**3.1. Apple pomace**

Traditional stabilization technology takes into account that apple pomace is an agroindustrial residue with a view to the fact that its disposal creates environmental problems of high cost. However, apple pomace is an interesting material that has attracted considerable attention as a potential source of dietary sugar, fiber, phenolic compounds and pectin.

These products can be used for many purposes in the pharmaceutical, cosmetics and food industries. The commercial production of apples in Brazil, based on only two cultivars, was designed to meet highly demanding customer requirements both with respect to quality and the retail prices of table fruit, and more recently the industrial product, either as juice or in a fermented form. Industrial apple pomace has in its composition all the constituents of the

fruit in varying amounts, more or less, and is the residue from the pressing of the grated mass of unfermented juice, cider, wines, brandies and distillates or vinegars 12.

Characterization of Apple Pectin – A Chromatographic Approach 329

25. Pectin must consist of at least 65% galacturonic

apple pomace in the recovery of native phenolic compounds associated with darkening, for use as antioxidants, resulting in a lighter color of the pectin obtained. These points to the growing trend of industry to find alternatives that promote the 'recycling' of waste, with maximum recovery and greater commercial exploitation of components previously

Pectin refers to a family of polysaccharides and oligosaccharides, which have common characteristics but are extremely diverse in their fine structure. The pectic skeleton is primarily a homopolymer of galacturonic acid bound in α(14), with varying degrees of

acid, according to the FAO (UN Food and Agriculture Organization) and EU (European

Pectin is one of the most important substances found in apples because it provides approximately 10% of daily fiber requirements. Pectin is a soluble fiber that is not absorbed by the intestine, i.e. the fibers are not degraded by digestive juices, but they increase the volume of fecal material, assist in the proper functioning of the intestine, retain water and various residual substances, facilitate the elimination of toxins along with stools, promote the protection of the intestinal mucosa and help in the treatment of diarrhoea. Pectin is also highly recommended for diabetics because it reduces the absorption of glucose. The daily consumption of approximately 2 small apples provides the required dose of pectin. Pectin also assists in reducing bad cholesterol because it forms a fiber barrier in the intestinal wall

In the industrial sector, pectic polysaccharides promote increased viscosity and act as a protective and stabilizing colloid in foods and beverages such as jams and jellies, fruit preparations for yoghurts, concentrated fruit juices and drinks, milk and fruit-based desserts, gelled dairy products, confectionery, and dairy products that are directly acidified or fermented. Other properties include the prevention of flotation in fruit preparations, stability of bread products, protein stabilization, softness in texture, increase in volume and

The first citation relating to pectin is found in an English article from 1750 about the preparation of apple jelly [26]. The process of extracting liquid pectin was recorded in 1908 in Germany and the process spread rapidly to the United States, where a patent was obtained in 1913 by Douglas (U.S. Patent no. 1,082.682) [27]. As for commercial production, in the 1930s Hermann Herbsthreith discovered the potential use and application of apple

The content of pectic substances varies depending on the botanical source of plant material. There are four by-products of the agricultural and food industries that are high in pectic

pomace, a hitherto discarded by-product of the production of fruit juice [28].

considered as by-products.

the control of syneresis 25.

*3.2.1. History of pectin* 

methyl esterified carboxyl groups 24;

preventing the absorption of cholesterol and other fats.

**3.2. Pectin** 

Union) 25.

In Brazil, 70% of apples produced are sold for fresh consumption, while 30% are considered 'industrial' fruit. One third of this fraction is considered to contain fruit of low quality, which must be discarded or used for the production of distillates such as alcohol or vinegar and the remaining two thirds are fruits that can be used for the processing of apple juice 13. On the global scene, in the 1980s, with reference to industrial fruit, 75% of the products consisted of apple juice or must and 25% was apple pomace, when 8 kg of raw material was required to produce the amount of 6 kg of apple juice necessary to make1 kg of concentrated juice. In 1996, the yield had risen by 85% due to the use of enzymes in the preparation of the must. Since that time, the yield has increased slightly depending on the variety of the fruit and the degree of maturity, which means that currently 7 kg of apples are required for 1 kg of concentrate. But it is possible to increase this by 1% with the use of the most up-to-date enzymes [14].

The drying of apple pomace seems to be the most economic technological approach to stabilize the product because it dramatically reduces the volume of water and makes transport cheaper. The yield of dried pomace at 60°C is about 50.0 g kg -1 in 10 hours, or 5% of the raw material. The appearance of the dried pomace is dependent on the adiabatic drying temperature. From 50 to 60°C, darkening enzymatic reactions are stimulated [15], while from 90 to 100°C Maillard reactions occur, with products appearing darker than those obtained in the range of 70 to 80oC. However, if the criterion of stopping the process is the time at which the temperature of the pomace starts to rise, that temperature will never rise to values higher than 52°C and thus the final temperature tends to be homogeneous.

The instability of apple pomace is related to its physical-chemical composition and the presence of some enzymes that are activated after the disintegration of plant tissues [16; 17; 14]. Apple pomace is composed of water (76.3%), soluble solids (23.7%) and is obtained from the epi-mesocarp (95.5%), seeds (4.1%) and stems (1.1 %). It has an average humidity of 80% and 14% of total soluble solids including glucose, fructose and sucrose. Its composition is related to the cultivar and processing [17]. The fiber content varies from 11.6 to 44.5%, and includes cellulose (12.0 to 23.2%), lignin (6.4 to 19.0%), pectin (3.5 to 18%,) and hemicellulose (5.0 to 6.2%). The average dietary fibers (35.8%) and sugars (54.4%) make up 91.2% of pomace and the other components are proteins, lipids and ash [18]. The chromatic characteristics L = 51.8, a = 5.4 and b = 18.2 have been determined in samples of apple pomace [19].

The use of apple pomace as a potential source of nutrients for the production of glucosidase by *Aspergil*l*us foetidus* was suggested by [20]. Ten years later [21] suggested its use for other technological purposes such as the recovery of phenolic compounds. Apple pomace is also recommended for biotechnological applications such as ethanol production [22], flavorings, citric acid, pectin, enzymes and molds for the extraction of dietary fiber and mineral coal [23].

The apple pectin that is derived from the extraction of apple pomace is dark brown in color compared to orange pectin. Studies are being carried out regarding the potential use of apple pomace in the recovery of native phenolic compounds associated with darkening, for use as antioxidants, resulting in a lighter color of the pectin obtained. These points to the growing trend of industry to find alternatives that promote the 'recycling' of waste, with maximum recovery and greater commercial exploitation of components previously considered as by-products.

## **3.2. Pectin**

328 Chromatography – The Most Versatile Method of Chemical Analysis

enzymes [14].

[16; 17;

pomace [19].

fruit in varying amounts, more or less, and is the residue from the pressing of the grated

In Brazil, 70% of apples produced are sold for fresh consumption, while 30% are considered 'industrial' fruit. One third of this fraction is considered to contain fruit of low quality, which must be discarded or used for the production of distillates such as alcohol or vinegar and the remaining two thirds are fruits that can be used for the processing of apple juice 13. On the global scene, in the 1980s, with reference to industrial fruit, 75% of the products consisted of apple juice or must and 25% was apple pomace, when 8 kg of raw material was required to produce the amount of 6 kg of apple juice necessary to make1 kg of concentrated juice. In 1996, the yield had risen by 85% due to the use of enzymes in the preparation of the must. Since that time, the yield has increased slightly depending on the variety of the fruit and the degree of maturity, which means that currently 7 kg of apples are required for 1 kg of concentrate. But it is possible to increase this by 1% with the use of the most up-to-date

The drying of apple pomace seems to be the most economic technological approach to stabilize the product because it dramatically reduces the volume of water and makes transport cheaper. The yield of dried pomace at 60°C is about 50.0 g kg -1 in 10 hours, or 5% of the raw material. The appearance of the dried pomace is dependent on the adiabatic drying temperature. From 50 to 60°C, darkening enzymatic reactions are stimulated [15], while from 90 to 100°C Maillard reactions occur, with products appearing darker than those obtained in the range of 70 to 80oC. However, if the criterion of stopping the process is the time at which the temperature of the pomace starts to rise, that temperature will never rise

to values higher than 52°C and thus the final temperature tends to be homogeneous.

The instability of apple pomace is related to its physical-chemical composition and the presence of some enzymes that are activated after the disintegration of plant tissues

The use of apple pomace as a potential source of nutrients for the production of glucosidase by *Aspergil*l*us foetidus* was suggested by [20]. Ten years later [21] suggested its use for other technological purposes such as the recovery of phenolic compounds. Apple pomace is also recommended for biotechnological applications such as ethanol production [22], flavorings, citric acid, pectin, enzymes and molds for the extraction of dietary fiber and mineral coal [23]. The apple pectin that is derived from the extraction of apple pomace is dark brown in color compared to orange pectin. Studies are being carried out regarding the potential use of

14]. Apple pomace is composed of water (76.3%), soluble solids (23.7%) and is obtained from the epi-mesocarp (95.5%), seeds (4.1%) and stems (1.1 %). It has an average humidity of 80% and 14% of total soluble solids including glucose, fructose and sucrose. Its composition is related to the cultivar and processing [17]. The fiber content varies from 11.6 to 44.5%, and includes cellulose (12.0 to 23.2%), lignin (6.4 to 19.0%), pectin (3.5 to 18%,) and hemicellulose (5.0 to 6.2%). The average dietary fibers (35.8%) and sugars (54.4%) make up 91.2% of pomace and the other components are proteins, lipids and ash [18]. The chromatic characteristics L = 51.8, a = 5.4 and b = 18.2 have been determined in samples of apple

mass of unfermented juice, cider, wines, brandies and distillates or vinegars 12.

Pectin refers to a family of polysaccharides and oligosaccharides, which have common characteristics but are extremely diverse in their fine structure. The pectic skeleton is primarily a homopolymer of galacturonic acid bound in α(14), with varying degrees of methyl esterified carboxyl groups 24; 25. Pectin must consist of at least 65% galacturonic acid, according to the FAO (UN Food and Agriculture Organization) and EU (European Union) 25.

Pectin is one of the most important substances found in apples because it provides approximately 10% of daily fiber requirements. Pectin is a soluble fiber that is not absorbed by the intestine, i.e. the fibers are not degraded by digestive juices, but they increase the volume of fecal material, assist in the proper functioning of the intestine, retain water and various residual substances, facilitate the elimination of toxins along with stools, promote the protection of the intestinal mucosa and help in the treatment of diarrhoea. Pectin is also highly recommended for diabetics because it reduces the absorption of glucose. The daily consumption of approximately 2 small apples provides the required dose of pectin. Pectin also assists in reducing bad cholesterol because it forms a fiber barrier in the intestinal wall preventing the absorption of cholesterol and other fats.

In the industrial sector, pectic polysaccharides promote increased viscosity and act as a protective and stabilizing colloid in foods and beverages such as jams and jellies, fruit preparations for yoghurts, concentrated fruit juices and drinks, milk and fruit-based desserts, gelled dairy products, confectionery, and dairy products that are directly acidified or fermented. Other properties include the prevention of flotation in fruit preparations, stability of bread products, protein stabilization, softness in texture, increase in volume and the control of syneresis 25.

## *3.2.1. History of pectin*

The first citation relating to pectin is found in an English article from 1750 about the preparation of apple jelly [26]. The process of extracting liquid pectin was recorded in 1908 in Germany and the process spread rapidly to the United States, where a patent was obtained in 1913 by Douglas (U.S. Patent no. 1,082.682) [27]. As for commercial production, in the 1930s Hermann Herbsthreith discovered the potential use and application of apple pomace, a hitherto discarded by-product of the production of fruit juice [28].

The content of pectic substances varies depending on the botanical source of plant material. There are four by-products of the agricultural and food industries that are high in pectic

substances (content over 15% on a dry basis): pomace from apples, citric albedo, sugar beet pulp and /sunflower rinds 29.

Characterization of Apple Pectin – A Chromatographic Approach 331

depending on the development, the type and number of simple sugars and oligosaccharides attached to this chain. The reason for this level of variation in RG-I is not known, but it

Rhamnogalacturonan II (RG-II) is the most structurally complex segment and comprises 10% of pectin. This structure, highly conserved in most plant species, consists of a homogalacturonan skeleton of approximately eight (probably more) monomeric units, containing side chains of up to 12 different types of sugars, some highly peculiar such as apiose, aceric acid, Docosahexaenoic acid (DHA) and 3-Deoxy-D-manno-oct-2-ulosonic acid (KDO). RG-II usually exists in cell walls as cross-linked dimers by a borate diol ester apiosil

Xilogalacturonana (XGA) is a homogalacturonan substituted with xylose linked to position 3. The degree of control of xylose may vary between 25% (watermelon) to 75% (apple). This xylose may be additionally substituted in 0-4 in conjunction with another xylose in β, which

Arabinogalactan I (ARA-I) is composed of skeleton β -D-Gal*p*; residues of α-L-Ara*f* may be linked to galactosyl units in position 3. Arabinogalactan II (ARA-II) is primarily associated with proteins (3-8%), also called arabinogalactan-proteins (AGPs). AG-II is composed of a β-D-Gal*p* 13 skeleton, containing short chains [α-L-Ara*f* (16) β-D-Galp (16)]n, where n = 1, 2 or 3. The protein part is rich in proline, hydroxyproline, alanine, serine and threonine [25]. Arabinan (ARA) is composed of an α-L-Ara*ff* skeleton in 15 links, where there may be side chains of α-L-Ara*f* (13) [35]. There is also another chain, not shown schematically, which is apiogalacturonan (API), HG substituted in O-2 or O-3 with D-apio*f* the 2-O-or D-3

The properties and application of pectin are affected by several parameters related to pectin structure, including the composition, presence and distribution of side chains, degree of methyl-esterification (DE), degree of acetylation (DA), molar mass, and the charge distribution along their backbone. Differently from proteins, whose structure is defined in relation to a template, pectin are a mixture of heterogeneous polymers and preparations that contain pectins that have been isolated by chemical and enzymatic treatments likely to cleave covalent bonds. The conditions used for pectin extraction can also release other cell wall polymers. Preparative chromatography can be used to separate homogeneous fractions

Determining the fine structure of pectins is a challenging task which encompasses the determination of molar mass, monosaccharide composition, configuration and ring size of monosacchrides, identification of glycosidic linkages, sequence of glycosyl units in the polymer, determination of DA and DE, as well as the distribution of the acetyl and methyl esters groups along the chain. A combination of chemical, spectroscopic and chromatographic methods is commonly used to fully characterize pectic polysaccharides. In recent years, X-ray diffraction, circular dichroism, light scattering, electron and atomic force

is more prevalent in reproductive tissues such as fruits and seeds [25;33].

apio*f*. It is present in aquatic monocots such as *Lemna* [33].

of pectic polysaccharides from a mixture.

**4. The application of chromatographic techniques** 

suggests a functional diversity.

between units in the side chain [34; 25].

The cell wall, a dynamic compartment of plants, can be divided into two layers called 'primary' and 'secondary'. The primary cell wall can be classified into: [1] type I, mainly composed of cellulose, xyloglucans, pectin and extensin, generally present in dicots and some monocots (non-comelinoides) and [2] type II, comprising mainly cellulose, glucoarabinoxylans and phenolic compounds with a lower proportion of pectin, found in Poaceae and in most monocotyledonous plants. The matrix of pectin controls, among other properties, porosity. The middle lamella consists of pectin molecules that are joined by cross-linked chains with homogalacturonan layers subsequently deposited in pectin of opposing cells 30**.**

Most of the pectin used by the food industry originates from such raw materials and is extracted under conditions of low acidity and high temperature, resulting in chains that are primarily homogalacturonan 25.

The ratio of raw materials and solvent in the extraction of pectin can be adjusted in order to separate the solid and liquid phases, the filtration of the extract and the costs of water evaporation in the process. Thus, it is possible to control the extraction of pectin to optimize its potential use 24; 31.

## *3.2.2. Structure and composition of pectin*

In 1934, citrus pectins were recognized as linear chains of galacturonic acid and since then it has been found that pectin is a highly complex molecule. The challenge of recent times has been to accommodate all the available information in a single structural model [25]. Pectin are formed by seventeen different monosaccharide, arranged in distinct polysaccharides from more than twenty different connections that form a network when joined together [25; 30] and they are grouped into different types of chains consisting of uronic acids, hexoses, pentoses and methyl pentoses. Several structural units may be replaced by methanol, acetic acid and phenolic acids. Sugars can exist in furanic or pyranic forms and with different anomers (α or β) with various types of linkages between monomers such as α(14), α(15), β(13) e β(14) e β(16) β(16) [32]. Recently there has been progress in the understanding of the very complex fine structure of pectic polymers.

Homogalacturonan (HG) is the most abundant pectic polysaccharide in the cell wall, equivalent to about 60-65% of the total pectin [25; 33]. It presents units of α-D- galacturonic acid in 14 links in a linear pattern. The carboxyl groups are partly methyl-esterified. The chains may be, depending on the plant source, partially O-acetylated at C-3 or C-2 [34; 33; 30].

Rhamnogalacturonan I (RG-I) has a chain represented by the disaccharide [4-α-D-GalA- (12)-α-L-Rha-(1]n [34; 25; 33; 30]. In summary, a variety of different glucan chains (mainly arabinan and galactan) are linked to rhamnose units. The chain length may vary considerably and the composition of RG-I sugars may be highly heterogeneous [34]. RG-I represents 20-35% of pectin, with a high degree of cell specialization and expression depending on the development, the type and number of simple sugars and oligosaccharides attached to this chain. The reason for this level of variation in RG-I is not known, but it suggests a functional diversity.

330 Chromatography – The Most Versatile Method of Chemical Analysis

pulp and /sunflower rinds 29.

primarily homogalacturonan 25.

31.

*3.2.2. Structure and composition of pectin* 

equivalent to about 60-65% of the total pectin [25;

opposing cells 30**.**

its potential use 24;

substances (content over 15% on a dry basis): pomace from apples, citric albedo, sugar beet

The cell wall, a dynamic compartment of plants, can be divided into two layers called 'primary' and 'secondary'. The primary cell wall can be classified into: [1] type I, mainly composed of cellulose, xyloglucans, pectin and extensin, generally present in dicots and some monocots (non-comelinoides) and [2] type II, comprising mainly cellulose, glucoarabinoxylans and phenolic compounds with a lower proportion of pectin, found in Poaceae and in most monocotyledonous plants. The matrix of pectin controls, among other properties, porosity. The middle lamella consists of pectin molecules that are joined by cross-linked chains with homogalacturonan layers subsequently deposited in pectin of

Most of the pectin used by the food industry originates from such raw materials and is extracted under conditions of low acidity and high temperature, resulting in chains that are

The ratio of raw materials and solvent in the extraction of pectin can be adjusted in order to separate the solid and liquid phases, the filtration of the extract and the costs of water evaporation in the process. Thus, it is possible to control the extraction of pectin to optimize

In 1934, citrus pectins were recognized as linear chains of galacturonic acid and since then it has been found that pectin is a highly complex molecule. The challenge of recent times has been to accommodate all the available information in a single structural model [25]. Pectin are formed by seventeen different monosaccharide, arranged in distinct polysaccharides from more than twenty different connections that form a network when joined together [25; 30] and they are grouped into different types of chains consisting of uronic acids, hexoses, pentoses and methyl pentoses. Several structural units may be replaced by methanol, acetic acid and phenolic acids. Sugars can exist in furanic or pyranic forms and with different anomers (α or β) with various types of linkages between monomers such as α(14), α(15), β(13) e β(14) e β(16) β(16) [32]. Recently there has been progress in the

Homogalacturonan (HG) is the most abundant pectic polysaccharide in the cell wall,

in 14 links in a linear pattern. The carboxyl groups are partly methyl-esterified. The chains

Rhamnogalacturonan I (RG-I) has a chain represented by the disaccharide [4-α-D-GalA- (12)-α-L-Rha-(1]n [34; 25; 33; 30]. In summary, a variety of different glucan chains (mainly arabinan and galactan) are linked to rhamnose units. The chain length may vary considerably and the composition of RG-I sugars may be highly heterogeneous [34]. RG-I represents 20-35% of pectin, with a high degree of cell specialization and expression

may be, depending on the plant source, partially O-acetylated at C-3 or C-2 [34; 33; 30].

33]. It presents units of α-D- galacturonic acid

understanding of the very complex fine structure of pectic polymers.

Rhamnogalacturonan II (RG-II) is the most structurally complex segment and comprises 10% of pectin. This structure, highly conserved in most plant species, consists of a homogalacturonan skeleton of approximately eight (probably more) monomeric units, containing side chains of up to 12 different types of sugars, some highly peculiar such as apiose, aceric acid, Docosahexaenoic acid (DHA) and 3-Deoxy-D-manno-oct-2-ulosonic acid (KDO). RG-II usually exists in cell walls as cross-linked dimers by a borate diol ester apiosil between units in the side chain [34; 25].

Xilogalacturonana (XGA) is a homogalacturonan substituted with xylose linked to position 3. The degree of control of xylose may vary between 25% (watermelon) to 75% (apple). This xylose may be additionally substituted in 0-4 in conjunction with another xylose in β, which is more prevalent in reproductive tissues such as fruits and seeds [25;33].

Arabinogalactan I (ARA-I) is composed of skeleton β -D-Gal*p*; residues of α-L-Ara*f* may be linked to galactosyl units in position 3. Arabinogalactan II (ARA-II) is primarily associated with proteins (3-8%), also called arabinogalactan-proteins (AGPs). AG-II is composed of a β-D-Gal*p* 13 skeleton, containing short chains [α-L-Ara*f* (16) β-D-Galp (16)]n, where n = 1, 2 or 3. The protein part is rich in proline, hydroxyproline, alanine, serine and threonine [25]. Arabinan (ARA) is composed of an α-L-Ara*ff* skeleton in 15 links, where there may be side chains of α-L-Ara*f* (13) [35]. There is also another chain, not shown schematically, which is apiogalacturonan (API), HG substituted in O-2 or O-3 with D-apio*f* the 2-O-or D-3 apio*f*. It is present in aquatic monocots such as *Lemna* [33].
