Tài liệu Advances in the extraction and preservation of anthocyanin from vegetables a review

School of Bioscience Master Science of Crop Biotechnology Research Project 3: Crop Biotechnology D24CB3 Dissertation “Advances In The Extraction And Preservation Of Anthocyanin From Vegetables: A Review” By Student name: Nguyen Di Khanh Student ID: 010162 Supervisor: Dr. Yin Sze Lim Date: 13th August, 2012 1 Abstract Nowadays, natural colourants have high demand for use in food industry rather than synthetic colourants which might cause adverse human health effects. Since the mid-1970s, anthocyanin extracted from fruits and vegetables have been found to be a great natural colorant. Anthocyanin pigments are used in chewing gum, yogurts, candies, jams, beverages, fruit preparation and confectionery. Depending on pH values, they account for colour of the plant leaves, flowers and fruits with red, pink, violet and blue. Besides giving colour to plants, anthocyanins also have antioxidant and antihyperglycemic properties; hence, they are used as therapeutic source for many treatments of diabetes, coronary heart disease and cancer. Many anthocyanin extraction methods such as conventional acidified water (CAW), ultrasound, microwave pre-treatment, supercritical fluid extraction and pulsed electric field (PEF) have been proposed by researchers and discussed in this review. Several drawbacks of the methods were reported such as time consuming, insufficient extraction rate, degradation of anthocyanin due to high temperature use, hydrolysis of anthocyanin by using acidified organic solvents; and no single extraction method could be applied for all plants. From the literature, ultrasound and microwave-assisted extraction are two putative methods for extraction of anthocyanins from vegetables. They have significant advantages such as cheap, easy to be manipulated, suitable for laboratory, domestic and large-scale industrial applications, less time- consuming, matrix independent, free sample particle size, less solvent used and long-term preservation. Importantly, with those properties, they help enhance the yield of anthocyanin and also suitable for application of most vegetables from nature. Furthermore, two putative methods could serve as a sound base for future large scale production of anthocyanin with high efficient and fast rate by further investigations, modifiers and optimizations. Apart from it, high pressure processing strengthen for the extracted anthocyanins with long-term preservation and industrial uses compared with the conventional preservation process, thermal processing. Generally, anthocyanins deserve to be deeply investigated for future use as natural colorant in the future. i Declaration I hereby declare that this thesis is, except where otherwise stated, entirely my own work and that it have not been submitted as a dissertation for a master degree at any other university. ---------------------------------August 13, 2012 Nguyen Di Khanh ii Acknowledgement After months of hard works in completing this research project, it finally comes to a day of expressing our gratitude to a number of people. First of all, I would like to thank my supervisor, Dr. Yin Sze Lim, lecturer in Nutrition research in School of Bioscience for guiding me and forinvaluable inputs to my research. I had some difficulties in doing this task, but she taught me patiently until I knew what to do. She have tried and tried to teach me until I understand what I supposed to do with the project work. Moreover, she helped me a lot with English consultation and grammar correction for my improved thesis write-up. Internet, books, computers and all that as my source to complete this project, they also supported me and encouraged me to complete this task so that I will not procrastinate in doing it. I thank to my family and friends for their encouragement and patience when I was doing this project. Without their support, I could not have done so much Last but not least, would also like to thank the University of Nottingham Malaysia Campus for giving a chance to conduct this project. From this project, I would able to gain more knowledge for my future in the bioscience world. iii Table of Contents Abstract .................................................................................................................................... i Declaration .............................................................................................................................. ii Acknowledgement ................................................................................................................. iii List of figures ......................................................................................................................... vii List of tables ........................................................................................................................... ix CHAPTER 1: INTRODUCTION ................................................................................................... 1 1.1. Research context ..................................................................................................... 1 1.2. Main objectives ....................................................................................................... 3 CHAPTER 2: BACKGROUND ..................................................................................................... 4 2.1. Phenolic compounds in vegetables ............................................................................. 4 2.1.1. Introduction ......................................................................................................... 4 2.1.2. Chemical properties of phenolic compounds from vegetables .......................... 4 2.1.3. Flavonoids............................................................................................................ 5 2.1.4. Other classes of phenolic compounds ................................................................ 6 2.1.4.1. Phenolic acid................................................................................................ 6 2.1.4.2. Tannins ........................................................................................................ 7 2.1.4.3. Stilbenes ...................................................................................................... 8 2.1.4.4. Lignans ......................................................................................................... 9 2.1.5. Synthesis and metabolic processes of phenolic compounds .............................. 9 2.1.6. Phenolic compounds in vegetables and their health-promoting properties .... 11 2.2. Anthocyanins from vegetables .................................................................................. 13 2.2.1. Introduction ....................................................................................................... 13 2.2.2. Vegetables – a great sources of anthocyanins .................................................. 13 2.2.3. Biosynthesis pathways of anthocyanins ............................................................ 13 2.2.4. Anthocyanins chemical properties and functions in nature ............................. 15 2.2.5. Anthocyanins stability ....................................................................................... 18 2.2.6. Anthocyanins biological properties ................................................................... 21 2.2.6.1. Antioxidant activities ................................................................................. 21 2.2.6.2. Other biological properties ....................................................................... 21 2.2.6.3. Anthocyanins and human health effects .................................................. 22 iv 2.2.7. Anthocyanins as natural colorants .................................................................... 23 2.2.8. Extraction and preservation processes of anthocyanins from vegetables ....... 24 CHAPTER 3: METHODOLOGY................................................................................................. 28 3.1. Searching method ................................................................................................. 28 3.2. Referencing ........................................................................................................... 28 CHAPTER 4: RESULTS AND DISCUSSIONS .............................................................................. 29 4.1. Methods for extraction of anthocyanins from vegetables ....................................... 29 4.1.1. Conventional extraction method - Soxhlet technique ...................................... 29 4.1.1.1. Introduction ............................................................................................... 29 4.1.1.2. Principles and mechanisms ....................................................................... 29 4.1.1.3. Practical design .......................................................................................... 30 4.1.1.4. Advantages and disadvantages ................................................................. 32 4.1.1.5. Potential applications ................................................................................ 32 4.1.2. Ultrasound-assisted extraction ......................................................................... 33 4.1.2.1. Introduction ............................................................................................... 33 4.1.2.2. Principles and mechanisms ....................................................................... 34 4.1.2.3. Practical design .......................................................................................... 35 4.1.2.4. Advantages and disadvantages ................................................................. 36 4.1.2.5. Potential applications ................................................................................ 38 4.1.3. Microwave-assisted extraction ......................................................................... 38 4.1.3.1. Introduction ............................................................................................... 38 4.1.3.2. Principles and mechanism ......................................................................... 39 4.1.3.3. Practical design .......................................................................................... 40 4.1.3.4. Advantages and disadvantages ................................................................. 41 4.1.3.5. Recent applications ................................................................................... 42 4.1.4. Supercritical fluid extraction ............................................................................. 42 4.1.4.1. Introduction ............................................................................................... 42 4.1.4.2. Principles and mechanism ......................................................................... 42 4.1.4.3. Practical design .......................................................................................... 43 4.1.4.4. Advantages and disadvantages ................................................................. 44 4.1.4.5. Potential applications ................................................................................ 45 4.1.5. Accelerated solvent extraction.......................................................................... 45 4.1.5.1. Introduction ............................................................................................... 45 4.1.5.2. Principles and mechanism ......................................................................... 45 v 4.1.5.3. Advantages and disadvantages ................................................................. 46 4.1.5.4. Potential applications ................................................................................ 47 4.1.6. 4.1.6.1. Introduction ............................................................................................... 47 4.1.6.2. Principles and mechanism ......................................................................... 47 4.1.6.3. Practical design .......................................................................................... 48 4.1.6.4. Advantages and disadvantages ................................................................. 48 4.1.6.5. Recent applications ................................................................................... 49 4.1.7. 4.2. Pulse electric field extraction ............................................................................ 47 Comparisons among methods for extraction of anthocyanins from vegetables 49 Preservation processes for extracted anthocyanins ................................................. 52 4.3.1. Introduction ....................................................................................................... 52 4.2.2. Thermal processing ........................................................................................... 52 4.2.2.1. Introduction ............................................................................................... 52 4.2.2.2. Principles of thermal processing ............................................................... 53 4.2.2.3. Advantages and disadvantages ................................................................. 53 4.2.3. High pressure processing .................................................................................. 54 4.2.3.1. Introduction ............................................................................................... 54 4.2.3.2. Principles and mechanism ......................................................................... 54 4.2.3.3. Advantages and disadvantages ................................................................. 54 4.2.4. Comparison between the thermal and high pressure processing .................... 54 4.2.5. Novel alternative techniques for preservation processes ................................ 55 CHAPTER 5: CONCLUSION ..................................................................................................... 56 List of references ................................................................................................................... 57 vi List of figures Figure 1.1: Chemical structure of acylated anthocyanins .................................. 2 Figure 2.1: Chemical structure of subclasses of flavonoids ................................ 6 Figure 2.2: Two sub-groups of phenolic acids, hydroxybenzoic and hydrocinnamic acids ................................................................................................................. 7 Figure 2.3: Chemical structure of two sub-groups of tannins - pro- anthocyanidins and gallotannins .................................................................... 7 Figure 2.4:Chemical structure of stibenes....................................................... 8 Figure 2.5: Chemical structure of Lignans ...................................................... 9 Figure 2.6: Biosynthesis pathways of anthocyanins ......................................... 14 Figure 2.7: General structure of anthocyanins................................................. 15 Figure 2.8: (a) General structure of the six common anthocyanidins;(b) Classification of anthocyanins with six common sub-groups .............................. 17 Figure 2.9: Thermal degradation of anthocyanins ............................................ 19 Figure 2.10: Chemical structures of anthocyanins in corresponding to pH values and the degradation reaction ........................................................................ 20 Figure 2.11: Mechanism of anthocyanins in preventing cancer .......................... 22 Figure 2.12: Steps involved in the whole extraction process of anthoyanins from vegetables ................................................................................................. 25 Figure 2.13: Two commonly used preserved processes: (a) thermal processing, (b) high pressure processing ........................................................................ 26 Figure 4.1: Schematically experimental apparatus of Soxhlet extraction technique: (a) laboratory Soxhlet extractor, (b) schematic diagram of Soxhlet extraction apparatus .................................................................................... 29 Figure 4.2: Two common ultrasound-assisted extraction apparatuses: (a) Ultrsound bath, (b) Ultrasound probe............................................................. 33 vii Figure 4.3: Laboratory and schematic illustration diagram of 3 litter volume ultrasound-assisted extractor........................................................................ 34 Figure 4.4: Industrial ultrasonic equipment with three different volumes 50L, 500L and 1000L .......................................................................................... 37 Figure 4.5: Schematic diagram of microwave-assisted extraction system ........... 38 Figure 4.6: Schematic diagram of supercritical fluid extractor ........................... 42 Figure 4.7: Schematic diagram of Accelerated solvent extraction ...................... 45 Figure 4.8: Sketch of the pulsed electric field treatment chamber...................... 47 Figure 4.9: Summery of the determination of anthocyanins including the extraction techniques have been used recently ............................................... 49 viii List of tables Table 2.1: Flavonoid group, their sub-groups, chemical characteristics together with some typical rich food sources ............................................................ 5 Table 2.2: Fruits and vegetable – great sources of phenolics ....................... 12 Table 2.3: Names and abbreviations of common varieties of anthocyanins .... 16 Table 2.4: Summary of functions of anthocyanins in some plants in nature ... 18 Table 2.5: Fruits and vegetables – common sources of anthocyanins with different indicated concentrations ............................................................. 24 Table 4.1: Advantages of ultrasound-assisted extraction technique compared with other techniques: MAE (microwave-assisted extraction), SFE (supercritical fluid extraction) and ASE (accelerated-solvent extraction) ........................... 36 Table 4.2: Summary of comparisons of the characteristics of the methods have been used for extraction of anthocyanins from vegetables…………………………………………………………………………………………………………….50 ix CHAPTER 1: INTRODUCTION 1.1. Research context Nowadays, natural colorants have high demand for use in food industry rather than synthetic colorants which might cause adverse human health effects (Zarena and Sankar, 2012). The neurological and behavioural effects caused by the synthetic dyes used in food industry are adverse to human health (Lu et al., 2010). Therefore, anthocyanins, with their high potential in terms of high stability, high colorant power and low cost, have been considered as a great candidate for this requirement of new sources of pigments. Since the mid-1970s, anthocyanins have been extracted from fruits, vegetables, cereals and flowers of a great variety of plants. Those have been comprehensively evaluated and accessed as potential sources for the extraction of anthocyanins. Depending on pH values, they account for colour of the plant leaves, flowers and fruits with red, pink, violet, blue and green (Lu et al., 2010; Naczk et al., 2011). The word anthocyanins came from Greek, in which anthos means flower and kyanos means blue. Anthocyanins, belong to the flavonoid family, are a group of phenolic compound that can be soluble in water. Chemically, they are glycosides of polymethoxy and polyhydroxy derivatives of flavylium or 2-phenylbenzopyrylium salts (Xu et al., 2010; Lu et al., 2010). Besides giving responsibility of colouration with bright colour to plants, anthocyanins also have antioxidant and antihyperglycemic properties (Arapitsas et al., 2008; Lu et al., 2010; Zarena and Sankar, 2012); hence, they are investigated as therapeutic source for many treatments of diabetes, coronary heart disease and cancer, preventing the process of aging (Arapitsas et al., 2008). Their antioxidant properties give health promoting benefits and protect against various oxidants by diverse actions on various enzymes and metabolic processes (Zu et al., 2010; Lu et al., 2010; Zarena and Sankar, 2012). Various health benefits associated with anthocyanins have been well studied such as antioxidant capacity, treatment of various blood circulation disorders based on capillary fragility, conservation of normal vascular porousness, augmentation of sight acuteness, radiation-protective agents, antineoplastic and chemo-protective agents, embarrassment of platelet combination, regulation for diabetes, vaso-protective and anti-inflammatory 1 properties (Zu et al., 2010). For instance, a total of twenty-four types of anthocyanins have been isolated and characterized in red cabbage. Most of them have cyaniding in the form of mono or di-glycoside, and acylated form of anthocyanins (see figure 1.1) (Arapitsas et al., 2008; Zu et al., 2010). Source: Xu et al., 2010. Figure 1.1: Chemical structure of acylated anthocyanins. The first problem associated with the use of anthocyanins in food systems is the method design for extraction process. So far, many anthocyanin extraction methods such as conventional acidified water (Soxhlet extraction), ultrasound, high pressure extraction with CO2 and co-solvents, microwave pretreatment, accelerated fluid extraction and pulsed electric field (PEF) have been proposed by researchers (Vera and Mercadante, 2007; Welch et al., 2008; Arapitsas and Turner, 2008; Gachovska et al., 2010; Ignat et al., 2010; Lu et al., 2010; Xu et al., 2010; Jacob et al., 2011). However, several drawbacks of the methods were reported such as time consuming, insufficient rate, degradation of anthocyanin due to high temperature use, hydrolysis of anthocyanin by using acidified organic solvents; and no single extraction method could be applied for all plants. For instance, in traditional techniques, the solvents such as ethanol, methanol, acetone or water which acidified with 2 hydrochloric acid or sulphure dioxide were commonly used. Moreover, the products extracted need further purification steps (Zarena and Sankar, 2012). Extracted anthocyanins not only need further purification steps but also a suitable preservation process for long-term uses and applications in food industry. Together with thermal processing, which is investigated and used for a long time ago, the high pressure processing is now taken advantaged for the preservation process of anthocyanins extracted from vegetables (Lu et al, 2010; Routray and Orsat, 2012; Idham et al., 2012). Anoptimum putative extraction and preservation method that enhances the yield of anthocyanin suitable for application to most vegetables is extremely essential. Hence, current study was carried out to review all of the methods that have been used for anthocyanins extraction and preservation with comparisons and contrasts to find out the best available method. 1.2. i. Main objectives To investigate methods used for extraction of anthocyanin from vegetables ii. To elucidate advantages and disadvantages of the methods used for the extraction of anthocyanin iii. To investigate the preservation techniques of extracted anthocyanin iv. To propose a putative method applicable for extraction and preservation of anthocyanin for vegetables 3 CHAPTER 2: BACKGROUND 2.1. Phenolic compounds in vegetables 2.1.1. Introduction Phenolic compounds consist of a diverse group of chemical molecules including lignin, lignans, tannins, stilbenes and most remarkably one, flavonoids (Laura et al., 2010). In plant, particularly in fruits and vegetables, more than 10,000 phenolic compounds were found with diverse chemical properties. Some are water-soluble, some are soluble in organic solvents and some are distinctively in-soluble polymers (Jay, 2008; Laura et al., 2010). 2.1.2. Chemical properties of phenolic compounds from vegetables Polyphenols, which are secondary metabolites produced by plants, are the highest antioxidant phenolic compounds antioxidants in human diets (Jay, 2008). One single compound is formed by an aromatic ring binding by some structural elements. They are normally hydroxyl mioties (Jay, 2008). Based on the number of phenol rings and those elements, polyphenols are classified into many sub-groups (Beecher, 2003; Jay, 2008; Laura et al., 2010). Flavonoids and non-flavonoids are two main groups of polyphenols. First, flavonoid group consists of anthocynidins, proanthocyanidins, flavan-3-ols, flavonols, dihydroflavonol, flavones, flavanones and isoflavones. They are chemically C6-C3-C6 struture (see table 2.1). Second, non-flavonoids group consist of simple phenols. secoiridoids, lignans, chalcones, xanthones, benzophenones, coumarins, cinnamic acids, phenylacetic and acetophenones, hydrolysable tannins and benzoic acids (Jay, 2008). This study mainly focuses on anthocyanins thereforethe flavonoid group will be more discussed in details. 4 Table2.1: Flavonoid group, their sub-groups, chemical characteristics together with some typical rich food sources. Source: Jay, 2008. 2.1.3. Flavonoids Flavonoids are glycosides formed from several small number of flavonoid aglycones (Jay, 2008; Huang and Cai, 2010; Laura et al., 2010). The chemical structu res of subclasses of flovanoids are introduced in figure 2.1. They are most water-soluble and can be found in the vacuoles of plant cells. Within plants, these compounds function as pigments which have chemically defence ability against attacked microorganisms as well as some particular insects (Jay, 2008). Moreover, flavonoids are also involved in some other biological interactions in plants (Laura et al., 2010). Nevertheless, the active researches recently which are mainly focusing on the antioxidant activities of flavonoids are their possiblehuman healtheffects (Huang and Cai, 2010). It is stated that highflavonoid consumption in human diets could contribute to mitigation in risks of some typical diseases and certain cancers (Liu, 2004; Huang and Cai, 2010; Laura et al., 2010). Particularly, fruits and vegetables are the great potential in such aspect. 5 Source: Huang and Cai, 2010. Figure 2.1: Chemical structure of subclasses of flavonoids. 2.1.4. Other classes of phenolic compounds 2.1.4.1. Phenolic acids Phenolic acids account for about one-third of the dietary phenols (Ignat et al., 2011). They can be found with free or bound forms within plants.Generally, the phenolic acids with bound forms are linked to some other components due to their acetal bonds and through ester and ether (Robbins, 2003; Zadernowski et al., 2009).Depend on certain extraction conditions and certain susceptibilities to degradation, phenolic acids will express in different forms (Ross et al., 2009; Ignat et al., 2011). There are two main sub-groups of phenolic acids (see figure 2.2), the hydroxycinnamic acids and the hydroxybenzoic acids (Ignat et al., 2011). 6 Source: Ignat et al., 2011. Figure 2.2: Two sub-groups of phenolic acids, hydroxybenzoic and hydrocinnamic acids. The hydroxycinnamic acids commonly have C6-C3 structure and consist of some representatives such as sinapic, p-coumaric, ferulic and caffeic acids. On the other hand, the hydroxybenzoic acids with C6-C1 structure consist of syringic, vanillic, protocatechuic, p-hydroxybenzoic and gallic acids (Ignat et al., 2011). 2.1.4.2. Tannins Tannins, which are high molecular compounds, constitute an important role in phenolic groups (Scalbert, 1991; Ignat et al., 2011). They consist of two sub-groups, pro-anthocyanidins (or condensed tannins) and gallotannins tannins (or hydrolysable tannins) (see figure 2.3). Although the tannins show their potential in functioning as biological antioxidants, protein precipitating agents and metal ion chelators, their biological activities are difficult to be predicted within one particular biological system (Ignat et al., 2011). The reasons explain for that is because of their enormous structural variation as well as their varied biological roles. Future work needs to study the relationships between structure and their activity inorder to predict the biological activities of tannins in any system (Scalbert, 1991; Ignat et al., 2011). 7 Source: Ignat et al., 2011. Figure 2.3: Chemical structure of two sub-groups of tannins - pro- anthocyanidins and gallotannins. 2.1.4.3. Stilbenes Stilbenes are present in human diet with very low quantities (Bavaresco, 2003). Resveratrol is the main representative of stilbenes, which is regularly in glycosylated forms (see figure 2.4). Resveratrol exists in both cis and trans isomeric forms (Bavaresco, 2003; Ignat et al., 2011). When the plants are infected by pathogens or are under some of stress conditions, they will produce stilbene compounds in response to those stresses. More than 70 plant species including peanuts, berries and grapes have been detected to exhibit that kind of reaction (Bavaresco, 2003; Delmas et al., 2006; Ignat et al., 2011). Source: Ignat et al., 2011. Figure 2.4: Chemical structure of stibenes. 8 2.1.4.4. Lignans Lignans are present in plants mainly as an aglycone (see figure 2.5). The glycoside derivatives of lignans in contrast, are mostly in minor forms. Lignans are normally constituted from oxidative dimerization of two phenyl-propane units (Ignat et al., 2011). Many efforts have been made to specialized studies of lignans as well as their synthetic derivatives due to their potential applications in various pharmacological capabilities and cancer chemo-therapy (Saleem et al., 2005; Ignat et al., 2011). Source: Ignat et al., 2011. Figure 2.5: Chemical structure of Lignans. 2.1.5. Synthesis and metabolic processes of phenolic compounds In plant metabolism, there are two different pathways named primary and secondary (Liu, 2004; Laura et al., 2010). Both of them can be found in all cells and in specialized cells, respectively. The primary pathways manipulate a group of basic compounds; while in the secondary pathways, a wide variety of unique compounds will be produced of this kind of metabolic mechanism (Laura et al., 2010; Ignat et al., 2011). In the primary pathways, the metabolism of nucleic acids, proteins, lipids and carbohydrates are taken under the help of various reactions such as the biosynthesis of nucleic, protein and lipid, the pentose phosphate shunt, the tricarboxylic acid cycles, and most importantly, glycolysis (Saleem et al., 2005). Differ from those metabolites, the products of secondary pathways which include coumarins, flavonoids, lignin, alkaloids, terpenes, phenylpropanoids and some other related compounds, are chemically produced by some specific 9 pathways such as the methylerythritol phosphate pathway or mevalonic acid pathway (Laura et al., 2010). The synthesis of phenolic compounds in plant can proceed due to various pathways and it leads to the diversity of metabolic sub-groups (Ignat et al., 2011). The roles of each phenolic compound in plant also varied due to their diversity (Liu, 2004; Laura et al., 2010). For instance, some play roles in protecting plants against excessive water loss and/or harmful ultraviolet solar radiation, whereas some are responsible for mechanical supports (Ignat et al., 2011). In addition, some of these compounds fascinate seed dispersers as well as pollinators for the plants (Laura et al., 2010). Some also can serve as signal molecules important for abiotic and biotic stress defense mechanisms (Liu, 2004). Moreover, some join in function of competition of the plant within the living conditions with others. Products of secondary metabolites usually present in plants with high amounts. Second to cellulose, phenolic compounds contribute about more than 40% of organic matter assemblage to the biosphere (Liu, 2004; Laura et al., 2010). The induced synthesis of phenolic compounds generally facilitate in growth and development of plants, especially vegetables (Laura et al., 2010; Ignat et al., 2011). However, the innate capacity, which helps the plants more adapted to natural environment by responding to various abiotic and biotic stresses, may induce metabolic responses. It can result in reduction of product quality (Laura et al., 2010). For instance, jasmonic and salicylic acid are products of lipid and phenolic metabolism, which has resulted from the responsibility of the plants to a multitude of stresses. Actually, when the plants respond to volatile stresses, some induced hormones such as jasmonic acid and ethylene can possess positive effect to the synthesis and accumulation of phenolic compounds up to high levels. In addition to that aspect of signalling pathway, plant hormones such as ethylene and abscisic acid have also been induced (Ignat et al., 2011). The excessive accumulation and/or over-production of constitutive synthesis of some particular phenolic compounds can cause reduction in product quality (Laura et al., 2010). Therefore, to improve the quality of food production using formulate techniques and management of cultural procedures, deeply insights in the synthesis and metabolism of phenolic compounds are extremely important. The main target is reducing the phenolic metabolism and its effect in product quality (Giovannucci et al., 2003). 10