 REFERENCES Adhikari, B., Howes, T., Bhandari, B.R. & Troung, V. (2003). 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