In Indonesia, people are familiar of using several herbal leaves for special purposes especially for condiments to act as natural colorants or natural flavors to improve color and flavors in food e.g. pandan leaves (Figure 1). Pandan leaves (Pandanus amaryllifolius Roxb) have been used in cooking and also as traditional herbal treatment for several illnesses in South East Asia Countries (Wongpornchai, 2004).
Figure 1. Pandanus amaryllifolius Roxb.
Classification of Pandan leaves are bellow
- Kingdom: Plantae
- Subkingdom: Tracheobionta
- Super Division: Spermathophyta
- Division: Magnoliophyta
- Class: Liliopsida
- Subclass: Arecidae
- Ordo: Pandanales
- Famili: Pandanaceae
- Genus: Pandanus
- Species: Pandanus amaryllifolius Roxb.
There are several herbs that have been investigated contain expressive amounts of several bioactive compounds which can decrease ageing and also prolong life span and living organism (Ferrari, 2013). Natural products, including essential oils and extracts are the main source of biologically active compounds that can give benefit for human health (Fernández-García et al., 2012). Many people said that pandan leaves are vanilla of the east since it is commonly used in several foods with the vanilla like aroma (Comax Flavors, 2011).
The genus name Pandanus is derived from the Indonesian name of the tree, pandan. In several Asia countries, pandan leaves, names given include pandan wangi (Malaysian), daun pandan (Indonesian), bai toey or toey hom (Thai), taey (Khmer), tey ban, tey hom (Laotian), dua thom (Vietnamese), and ban yan le (Chinese) (Wongpornchai, 2004). The distribution of pandan leaves is found over Southern India, the Southeast Asia peninsular, Indonesia and Western New Guinea (Wongpornchai, 2004).
The plants grow in clumps and have thin and sharp leaves at the edge where the form is like sword, fragrant odor. Pandan leaves, commonly known as pandan, are often used to give a refreshing, fragrant flavor to both sweet and savoury South-East-Asian dishes (rice, chicken, jellies, drinks, puddings, custard or sweets).
Pandan leaves are also used in cooking ordinary non-aromatic rice to imitate the more expensive aromatic Basmati and Jasmine rices (Nor, Mohamed, Idris, & Ismail, 2008). Since the flavour of pandan leaves is similar to that possessed by some famous aromatic rice varieties, the leaves often find their way into the rice pot to enhance the aroma of lesser rice varieties. By increasing the aroma in lesser rice varieties, it can increase the consumer acceptance by enhance the flavour perception in customer where the non-aromatic rice has similar flavour with the aromatic rice e.g. Basmati and Jasmine rice. Flavour perception is interesting subject. The flavour of food is ultimately a product of the brain. The brain combines sensory information from taste, smell and touch to generate our perception flavour, and how it does this is currently a hot topic in psychology and neuroscience (Stevenson & Richard, 2013). The study of the mechanism of important flavour during cooking rice is quite complex, where the absorption of important flavour by rice in both optimal and excess water cooking was highly dependent on the presence of water, moisture content of rice, water to rice ratio, starch gelatinization process as well as temperature and time of cooking (Yahya, 2011).
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Rice grains with the popcorn like fragrance are very popular among several Asian countries. In particular, Basmati in India and Pakistan; Khao Dawk Mali 105 in Thaliand, Pandan rice in Indonesia are very popular (Bryant & McClung, 2011; Kawakami et al., 2009). These aromatic rice are more expensive and also more valuable than non aromatic one. Since fragrant rice is very expensive and pandan leaves that have aromatic rice like flavour. Nowadays, since the interest of customer flavour companies have come out with a number of mimetic rice flavour oils. 2-Acetylpyroline (2 ACPY) as one of the main compounds in rice also will give the popcorn like aroma like fragrance (F. Yahya, Fryer, & Bakalis, 2011). Because of that, nowadays the encapsulated process of pandan aroma had been developed. Spray drying is the most common and cost effective way to perform encapsulation of flavors. The encapsulated flavour of pandan leaves by using gum Arabic and maltodextrin had been developed (Kawakami et al., 2009).
Pandan leaf extract has been used for food industries as dye materials, and also soya beverage and coconut milk. As a traditional herbal this leaves are generally used for traditional medicine especially to encounter the typhus illness in Indonesia (Roosita, Kusharto, Sekiyama, Fachrurozi, & Ohtsuka, 2008). The effect of antimicrobial effect of pandan leaves has been investigated on the preservation of stored milk (Khusniati & Widyastuti, 2008).
Sometime, pandan leaves are also used to wrap food for cooking, such as chicken wrapped in pandanus leaves and are neatly folded into small baskets for filling with puddings and cakes (Wongpornchai, 2004). The leaves are sometimes also can be put into frying oils to impart flavour to fried food. Pandan extracts also capable of retarding oxidation in palm olein during deep frying process than as effectively other antioxidant which is BHT (Butyl Hydroxy Toluene). In sensory evaluation, the extract also was able to maintain sensory quality of French fries. The delightful flavour characteristic from pandan leaves, which is well-known throughout the world as an important component in Asian cookery, has made the industrial production of both natural extracts and artificial flavourings containing green food colors for use as food additives in Southeast Asian countries enlarge during the past two decades).
Like other green leafy vegetables, pandan leaves are also known as potential source of several lipophilic antioxidant e.g. β-carotene, vitamin E, phenolic compounds, ascorbic acid (Isabelle et al., 2010; Lee, Su, & Ong, 2004). Leafy vegetables are nutrients dense sources. They possess antioxidant activity and thus have the potential to be used as cheap natural sources for reducing cellular oxidative damage and reduce degenerative conditions such as cardiovascular diseases and cancers. The consumption of several leafy vegetable are encouraged enough to fulfill nutrient especially in developing countries (Uusiku et al., 2010). Investigation of nutritional value of plants are essential especially to develop strategies to promote the utilization, cultivation and commercialization on these sources of nutrients which could be promoted a new source and other developing countries to assist in promoting biodiversity and combating malnutrition (Schönfeldt & Pretorius, 2011; Uusiku et al., 2010).
The delightful flavor characteristic from pandan leaves, which is well-known throughout the world as an important component in Asian cookery, has made the industrial production of both natural extracts and artificial flavorings containing green food colors for use as food additives in Southeast Asian countries enlarge during the past two decades (Wongpornchai, 2004). Pandan leaves which is known as one aromatic plants has been used in several Southeast Asia countries to confer aroma and flavors in several traditional food. Application of pandan leaves flavor have been used in rice, where rice-starch coating containing natural pandan extract produced non-aromatic rice with aroma compounds similar to that of aromatic rice (Laohakunjit & Kerdchoechuen, 2007). Supercritical carbon dioxide extraction from pandan leaves also have been investigated as a novel applications in food flavorings (Bhattacharjee, Kshirsagar, & Singhal, 2005; Laohakunjit & Noomhorm, 2004).
Nowadays pandan leaves have been investigated also as waste treatment. The performance of extracted pandan leaves was investigated towards treatment of textille wastewater by using flocullation process (Ngadi, N. , Yusoff, 2013). This give such a promissing to develop several process by using natural source e.g. pandan leaves for several purposes.
The color of food is perhaps the first attribute that consumers assess when determining the quality and appearance of a product, and therefore conditions its acceptability. Color becomes a measure of quality and also an indication of deterioration. More than 700 naturally occurring carotenoids have been identified (Britton et al., 2004). Carotenoids are widely distributed whereas C40 isoprenoid pigments with polyene chains contain up to 15 conjugated double bonds. They furnish flowers and fruits with distinct colors (e.g., yellow, orange, and red), which can attract pollinators
In addition carotenoids play important roles in photosynthesis, light harvesting, and prevention of photooxidative damage (Britton et al., 2004). Carotenoids can be classified as carotenes (oxygen-free; e.g β-carotene) and xanthophylls (oxygen-containing; e.g. lutein, zeaxanthin, neoxanthin, violaxanthin, and antheraxanthin (Fig. 2).
Fig. 2. Chemical structures of selected carotenoids
The polyene chain of carotenoids is responsible for the color of plants and fruits. The length of the chromophore influences the color, for example from the colorless phytoene, via the orange color of β-carotene to the red of capsaxanthin (due to the increasing number of double bonds). Besides the color, the polyene chain is responsible for the instability against several environmental factors e.g. oxidation, heat and light or oxidizing chemical (Britton et al., 2004).
Carotenoid pigments are group of bioactive compounds that are of interest to the food scientists, nutritionists and food industries due to their positive impact on human health and their economic benefits. Carotenoids are responsible for the attractive color of most fruit and vegetables, having diverse biological functions and activities. An extensive number of factors determine the efficient incorporation of these phytochemicals from the diet In particular, an interest in increasing the consumption of carotenoids has been evident since the health effect of carotenoids, e.g. β-carotene consumption reduces the incidence of some types of cancer, and further evidences were obtained in subsequent studies (Britton et al., 2004).
In animals, carotenoid pigments have several important biological activities from nutritional and physiological standpoints. Animals and humans cannot synthesize carotenoids de novo although they can metabolize some of them into vitamin A (retinol). Approximately 10% of carotenoids meet the main structural requirement for acting as vitamin A precursors, i.e., contain a β-type non-substituted ring, being β-carotene and β-cryptoxanthin the most representatives (Fernández-García et al., 2012; Rodriguez-Amaya, 2010). The extensive presence and distribution of carotenoids in nature, where mainly are found in fruits and vegetables (foods that occupy or should occupy an important place in our diet), make carotenoids with provitamin A activity the most important source of retinol. Some groups of people, the vegetarians, even depend almost exclusively on fruits and vegetables as a source of retinol in the form of its precursors. In mammals, therefore, the unique and important biological function of carotenoids with retinol equivalence is their role as vitamin A precursors, which is necessary for vision, growth, cell differentiation, and other physiological processes (Fernández-García et al., 2012).
Data published in the study “Global prevalence of vitamin A deficiency in populations at risk 1995–2005” published by the World Health Organisation in 2009, indicate that 190 million preschool-age children and 19.1 million pregnant women had levels of serum retinol less than 0.7 μmol/L, which is the lower limit of normal, and below which is considered a state of vitamin A deficiency. The deficient population is distributed in countries whose gross domestic product (GDP) is less than US$15,000 and in those with 92% of the world’s population (WHO, 2005). Fortification in several foods is one alternative for reducing the vitamin A deficiency (VAD)
Unfortunately, in developing countries e.g. in Indonesia potential knowledge to find indigenous plant resources to fulfill provitamin A requirement as essential nutrition have not established enough. Vitamin A deficiency (VAD) is one of major public health concern in Indonesia. Lack of intake of Vitamin A can cause this VAD and other degenerative disease (Fernández-García et al., 2012). Several biochemical studies have proved that intake of sufficient carotenoids may give a protective effects from several diseases e.g. cancer, cardiovascular disease, cataracs, etc (Meléndez-Martínez, Vicario, & Heredia, 2007).
In Indonesia, several program have been developed to give sufficient intake of pro vitamin A e.g. fortification in several in foods, supplementation and diversification of food which mean finding a new potential provitamin A source (S. G. Berger, de Pee, Bloem, Halati, & Semba, 2007; de Pee, West, Muhilal, Karyadi, & Hautvast, 1995; Muslimatun et al., 2001; Robert & Karyadi, 1988; Wieringa et al., 2003). The vitamin A capsule distribution program in Indonesia was more widely expanded in the 1980s to overcome VAD. Indonesia has one of the strongest vitamin A capsule distribution program for child survival and the intended coverage is for all infants 6-12 months and all preschool children 12-59 month of age. Universal periodic vitamin A supplementation is known as an effective intervention to increase child survival in Indonesia as one of developing country (S. G. Berger et al., 2007). Giving vitamin A to children with measles, serious malnutrition, diarrhea, or other illnesses protects against death and blindness. Besides supplementation, another effort to overcome VAD is fortification. Fortification of foods commonly consumed by children is a viable strategy in developing countries. Margarine, dairy products, sugar, wheat flour, and monosodium glutamate (MSG) have been fortified with vitamin A in different countries. Finally, diversification of vitamin A rich food or provitamin A rich foods is another approach to overcome VAD (Pollard & Favin, 1997).
Learning from several developed countries, food fortification program has proven an effective and low-cost way to increase the micronutrient supply and reduce the consequences of micronutrient deficiencies. It has been rarely used in the developing world, but general conclusions can be drawn. The biological efficacy, but not the effectiveness, of fortifying oil and hydrogenated oil products as well as cereal flours and meals with vitamin A has been shown. Sugar has been fortified with vitamin A in Central American countries for years, and biological efficacy and program effectiveness are well established. Efficacy of fortifying monosodium glutamate with vitamin A was demonstrated but a program has not been established (Dary & Mora, 2002).
Fortification with vitamin A in the developing world should satisfy certain elements for success. Firstly, a potential food matrix a food regularly consumed, produced by a few centralized factories, without sensorial changes compared with the nonfortified equivalent, and nutrient remains bioavailable and in a sufficient amount) is required. Second, fortified foods should provide at least 15% of the recommended daily intakes for the target group (e.g., individuals consuming the lowest amount of the fortified food). Third, voluntary fortification of processed foods should be regulated to prevent excessive consumption of vitamin A. Forth, the neighboring countries should harmonize technical standards, facilitate compliance and minimize conflicts over global trade laws. Fifth, a practical monitoring system should be instituted. Six, Social marketing activities should be permanent and aimed at industry, government and consumers. Seven, food fortification should be combined with other strategies (e.g., supplementation) to reach those not adequately covered by fortification alone. Infants and small children, whose dietary habits differ from those of adults, require special attention. Fortification of food commodities is a very attractive and economic way to prevent and control vitamin A deficiency. Effective food fortification might make supplementation of postpartum women and older children unnecessary (Dary & Mora, 2002).
Degradation of carotenoids yield to apocarotenoids which can exhibit powerful aroma properties (Winterhalter & Rouseff, 2002). Examples of volatile breakdown products of carotenoids are compounds with 13, 11, 10 or 9 carbon atoms, and the terminal group of their carotenoid parent as illustrated in Fig.3.
Fig. 3. (a) Formation of (i) 2,2,6-trimethylcyclohexene-1-one, (ii) β-cyclocitral, (iii) dihydroactinidiolide/ DHA and (iv) β-ionone from β-carotene; (b) Chemical structures of carotenoid derived aroma compounds with the megastigma structure
The C13 compounds are the most abundant carotenoid derived aroma components in nature. They can be divided into: (1) compounds with the megastigmane structure, including the families of ionones and damascones with oxygen at C9 position in ionones or at C7 as in β -damascenone and (2) compounds with the megastigmane structure without oxygen in the lateral chain, e.g. megastigma-4,6,8-triene (Winterhalter and Rouseff, 2001). 2,2,6-Trimethylcyclohexen-1-one, β-cyclocitral and dihydroactinidiolide (DHA) are examples of C9, C10, C11 norisoprenoids, respectively (Winterhalter and Rouseff, 2001). Carotenoid derived aroma compounds are wide spread in nature where they occur in: (1) leaf products e.g. tea and tabbacco; (2) fruits e.g. grapes, starfruit, quince, and citrus fruits; (3) vegetables e.g. spinach, tomato, melon; (4) spices e.g. saffron, red pepper, and also in essential oils e.g. Osmanthus fragrans, Boronia megastigma, Rosa damascena (Winterhalter & Rouseff, 2002).
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Several carotenoid derived aroma compounds are extremely powerful, e.g. the fruity signature of β-ionone is recognizable even at concentrations as low as 0.007 ppm, and the rose and raspberry-like aroma of β-damascenone is recognizable at even lower concentrations of 0.002 ppm (Winterhalter & Rouseff, 2002). Volatiles in plants can be beneficial for humans. Recently, damascenone as one of norisoprenoids and related compounds were identified as potential cancer prevention phytochemicals. It was found that these compounds can both up-regulate the phase 2 cytoprotective enzymes and inhibit the induction of pro-inflammatory enzymes (Gerhäuser et al., 2009). The damascones and related species showed significantly higher activities than ionones and their derived compounds. Besides damascenone, β-ionone has been shown to hold potent anti-proliferative and apoptosis induction properties in vitro and in vivo (J.-R. Liu et al., 2004). These results showed that the enzymatic reaction products of carotenoids have a good positive effect for human health that very promising for future application.
HS-SPME for Flavor Analysis
One of the primary goals in flavor research is to identify several flavor constituent in various sources (Linskens, 1996). The characterization of aroma compounds from natural sources is still a challenge despite the sophisticated techniques now available (Roe, 2005). Flavor components are usually present in a very low concentration (ppm or ppb). In addition, they have a wide range of polarity, solubility, volatility, and thermal and pH stability. The sources may be very complex and cause interference with the isolation techniques. Therefore, there is no single and simple method for the identification of aroma compounds from several natural sources (Roe, 2005).
In order to study the flavor, it is first necessary to isolate volatiles from the complex non volatiles material. There are several methods for analysis of volatile constituent in plants and always have been developed from time to time for their efficiency and reproducibility. One of the other popular methods for analysis of volatile constituents in plants are headspace sampling techniques. Headspace sampling is probably the easiest way to capture and detect aroma compounds, since they exist in the space above the sample (Roe, 2005). It is simple and convenient and it has been used for all kinds of materials. It is especially useful for several sources that give of a lot of odor such as flowers and fruit. For samples that do not have odors, gentle heating can be accepted to help the release of volatiles. Due to the fact that these techniques detect highly volatiles compound, these techniques can be used to help to identify compounds that may be hidden in solvent peaks in liquid extracts. The advantages examples are: (1) simple and quick; (2) solventless technique; (3) low amount of sample; (4) no artifacts are formed and no contaminants introduced (Roe, 2005). Some disadvantages of these techniques as examples are: (1) relative concentration of component in headspace does not reflect the concentration in the sample due to the difference in volatility of aroma compound. This methods can be classified to: (1) static headspace sampling where the sample is put into a sealed headspace vial and left to equal and atmosphere above the sample and (2) dynamic headspace where method the volatiles above the sample are swept away by carrier gas, onto a trap such as TENAX (Roe, 2005). The headspace volatiles are purged by air or nitrogen and are trapped by adsorption on porous polymer traps. Various trapping materials have been used such as charcoal, the Porapak series, the Chromosorb series, and Tenax. In a second step the volatiles are recovered by solvent or heat desorption (Linskens, 1996).
Sorptive techniques allow rapid and solvent less extraction and pre-concentration of aroma compounds. They are based on the partitioning of organic components between aqueous or vapour phase and thin polymeric films (Roe, 2005). This technique group includes SPME (Solid Phase Microextraction), HSSE (Head Space Sorptive Extraction) and SBSE (Stirrer Bar Sorptive Extraction). SPME has been widely used a fused silica fibre coat with polymer film to collect the volatiles from the sample. In the mean time range of polar, non-polar and mixed fibers are available in the market. The fibre is inserted within a needle which is placed into a SPME holder for sampling and desorbing purposes. The sample is placed in a SPME vial then sealed by a septum cap.
Mechanism for Enzymatic Formation of Norisoprenoids
Carotenoid derived aroma compounds can be formed via an enzymatic or chemical degradation. The primary oxidative unspecific cleavage can be initiated by peroxides, photo-oxidation, or by thermal degradation (Winterhalter & Rouseff, 2002). The specific enzymatic degradation of carotenoids is catalyzed by CCDs (Carotenoid Cleavage Dioxygenases) and leads to the production of particular carotenoid derived aroma that are more environmental friendly which is suitable to the green technology approach. CCDs have the capability to cleave a broad spectrum of carotenoids, leading to the production of carotenoid derived aroma compounds e.g. tomato, maize, rose (Huang, Horváth, et al., 2009; Simkin, Schwartz, Auldridge, Taylor, & Klee, 2004; Vallabhaneni, Bradbury, & Wurtzel, 2010) In rose, CCD has the potential to cleave different substrates specifically at 9,10 (9`-10`) double bonds (Fig. 6) (Huang et al., 2009).
Fig.4. Cleavage sites and volatile reaction products of recombinant RdCCD1 enzymes from Rosa damascena
The aim of this research is to investigate the carotenoid profiles in pandan leaves, the flavor compounds which is derived from carotenoids and the mechanism of flavor compounds from carotenoids in pandan leaves. The results from this research could be useful for studying the chemical and biochemical characteristics of flavor formation from carotenoids in model plant e.g. pandan leaves. In detail the objectives of the research are explained point by point bellow :
- Characteristic of carotenoids in pandan leaves by RP-HPLC (Reversed Phase High Performance Liquid Chromatography)
- Characteristic of flavor profile in pandan leaves by HS-SPME GC-MS (Headspace Solid Phase Microextraction Gas Chromatography Mass Spectrophotometry)
- Carotenoid Cleavage Activities by crude enzymes from Pandan Leaves including the characterization of enzyme activity in different carotenoid substrates, optimum pH and optimum temperature.
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