THE EFFECT OF SIZE AND DRYING METHOD ON RETENTION OF BETA-CAROTENE IN CARROT

CHAPTER ONE

INTRODUCTION

 Extrusion Cooking

 Extrusion cooking is a process in which processing of feed material is achieved under the combined effect of temperature, pressure and shear. In recent years, extrusion process draws interest in food industry especially in the production of snacks, cereals and pasta as due to the advantages it offers. While short residence time provides the quality of extrudates; opportunity to form variously shaped products creates appetizing foods. Moreover; too little waste production during extrusion makes it possible to overcome the environmental or government restrictions and also lowers the cost of final product (Riaz&Rokey,2011a).

Food extrusion can be thought as a high temperature-short time process as barrel temperatures are adjusted to not to exceed 180°C and residence time in extruder usually changes in the range of 20-200 seconds (Martínez-Bustoset al., 2012). This time- temperature combination is adequate to the physical changes or chemical reactions like starch gelatinization, protein denaturation, enzyme inactivation and destruction of microorganisms (Wolf, 2010) whereas formation of flavours or color change reactions may not take place mainly as a result of limited time (Chessari&Sellahewa, 2001; Martínez-Bustos et al., 2012).

A typical extruder is composed of engine, feeder, barrel, screw, die and control panel. In the barrel section, with the help of the screws’ rotating action shear energy is generated. In consequence of the effect of shear energy and high temperature of extrusion, pressure inside the barrel rises and causes food material to undergo chemical and rheological changes. When leaving the barrel through a die the rapid pressure drop induces evaporation of water and expansion of extrudate, which result in a puffed and porous product. Die also gives the extrudate its final shape. The smaller the diameter of the hole on die, the higher the resistance that food material encounter at the exit, which increase the inside pressure and also causes more back flow of food material (Moscicki&Zuilichem,2011).

With regard to screw number, extruders are classified as single screw extruders or twin screw extruders. Extruders whose barrel contains two screws with equal length named as twin screw extruders, whereas if only one screw is located it is called as single screw extruder. According to the configuration and rotation of screws, twin screw extruders are further grouped as intermeshing/non-intermeshing and co- rotating/counter-rotating, respectively. If the screws penetrate each other, the type of extruder is intermeshing twin screw extruder; otherwise it is non-intermeshing. Furthermore; if the screws rotate in the same direction, it is termed as co-rotating. The extruders with the screws rotate in opposite direction are named as counter- rotating twin screw extruders (Giles et al., 2004; Riaz&Rokey, 2011b). Main drawback of single screw extruders is poor mixing ability, for this reason additional blending may be required prior to feeding especially for multi-component food materials. Apart from mixing efficiency, twin screw extruders are superior to single screw extruders in terms of self-cleaning opportunity, high productivity and flexible feed material handling (Moscicki&Zuilichem, 2011; Riaz, 2001). Ingredients which have too high moisture content and are sticky that cannot be handled in single screw extruders can be processed in twin screw extruders. More complex design and high cost can be counted as adverse sides of twin screw extruders. Twin screw extruders are used both for the pet food industry and for human consumption such as in the production of breakfast cereals, pasta, potato pallets, candy, chocolate and snack foods (Berk,2013).

1.2.   Snack Foods

By the application of modern extrusion techniques, it has become widespread to market cereals as snacks so that snack foods are gaining popularity as much as breakfast cereals or corn flakes in recent years. Statistics confirm this as consumption of snack food rose threefold in the United State from 1977 to 1994 (Camire, 2001; Putnam &Gerrior, 1999). Moreover, US Department of Agriculture drew up a report in 2000 indicating that snack foods meet 20% of energy and some nutrient requirements of American children (Camire, 2001; USDA, 1999). As much comparable data is not available for other regions and nations, this statistics can give us a general view for the consumption trends for snacks. However; assumption of the snack foods as unhealthy acts as a barrier and limits the consumption of them (Camire, 2001; Dinkins, 2000). Therefore, fortification of snacks is a considerable parameter.

Cereals are used as major ingredients in extrudates because of good expansion ability as a result of high starch composition (Dar et al., 2014). Corn flour and corn grit are commonly used ingredients in the extruded snack food production; however, using corn as a single ingredient result in a snack with high glycemic index and unsatisfactory nutritional value. Therefore corn based snack foods need to fortify with fruit or vegetables as a minor ingredient (Brennan et al., 2013; Caltinoglu et al., 2014; Conti-Silva et al., 2012). Inferences of the surveys and health concern of public direct the researchers to enhance nutritional quality of snack foods and develop functionalfoods.

1.3.   Functional Foods

  Variations in the physical conditions, health status, lifestyle, culture and nutritional requirements of people prevents to form a standard foodstuff fit everyone and directs food scientists to produce food enriched with specific ingredients. For this reason, as well as providing basic nutritional requirements, the food products that have a positive impact on health or developed to be beneficial on physiological means are defined as functional foods (Diplock et al., 1999; Pang et al., 2012; Thompson &Moughan, 2008). Genetic engineering is one of the methods used in creating functional foods. Simpler and easier development of functional foods can be achieved via either addition of functional ingredients to food or alteration the composition of unprocessed food. Today, functional food market turns into a big industry worth annually 60 billion dollar and gradually increases its market presence (Consumer Reports on Health, 2007; Edlin&Golanty, 2012; Thompson &Moughan,2008).

1.4.   Carrot

 Carrot is a kind of vegetable whom composition is dominated by water (86-90%) and remaining is composed of carbohydrate (7-10%), fiber (3%), protein (1%), ash (1%) and fat (0.2%). Fiber and carbohydrate contents are formed mainly from cellulose and simple sugars, respectively (Arscott&Tanumihardjo, 2010; Sharma et al., 2012a). Carrot contains adequate amounts of thiamin, riboflavin and niacin. Potassium is the dominant mineral in it (Nicolle et al., 2004). Carrot is also known for the rich beta carotene content which is responsible for the orange color (Arscott&Tanumihardjo, 2010). Carrot contains approximately 9700 µg β-carotene/100 g, 3140 µg α-carotene/100 g and 220 µg lutein/100 g (Stahl &Sies, 1999). High β- carotene composition is crucial as β-carotene is the precursor of Vitamin A. Accordingtodatagivenin2002,approximately 140 million pre-school children and 7 million pregnant woman face vitamin A deficiency, thereupon up to 3 million of that population die in a year (Haddad et al., 2004). World Health Organization defines Vitamin A deficiency as a public health problem that affects 122 countries worldwide (Arscott&Tanumihardjo, 2010; World Health Organization, 2009). β-carotene content also contributes the antioxidant potency of carrot as phenolic compounds do. Carrots have antioxidant activity because of the phytonutrients and phenolic compounds they include. Chlorogenic acid is the dominant phenolic acid found in carrot (Sharma et al., 2012a; Zhang &Hamauzu,2004).

1.5.   Antioxidants

  A great majority of lipids in food materials are in the form of fatty acids esters and glycerol. As a result of the reaction of fatty acid esters with oxygen, lipid deterioration and fat rancidity take place. Other than fat rancidity, oxidation also results in nutritional reduction, undesirable flavor formation and color change in foods. Antioxidants are the molecules that contribute to extension of the shelf life of food substance by lessening these negative impacts of oxygen (Miková, 2001; Yanishlieva-Maslarova, 2001). As well as preventing the deleterious effect in foods, some of the antioxidants are to be believed to be beneficial on human body by preventing the harmful effects of free radicals, nucleic acid damage and also helps to fight the cardiovascular disease to some extent (Molyneux, 2004; Pisoschi&Negulescu, 2011; Pokorný& Schmidt; 2001).

Although it is possible to gain foods antioxidant property with the addition of synthetic antioxidants like BHT, BHA and TBHQ, safety concerns and governmental restrictions limit the usage of them, which results in a growing interest on the natural antioxidants (Balasundram et al., 2006; Frankel, 1998; Pokorný& Schmidt; 2001; Wanasundara&Shahidi, 1998). There are several natural antioxidants found in foods such as phenolic acids, carotenoids, flavonoids, tocopherols, benzoic acid derivatives, etc. As there is a wide range of compounds that show antioxidant property and as the antioxidant activity changes according to the several factors such as polarity, solubility and temperature; a standard method determines the certain antioxidant activity or antioxidant capacity of a single compound is not available (Litescu et al., 2010; Ou et al., 2002; Pisoschi&Negulescu, 2011). Antioxidant activity and antioxidant capacity are two different terms related to each other. Antioxidant activity refers to the reaction rate of a single antioxidant with free radical, however; antioxidant capacity is a more general term, which regards the reaction between the antioxidant solution includes the mixture of antioxidant compounds and free radical (Litescu et al., 2010; Prior & Cao, 1999). Generally accepted antioxidant activity determination methods are based on reaction between the antioxidant and organic radical then followed by the observation its absorbance spectrophotometrically. DPPH (2,2-diphenyl-1-picrylhydrazyl) method, ABTS method, TEAC (Total Radical Trapping Antioxidant Parameter) assay, ORAC (oxygen radical absorption capacity) assay, FRAP (ferric reducing antioxidant power) assay can be counted as the most common antioxidant activity determination methods (Litescu et al., 2010; Pisoschi&Negulescu,2011).

1.6.   Phenolic Compounds

Phenolic compounds involve a large variety of structures from simple phenolic acids to polymer polyphenols. They are available in plants and play a role in enzymatic browning, color and flavor of fruits (Cheynier, 2012). Their anti-allergenic, anti- artherogenic, anti-inflammatory, anti-microbial, anti-thrombotic and anti-oxidant properties make them beneficial for health (Balasundram et al., 2006; Middleton et al., 2000). Vanillin, gallic acid, tannins, flavonoids and hydroxycinnamic acids can be counted as examples for the natural phenolic compounds (Balasundram et al., 2006; Middleton et al., 2000). Butylatedhydroxyanisol (BHA) and butylatedhydroxy-toluene (BHT) are the most common synthetic phenolic compounds used for their antioxidant property (Yanishlieva-Maslarova,2001).

For the quantification of phenolics both spectrophotometric and chromatographic techniques are available. High performance liquid chromatography (HPLC) and gas chromatography (GC) are the main chromatographic techniques can be used for the separation and quantification of phenolics. If not a specific phenolic group is searched, the Folin–Ciocalteu method is the most widely used procedure for the total phenolic content determination (Naczk&Shahidi, 2004).

1.7.   Carotenoids

Carotenoids are the colored lipophilic compounds function as pigments in fruits and vegetables (Rodríguez-Bernaldo de Quirós, & Costa, 2006; Su et al., 2002). Their lipophilic nature gives them a soluble characteristic in organic solvents and makes them insoluble in water (Rodriguez-Amaya, 2001). Carotenoids synthesized in plants are obtained by animal and humans via diet (Rodríguez-Bernaldo de Quirós, & Costa, 2006). Nowadays, more than 600 carotenoids have been identified (Shegokar&Mitri,2012).

According to their structures carotenoids classified into the two main groups namely, carotenes and xanthophylls. Carotenes also called as hydrocarbon carotenoids are formed only from carbon molecules, whereas xanthophylls or oxy-carotenoids also contain oxygen. The specific examples of hydrocarbon carotenoids are β-carotene (C₄₀H₅₆) and lycopene (C₄₀H₅₆). Lutein (C₄₀H₅₆O₂) and zeaxanthin (C₄₀H₅₆O₂) are classified as oxy-carotenoids (Donhowe& Kong, 2014; Rodríguez-Bernaldo de Quirós, & Costa, 2006; Su et al., 2002; Yonekura& Nagao, 2007).

Beta-carotene is an orange colored carotenoid found in red-orange fruits and vegetables like carrot. As humans are not be able to synthesize Vitamin A, getting the sufficient amounts in the diet is important. Beta-carotene, alpha-carotene, gamma-carotene and beta-cryptoxanthin are the precursor vitamin A carotenoids, meaning that they are in inactive form to Vitamin A. Among them, beta-carotene comes to the forefront with its symmetrical structure, which result in the better conversion to vitamin A and makes β-carotene having the highest vitamin A activity (Donhowe& Kong, 2014; Grune et al., 2010; Handelman, 2001; Shegokar&Mitri, 2012; Yonekura& Nagao, 2007).

Lutein is mainly found in human serum and also located in retina on eyes. It is beneficial for vision function and skin health (Bone et al., 1997; Handelman, 2001; Shegokar&Mitri, 2012). Lutein protects the eye by filtering the blue light and also contributes to prevent diseases like age-related macular degeneration and cataract (Kalariya et al., 2012; Kijlstra et al., 2012). Green leafy vegetables are the main source of lutein. Recommended daily amount of lutein is 5-12 mg. (Shegokar&Mitri, 2012).

During food processing or storage period of foodstuffs, loss of carotenoids may be seen due to several reasons such as enzymatic degradation, thermal destruction, oxidation, prolong exposure to light, being in contact with acid and isomerization from more stable trans form to cis-isomers (Rodriguez-Amaya & Kimura, 2004; Rodriguez-Amaya, 1997,1999,2002).

Although it is possible to determine carotenoid content by spectrophotometric methods, chromatographic techniques are more widely used as isomers of carotenoids, especially cis and trans isomers of β-carotene cannot be separated spectrophotometrically (Godoy & Rodriguez-Amaya, 1994; Schierle et al., 2004). HPLC is the widely used method for the quantification of carotenoids. In the HPLC analysis, separation and determination of carotenoids depend on the comparison of retention times with the authentic standards. As retention times change with type of column, mobile phase and wavelength; selection of these three parameters is crucial. C-18 and C-30 reverse phase columns are the most common carotenoid columns that are able to identify different carotenoids; however, C30 column is superior as it can also separate the cis-trans isomers of carotenoids (Takaichi, 2000, 2014).

1.8.   Bioavailability & Bioaccessibility

 Carotenoids play critical roles in the biological functions by means of their health promoting effects that contributes to fighting of diseases like cancer or cardiovascular diseases. As mammals cannot synthesize carotenoids, humans should obtain appreciable amount of carotenoid through diet (Astorg, 1997; Krinsky, 1993; Palmero et al., 2014). However; to be able to take advantage of carotenoids’ favorable effect, digestion and absorption of maximum amount of carotenoids poses crucial importance as well as consuming them via diet. This concept introduces two key terms; bioavailability and bioaccessibility (Verrijssen et al., 2014). Bioaccessibility represents the amount of nutrients released from food matrix and ready for intestinal absorption in pursuit of digestion. Bioavailability is defined as the actual amount of the nutrients absorbed into the circulation system and ready for body utilization (Amorim-Carrilho et al., 2014; Colle et al., 2010; Duchateau&Klaffke, 2008; Palafox-Carlos et al., 2011; Parada& Aguilera, 2007; Sensoy, 2013; Van Loo-Bouwman et al.,2014).

Beginning from ingestion to entrance body circulation, food material faces mainly two steps; digestion and absorption. Digestion starts mechanically in the mount and continues in the esophagus, stomach and small intestine, respectively. It includes breaking down food material into smaller pieces with the help of digestive juices and enzymes. Crushing food material makes the functional compounds more easily absorbed in the small intestine. In the course of absorption; some nutrients, water and minerals are entered into the lymph and transported into the blood stream through the walls of small intestine (Norton et al., 2007; Sensoy, 2013).

In the particular case of absorption of carotenoids, carotenoids are released from disrupted food matrix through the cell wall. Apart from cell wall, location of carotenoids inside the chromoplast organelle is another factor that affects the release of carotenoids. This step proceeds with solubilization of carotenoids in oil droplets in the stomach and micelle formation. Thus, oil-reach nature facilitates the solubility of carotenoids. Then, absorbable carotenoids are taken into small intestine. For the provitamin-A carotenoids, conversion of vitamin A into retinol takes place in this step. Finally carotenoids secreted into lymph and blood to join the circulation system (Castenmiller& West, 1998; Palmero et al., 2013, 2014; Rodriguez-Amaya, 2010; Van Loo-Bouwman et al., 2014). Therefore, bioaccessibility and bioavailability of carotenoids depend on both food-related factors such as type and location of carotenoid, destruction of food matrix, presence of other compounds like fat and fiber, food processing method and human-related factors like genetic background and general health status of human (Erdman et al., 1993; Furr& Clark, 1997; Pugliese et al., 2014b; Rodriguez-Amaya, 2010).

Quantification of bioavailability/bioaccessibility can be achieved through two different procedures namely; in-vivo and in-vitro methods. In-vivo approach for bioaccessibility estimation is based on the animal or human studies. However;  humanmodelled studies are not always satisfactory as human-related factors like genetic and health condition of test subjects may affect the subjectivity of results. Moreover; if the studies done on animals, plausibility of the results remains questionable as it is controversial that whether animal models fit completely to human metabolism or not. Additionally, even if animals are accepted to meet human models exactly, cost and ethical considerations still remain as challenges to in-vivo studies (Carbonell-Capella et al., 2014; Fernández-García et al., 2009; Parada& Aguilera, 2007; Pugliese et al., 2014b; Sensoy, 2013; Van Loo-Bouwman et al., 2014). Therefore, to gain information about bioaccessibility/bioavailability, in-vitro approaches are more commonly used in the literature. In-vitro bioaccessibility procedure includes the modeling gastric and intestinal digestion with artificial membranes in the laboratory conditions. Provided that the digestion simulation is followed by Coco-2 cells (polarized human colon carcinoma cells) uptake procedure, bioavailability quantification can be estimated (Carbonell-Capella et al., 2014; Parada& Aguilera, 2007; Sensoy, 2013). In vitro methods can be applied either  static or dynamic approach. In static approach the chemical digestion stimulated but mechanical forces and fluid motion are not included. In this manner, dynamic in- vitro approach can characterize in-vivo conditions and therefore; is superior to static model (Amorim-Carrilho et al., 2014; Ferrua& Singh,2010).

1.9.   Research Generalization

Starch is carbohydrate molecule composed of amylose and amylopectin units. Main sources of starch can be counted as potatoes, wheat and corn (Souza & Andrade, 2002). Starch is widely consumed in human diet after being exposed to food processing techniques which may result in the gelatinization of starch during heating and retrogradation during cooling (Wang & Copeland, 2013). The main effect of extrusion on the starch is gelatinization, which includes the swelling and losing the crystalline structure of starch molecule and formation of a viscous paste (Berk, 2013; Kitabatake&Doi, 1992). Starch gelatinization is crucial as extend of it influences the digestivity (Wang & Copeland, 2013). Enzymes can show higher activity on readily gelatinized starch (Berk, 2013). To investigate the starch characterization several methods have been applied such as, Differential Scanning Colorimeter (DSC), BrabenderViscoamylograph (BV) or Rapid Visco-Analyzer (RVA). DSC instrument is widely used to determine starch gelatinization in food industry as it offers fast and precise results and requires small amount of sample. With the help of DSC method it is possible to determine the gelatinization temperature and transition enthalpies (Péret et al.,1998).

THE EFFECT OF SIZE AND DRYING METHOD ON RETENTION OF BETA-CAROTENE IN CARROT

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