6 pectina 1901 1925

Russian Chemical Bulletin, International Edition, Vol. 63, No. 9, pp. 1901—1924, September, 2014

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Pectic polysaccharides: structure and properties O. A. Patova, V. V. Golovchenko, and Yu. S. Ovodov† Institute of Physiology, Komi Science Center, Ural Branch of the Russian Academy of Sciences, 50 ul. Pervomayskaya, 167982 Syktyvkar,Russian Federation.

Fax: (821) 224 1001. E mail: patova_olga@mail.ru This review summarizes accumulated data about the structural organization of pectic macro molecules, including the latest developments in structural investigations of the molecular de terminants of pectins, the existing models of pectic macromolecules, and several examples of pectins structure. Biological functions of pectic polysaccharides in a plant cell are described.

The role of pectic polysaccharides as physiologically active components of the dietary fiber in nutrition is discussed and the data is given on their transformations in the digestive process. The effect of the structure of pectic polysaccharides on their gel forming ability is surveyed. The key issues are reviewed, which account for the importance of the further investigation of the struc ture and functional properties of pectins.

Key words: pectins, zosterin, pectins of duckweed, sugar beet, olive fruits, sunflower capitu lum, structure, biological functions, physiological activity, gelation, dietary fiber, degree of methyl esterification.

Introduction Pectic compounds (pectic polysaccharides) form a group of complex and variable natural compounds. Pec tins are an important component of primary plant cell wall and intercellular spaces, where they fulfil a vast array of biological functions.1,2 Pectins are contained in dietary fiber, which consti tutes a significant part of the human plant ration. They are received by the human organism as a part of vegetables and fruits, and also in the form of functional food ingredi ents and biologically active food additives. Pectic polysac charides are physiologically active compounds, they facil itate the removal of toxins, salts of heavy metals, radioiso topes and have immunomodulating and anti inflammato ry effect.3 Gel forming ability of pectins facilitated their application in pharmaceutical and food industries. Tradi tionally, they are used as gelling agents, stabilizers and thickeners.4 Structural organization of pectic polysaccharide mac romolecules determines their functional properties, bio logical and physiological activity. Up to date a large amount of data has been accumulated on structural deter minants of pectins, which account for the unique proper ties and multifunctionality of these compounds.5—9 Carbo * Based on the materials of the VIII All Russian Conference "Chemistry and Technology of Plant Substances" (October 7—10, 2013, Kaliningrad). † Deceased.

hydrate chains of pectins comprise linear and branched fragments, such as linear galacturonan (HG), branched xylogalacturonan, apiogalacturonan, rhamnogalactur onan I (RG I) and rhamnogalacturonan II (RG II).10,11 Although significant progress has been achieved in the field of structural studies of pectic polysaccharides, many issues concerning structure of these macromolecules still remain to be clarified.

In the present review modern knowledge is expounded on structure and properties of pectic polysaccharides and key issues are outlined, which substantiate the necessity the further investigations in this field of carbohydrate chemistry.

1. Pectic polysaccharides as components of a plant cell wall Plant cell wall is characterized by the vast diversity of components and strictly defined arrangement. Cell wall matrix contains 75% of water and resembles thick hydrogel,1 which is formed basically of pectic polysaccharides. Pec tic polysaccharides are found in all higher land and aquat ic plants,11—15 marine sea grasses11,16—18 and freshwater algae.19—21 They form one of the basic groups of plant cell wall polysaccharides and make up as much as 35% of the cell wall total dry weight in dicotyledons.22 In monocotyl edons the content of these compounds is decreased to 10% of the total weight of cell wall polysaccharides.2 For the first time pectins were isolated from tamarind fruits in the 13 th century, and the term pectin (from An

Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 9, pp. 1901—1925, September, 2014.

1066 5285/14/6309 1901 © 2014 Springer Science+Business Media, Inc.

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cient Greek pektikos) for pectic polysaccharides was in troduced by French chemist Henry Braconnot as late as in the 19 th century, after he demonstrated, that these com pounds are responsible for the gelating properties of fruits.23 In 1944, the American Chemical Society committee adopted formal internationally recognized nomenclature of pectic compounds, which gives the following defini tions of the basic terms: pectin is a water soluble com pound, devoid of cellulose and built up from galacturonic acid residues with partial or total methyl esterification; pectic substances are the mixtures of pectins with concur rent arabinans, galactans, arabinogalactans; pectinic acid, which is a galacturonan with partially or totally methyl esterified galacturonic acid residues and corresponding pectinates; pectic acid, which is a polygalacturonan, which does not contain methyl esterified galacturonic acid resi dues in the main chain and the corresponding pectates.4 Pectins are biopolymers exclusively of the plant origin.

In the plant cells they are present in soluble and insoluble forms. Insoluble pectic polysaccharides are referred to as protopectin, and the soluble ones were named hydropectin or pectin.4 Under the action of enzymes or when boiled with diluted acids, protopectin is transformed to water soluble pectin, which can be extracted from the plant tis sues in the form of sodium, potassium, and ammonium salts.24 Protopectin is a part of the protopectin complex, which is the insoluble high molecular mass complex con structed from pectins, cellulose and hemicelluloses by co valent and hydrogen bonds. There are only scarce data regarding protopectin structure.25,26 In 1948 it was sug gested that protopectin in a swollen form preserves the plant cell protoplast from dehydration and provides sur vival in unfavorable environmental conditions.27 The chemistry of pectic substances began in 1917 when Ehrlich28 described D galacturonic acid as one of the ba sic components of all pectic polysaccharides.10 Difficul ties, which arose during the studies of chemical properties and molecular structure of pectins, postponed the investi gation of synthesis and modification of these compounds in plants and elucidation of biological role of these biopoly mers in vital functions of plants.27 In 1936—1937 it was shown that pectins constitute a group of polymer homologs with a long linear carbohydrate chain, which consists of 1,4 linked  D galactopyranosyluronic acid residues.24 To date, about 17 different monosaccharides were found in pectin carbohydrate chains, and the most abun dant are the residues of D galacturonic acid, D galactose and L arabinose.29,30 1.1. Structure of pectic polysaccharides The term pectic compounds means the group of oligo saccharides and polysaccharides, which have similar prop erties, but immensely diverse fine structure.9 Pectic poly saccharides contain the following structural elements: ho

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mogalacturonan (HG), rhamnogalacturonan I (RG I), rhamnogalacturonan II (RG II), apiogalacturonan (AGA), xylogalacturonan (XGA), arabinan, galactan, arabinoga lactan.5,6 Presumably, these structural elements are linked covalently. In several pectic polysaccharides the presence of covalent bonds between the carbohydrate backbone and galactan, arabinan or arabinogalactan (in RG I) side chains was established; however, the assumption about the covalent bonding between HG, RG I, RG II, XGA and AGA is based solely on their simultaneous elution during chromatography and no strong evidence of the pres ence of these bonds was shown.9,29,31 Homogalacturonan (HG) is a polymer, which is formed by 1,4 linked residues of  D galacturonic acid. Homo galacturonan is the prevalent structural element of pectic polysaccharides. The content of HG in the molecules of pectins can exceed 80%.2 According to the results of the immunohistochemical analysis, the primary cell walls and the middle lamella of potatoes Solanum tuberosum include vast regions of HG.32 Carbohydrate chains of pectins from the leaves of Arabidopsis thaliana contain 23% of HG (see Ref. 33). Pectins of the cell wall of the sycamore Acer pseudoplatanus suspension cells comprise as high as 10% of HG (see Ref. 34). Molecular mass of HG isolated from apple, sugar beet and citrus pectins was found to be 21, 19 and 24 kDa, which corresponds to chain length of approxi mately 72—100 galacturonic acid residues;35 also, shorter HG chains were found.36 Galacturonic acid residues in HG can be methyl esterified at O(6) or acetylated at O(2) and / or O(3) (see Ref. 37). Degree of methyl esteri fication (DM) and degree of acetylation (DA) strongly influence functional properties of pectins. Depending on degree of methyl esterification, pectins can be referred to as high (>50% of esterified galacturonic acid residues) or low esterified (less than 50% galacturonic acid residues are esterified) pectins.38 It was found, that degree of methyl esterification of galaturonic acid residues varies depending on the origin and is strictly regulated in the life cycle of plants.39 Another important parameter of HG, along with the number of methyl groups, is their distribution in the molecule. Blocks, which contain more than 10 non ester ified galacturonic acid residues can form Ca2+ complex es, which play an important role in the plant cell.40 It is generally accepted that HG forms not only the linear backbones of pectins, but also the main chains of other structural elements of pectic macromolecules: RG II, AGA and XGA, which form the branched area.

However, in most pectic polysaccharides the branched area consists of RG I. Rhamnogalacturonan I (RG I). The carbohydrate backbone of RG I is built of disaccharide repeating units: 4  D GalA 12  L Rha 1]n.

RG I macromolecules are structurally diverse due to monosaccharide residues of their side chains, type

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of linkages between them, and the presence of branch es.9,41 There is a view, that even within one cell wall, the RG I molecules are different (see Ref. 1). The origin of structural diversity of RG I is not understood so far; it indicates to multiplicity of its biological functions in the plant cell.

Rhamnogalacturonan I is considered to be a com ponent of the primary cell wall,9,42 though the presence of polymers of this type was established also in the sec ondary cell walls of the gelatinous type.43—45 Content of RG I in the primary cell wall of different plants var ies. For example, RG I accounts for 36% of the mass of potatoes cell walls46 and only 7% of the mass of sycamo ra.34,42 Content of RG I in pectins usually varies from 5 to 48% (see Ref. 47, 48), and its quantity is not as large as that of the linear HG. At the same time, RG I is a prevail ing structural element in some pectic polysaccharides49 and almost the only pectic component of some types of mucilage.50,51 It is difficult to estimate the length of RG I due to its relatively high heterogeneity.49,52,53 Nevertheless, it was shown, that RG I of the sycamore pectic polysaccha ride can be built of 100—200 repeating disaccharide units,42 soybean pectin contains 15—100 units of this type,49 while pectins from citrus have only 15—40 (see Ref. 54).

Galacturonic acid residues in RG I, similarly to HG, can be acetylated at atoms O(2) and O(3);55 but, in most cases, they are not esterified with methanol. However, RG I from the cell wall of flax was shown to contain both acetylated and non acetylated galacturonic acid re sudues.56 In most cases, rhamnose residues are the branching points. The residues of galacturonic acid of RG I are usu ally not substituted, though there is indirect evidence that 2% of galacturonic acid residues in RG I in sugar beet cell walls are substituted at O(3) with single residues of  D glucuronic acid.57 From 20 to 80% of rhamnose residues in RG I are substituted mostly at O(4) and / or O(3) (in minor quantities) with linear and/or branched oligo and polysaccharide chains of different types, which are: 1,5 linked  L arabinans, 1,4 linked  D galactans, ara binogalactans I (AG I), arabinogalactans II (AG II) and, presumably, galactoarabinans.8,15 Moreover, RG I side chains can include residues of L fucopyranose,  D glu curonic and 4 O Me  D glucuronic acids.58 Depending on the origin, the length of side chains can substantially vary.59 In side chains  L arabinose residues at positions O(2) , O(3) , O(5) and  D galactose residues at position O(6) can contain esters of ferulic acid.60 Degree of poly merization of side chains, as a rule, does not exceed 50 glycosyl residues; however, galactan with degree of polymerization 370 (Mw ~ 60 kDa)61 was isolated from the cell wall of the tobacco plant by extraction with EDTA solution.

Structure of RG I is being constantly revised. The ob stacles for its investigation are connected primarily with

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limitations of analytical methods. Moreover, structur al details of RG I are still unknown because labile bonds of side chains of RG I can be significantly destroyed during the extraction; structural studies of RG I with in the cell are not possible10 to date. Immunocytochemi cal studies evidence that the structure and position of side chains in RG I correlate with the stages of develop ment of cells and tissues, evidencing that RG I can have different functions in the cell.10 It was found that ap pearance of galactan and reduction of arabinan con tent in the carrot cell wall are connected with the transi tion of the cells from division to elongation.9 This is the reason why the relative elasticity and flexibility of cell membranes is probably dependent not only on the carbo hydrate backbone, but on the side chains of RG I as well (see Ref. 62).

Xylogalacturonans (XGA) were first isolated from the pollen of the mountain pine.63 Residues of galacturonic acid of the main carbohydrate chain of XGA are partially substituted at O(3) with single residues of  D xylose and/ or oligosaccharide units, which are built of 1,2 , 1,3 , 1,4 linked, 3,4 and 2,3 disubstituted residues of  D xy lose (degree of polymerization 2—8).

In zosteran, which is a pectin from sea grass of Zoster aceae family, xylose residues are connected to the residues of galacturonic acid of the carbohydrate backbone at O(2);64 and in pectin from the cultured carrot cells they are connected to O(2) and O(3) atoms.65  D Xylan side chains of XGA are frequently substi tuted with residues of  L arabinofuranose,  L fucopyra nose and -D-galactopyranose, and the latter can be sub stituted with -D-glucuronic acid66 at atoms O(4) and O(6).

XGA fragments were found in the extract of water soluble soybean pectins.36 Depending on the origin, the degree of substitution of the galacturonan core with xylose residues varies from 20 to 100%, and degree of methyl esterifica tion of the residues of galacturonic acid is between 40 and 90%. Functions of xylogalacturonans in the plant cell are supposed to be connected with reproduction, as they are usually found in flowers, fruit and seeds;15 however, their presence in sea grass of the Zosteraceae family, roots, stems and leaves of carrot, wheat, mulberry, cotton, pine64,67—73 suggests a wider range of functional properties of xyloga lacturonans.

Apiogalacturonan (AGA), as well as XGA, is a rare struc tural element of pectic polysaccharides. Plant sources, in which this compound was found, are limited to the plants of the duckweed family (Lemnaceae)13,14,74 and sea grass es of the eelgrass family (Zosteraceae).64 In AGA the resi dues of galacturonic acid of the backbone are substituted at positions 2, 3 and 2,3 with side chains, which are built of -D apiose residues. It should be mentioned, that side chains of AGA from sea grasses consist of single apiose residues,64 and in duckweed pectin, they consist of single  -D apiose residues together with oligosaccha

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ride chains, in which -D apiose residues are linked by 1,5 bonds.13,14,74 Proportion of galacturonic acid and apiose residues in zosteran was 4 : 1 (see Ref. 64). AGA content in cell walls of duckweed buds and green leaves varies within the broad range and can constitute from 0.2 to 20% of the mass of non cellulose polysaccharides.75 It can be assumed that there should be a reason why apoigalactur onan was found only in pectins of the freshwater duck weed and sea grasses, which are higher aquatic flowering plants.2,76 Though carbohydrate chains of pectic poly saccharides from duckweed and eelgrass have the low content of methyl esterified residues of galacturonic acid, the high resistance of these plants to the natural process of putrefaction and decomposition is well known, which provides efficient preservation of these plants in aqueous medium. AGA, which is the main fragment of the carbohydrate chain of these pectins, is responsi ble for their resistance to the action of pectinolytic en zymes, which are generated by fungal and bacterial phyto pathogens.2,64,76 Rhamnogalacturonan II (RG II) has the most complex structure among other pectin elements.58,73,76—78 RG II is composed of 12 different monosaccharide residues. Al though the degree of polymerization of RG II is 60, 20 different types of linkages can be found in its carbohydrate chains.1,79 Content of RG II in pectic polysaccharides is up to 10%80 and 0.2—3.6% of dry mass of the plant material. 81 However, taking into consideration its comparatively low molecular mass, the number of mole cules of this polymer is close to those of other pectin structural elements.1 Despite its complexity, structure of RG II is rather conservative,82 evidencing the impor tance of biological functions of this polysaccharide in plant cell walls.

Rhamnogalacturonan II is resistible to the action of 1,4 endo--D polygalacturonase, and it can be isolated after the preliminary treatment of pectins with this en zyme. In carbohydrate chains of RG II both the resi dues of abundant monosaccharides (D galacturonic acid, L rhamnose, D galactose, L arabinose, D xylose, D glu cose, L fucose, D mannose and D glucuronic acid), and the unusual and rare monosaccharides (D apiose, 2 О methyl L fucose, 2 keto 3 deoxy D manno octonic acid (KDO), 2 О methyl D xylose, 3 deoxy D lyxo heptulo saric acid (DHA), aceric acid (3 С carboxy 5 deoxy L xylo furanose, AceA))73,83 are found. Aceric acid, as a monosaccharide component of RG II, was first found in cell walls of the suspension culture of sycamore.84 3 Deoxy D lyxo heptulosaric acid was identified with the use of chromato mass spectrometry and proton resonance spectrometry in 1988 (see Ref. 82). The first evidence of the presence of KDO in pectins was reported in 1978,85 and later82 these data were totally confirmed. Before that time, KDO was found only in bacterial lipopolysaccha rides. It was recently suggested, that three mentioned above

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acidic monosaccharides (АсеА, DHA and KDO), are the obligatory components of RG II and must be present in any plant.73 First RG II was isolated from the sycamore Acer pseudoplatanus86,87 cell walls. The presence of RG II was shown in rice cell membranes,88 Douglas fir,89 onion,90 kiwi fruit,91 reddish,92 roots of Bupleurum falcatum,93 and leaves of Arabidopsis thaliana.94 Also, RG II was isolated from the pulp of sugar beet,95 from the products of grape processing, as a main polysaccharides component of the juice from red grape fruit, apples Malus domestica, carrots Daucus carota, tomatoes Solanum licopersicum after en zyme processing.76 RG II is present in pectic polysaccha rides of currant and blueberry (in cell walls, in juice and in press cake) as a dimer.96 As it was shown by immu nochemical methods, the galacturonic residues of the main carbohydrate chain of RG II, which are located near the plasmatic membrane, are not esterified with methanol, while RG II molecules, which are located in primary cell walls, contain large number of methoxyl groups.97 The largest quantity of RG II is present in vegetable and fruit juices.96,98 Total structure of RG II has not been established so far, though the suggested partial structure correctly out lines its basic features.77 It was found that RG II from the reddish contained boron in the form of borate diol esters, which cross link RG II molecules to form a dimer. 80,99 Similar dimer was also found in RG II from the sugar beet.95 Although the role of boron in plants is not totally es tablished, it was shown, that boron is a microelement, which is important for plant growing: deficiency of this element at the beginning of growing results in forma tion of cell walls with abnormal morphology.100,101 Rham nogalacturonan II in the form of dimeric boron esters was isolated from a suspension sycamore culture and from the peanut sprouts.87 This phenomenon was also observed for RG II obtained from the currant and blue berry fruits.96 It was shown that borate ester linkages, which connect two RG II molecules, are formed between hydroxyl groups at С(2) and С(3) of the apiofuranose residues,80 which, therefore, play an important role in plant tissue growing along with boron. Formation of borate ester linkages be tween the apiose residues and RG II was studied in vitro.

It was shown, that methyl -D apiofuranoside gives more stable borate diol esters with sodium borate, than methyl -L apiofuranoside does.102 Interaction of methyl -D ap iofuranoside with sodium borate at рН 8 gives two diaste reomers (A and B).102 The possibility of in muro formation of this type of diastereomers in the plant cell wall has not been described so far.83 Structural analysis showed that formation of the А dimer in a plant cell is more proba ble.103 Nevertheless, possibility of the formation of dimer В104 should also be considered. In vitro studies of the reac

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tion of sodium borate with RG II, which was isolated from cell walls of suspension sycamore culture, peanut stems, and red wine, showed that the optimal рН value for in vitro formation of the dimer was 3.0—4.0 (see Ref. 83).

As methyl -D apiofuranoside does not form borate diol esters at рН < 5 (see Ref. 102), it can be anticipated that anionic character and structure of RG II are the key fac tors, which influence the dimer in vitro formation. It is noteworthy that the dimer in vitro formation takes place in the absence of catalytic proteins. The rate and the yield of RG II dimer production in vitro increase in the presence of two and three valent cations with ionic radii greater than 1.0 Å (see Ref. 102). Calcium cations in low concentrations (1 mmol L–1) are less effective, than larger cations.102 Nevertheless, higher concentra tions (10 mmol L–1) of calcium and boronic acid increase the rate of the dimer formation in vitro.105 The role of Са2+ in the dimer formation in cells is not understood so far, but there is a view, that its presence stabilizes the RG II106 dimer.

It is known, that RG II is the only polysaccharide in primary cell walls,83,95,99,100,102 which is connected by bo rate bridges. The resulting dimer has acid labile borate ester linkages, which are hydrolyzed, when the рН value in the cell wall decreases during the auxin induced cell growth.100 Mutations, which cause even minor changes of RG II structure, and, consequently, reduction of the quantity of RG II dimer, result in severe growth defects, for example, dwarfing, and hence evidence that RG II dimerization has a crucial importance for normal growth and development79 of plants.

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Borate complex of RG II is a part of a macromolecu lar pectin complex, which consists of HG, RG II and RG I, and the borate esters form a cross linked RG II containing macromolecular pectin.107 In order to corro borate this statement, high molecular mass pectin was solubilized by treatment of the sugar beet cell walls with imidazole in the presence of HCl. Treatment of the pectin with endo polygalacturonase destroyed HG and result ed in formation of RG I, Mw = 5—10 kDa. Pectin of Mw = 150—200 kDa contained 69 g of boron/1 g, while Mw of the borate complex is 10 kDa, and it contains 1 mole of boron.95 Three aforementioned polysaccharides were solubilized from non soluble cell walls by treatment of the latter with aqueous buffers and calcium chelating agents. Polysac charides could not be separated by gel permeation chrom atography as they were eluted within the void column vol ume (Mw > 200 kDa). Fractions, which contained RG I, RG II, and oligogalacturonides, were obtained by treat ment of high molecular mass compounds with endo po lygalacturonase.73 HG, RG I, and RG II are likely linked to each other covalently,9,41,108,109 though there are no strong evidences of the presence of these bonds. An alter native structure of pectic polysaccharides has been pro posed,29 which the authors consider to be in better agree ment with the structural data for pectins available to date.

They anticipate, that the backbone of pectins is formed by RG with alternating residues of galacturonic acid and rhamnose, and the side chains are formed with not only arabinans, galactans and arabinogalactans, but also with HG, AGА, XGА, HG with parts of RG II (Fig. 1).29

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AG II

A

AG I

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RG II A

RG II

A

RG II

RG II

RG II RG II

Homogalacturonan

Xylogalacturonan

Rhamnogalacturonan I

Rhamnogalacturonan I

Arabinan

Arabinogalactan I

RG II AG I

AG I A

 L AcefA

 D DhapA

 D Apif  L Araf

A

 D GalpA

 D Xylp

 L Fucp

 D GlcpA

 D Xylp  D GalpA methyl esterificated

 L Glap

Kdop

 L Araf

 D Galp

 L Rhap

2 O Ac  D GalpA

 L Arap

 D GalpA

 L Rhap

2 O Me  D XylpA

Apiogalacturonan II

AG II

Fig. 1. Approximate macromolecular structure of pectic polysaccharides (see Ref. 29).

1.2. Representatives of pectic polysaccharides Zosteran is a pectic polysaccharide, which was isolated from sea grass of the Zosteraceae110 (eelgrass) family. It was found, that its carbohydrate chains contain residues of galacturonic acid and D apiose residues as the major neutral monosaccharide, together with trace quantities of D galactose, L arabinose, L rhamnose, D xylose and 2 О methyl D xylose resudues.18 A characteristic feature of zosteran is the low content of methyl esterified residues of galacturonic acid (>1%); it can be connected with its osmoregulating function in sea grass cells. It was shown that the backbone of zosteran is built of 1,4  D galactur onan.111 The main component of side chains is apiogalac turonan, which accounts for 25% of the zosteran macro molecule and is built of residues of D apiofuranose and D galacturonic acid in proportion ~5 : 4. 64 Side chains of apiogalacturonan are built of single res idues of D apiofuranose, which are connected to the carbo

hydrate backbone at positions 2, 3 and 2,3 of the D galac turonic acid. Along with apiogalacturonan, minor regions of xylogalacturonan were found, which have side chains built of 1,3 and 1,4 linked xylose residues, which, in turn, in some cases have branches at position 4. At the non reducing end of the xylogalacturonan side chains, xylose, arabinose, and 2 О methyl xylose residues were found.64 Besides, regions of RG I were unveiled within carbohy drate chains of zosteran, which bear sparse side chains consisting of 1,3 linked galactose residues, which are 1,4 linked to the rhamnose residue of the carbohydrate backbone through a galacturonic acid residue. Methyla tion analysis data showed the terminal position of arabinofuranose and galactose residues. Approximate structure of the zosteran macromolecule is presented in Scheme 1.

Duckweed pectin (lemnan), along with zosteran, con tains apiogalacturonan as the major component in the branched area. However, differently from zosteran, the

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side chains in apiogalacturonan of the pectin from the duckweed Lemna minor are formed by not only single, but also by 1,5 linked  D apiose13,14,74,112,113 residues. High yield of 1,4  D galacturonan obtained by the acid hy drolysis procedure demonstrates, that carbohydrate back bones of both branched and linear regions of lemnan are built of 1,4 linked  D galactopyranosyluronic acid resi dues. NMR analysis of apiogalacturonan from duckweed pectin showed the presence of 3,4 di О substituted resi dues of galacturonic acid.74 Heteroglycanogalacturonan was isolated as a minor component of the branched region of the duckweed pec tin, which contained, along with D galacturonic acid resi dues, 1,5 linked residues of  L arabinose, 1,4 linked  D xylose, 1,3 , 1,4 and 1,6 linked  D galactose. Xy

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lose residues, which were found in the duckweed pectin, can be structural elements of either xylogalacturonan or apiogalacturonan. Earlier, application of isotopic labeling technique showed that apiogalacturonan, which was syn thesized in vitro using enzymes from L. minor, contained xylopyranose residues (4.8%).113 This observation suggests that xylose residues are the inherent part of duckweed cell wall apiogalacturonan.113 Monosaccharide content of the minor component suggests that, as in the case of zosteran, duckweed pectin can include RG I. Reliable information on structural details of the L rhamnose residues and their position is not available because of the small number of these residues.74 Structure of the duckweed pectin macromolecule is presented on Scheme 2.

Scheme 1

Galacturonan Apiogalacturonan

Rhamnogalacturonan I

Galacturonan

Xylogalacturonan Approximate number of monosaccharide residues: D GalpA, 270; D Xyl, 65; L Rhap, 20; D Api, 55; L Ara, 5; D Gal, 30; 2 O Me Xyl, 5. TSR is terminal monosaccharide residue (Xyl or 2 O Me Xyl or Ara).

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Scheme 2

3,4 di O substituted D Galp 2,4 di O substituted D Xylp 2,5 di O substituted L Araf

Sugar beet pectin carbohydrate backbone contains galacturonic acid (55—75%), rhamnose, arabinose, ga lactose residues and small quantities of xylose, mannose and glucose residues. Besides, it bears methoxyl groups (7—8%), acetyl groups (7%) and small quantities of feru lic acid residues (~0.5%).114 High percentage of acetyl groups causes its low, as compared to apple and citrus pectins, gelation ability.115 Acetyl and methoxyl groups are linked to the residues of D galacturonic acid of the main carbohydrate chain and are totally removed during alkali treatment.

Fragments of the carbohydrate backbone, which con tain residues of galacturonic acid and rhamnose, were obtained during acid hydrolysis of de esterified sugar beet pectin and separated using ion exchange chromato graphy.50 The most abundant fraction, which was obtain by separation, contained equal quantities of rhamnose and galacturonic acid. NMR analysis showed the presence of the following repeating unit 4)  D GalpA (12)  L Rhap (1…. Oligomers were obtained from the sugar beet pectin, which contain rhamnose <1 mol.% with aver age degree of polymerization 91—108,34 together with longer chains with alternating residues of galacturonic acid and rhamnose. Average degree of polymerization of oligo mers of this type is ~20 (see Ref. 54). Treatment of this pectin with rhamnogalacturonase gives oligosaccharides

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with the residue of galacturonic acid on the reducing end and rhamnose residues, which are partially substituted with galactose.116 It was shown, that side chains of the sugar beet pectin are attached to the RG backbone and can have the arabinan or the galactan structure (see Ref. 117). Be sides, a small number of galactose residues is linked di rectly to arabinan side chains, which are in turn, linked to rhamnose residues of the RG backbone (see Ref. 117).

Arabinose is included in side chains as terminal, 1,3 linked, 3,5 di О and 2,3,5 tri О substituted residues. Earlier it was shown, that rhamnogalacturonan is located on the reducing end of arabinogalactan in sycamore pectin, as well as in sugar beet pectin.117 1,2 Linked and 2,4 di О sub stituted rhamnose residues are present in almost equal quantities to evidence the equal length of the regions of linear RG and RG I in sugar beet pectin. Xylose is mostly found in the form of terminal and 1,4 linked residues, with the exception of pectin, which is extracted with aqueous 4 М NaOH, where it is present as terminal and 1,2 linked residues. In the majority of extracted pectins terminal and 1,4 linked galactose residues are found, as well as 1,6 link ed, 4,6 di О substituted galactose residues and 1,4 linked mannose residues. Glucose is present mostly as 1,4 linked residues, though some quantity of 4,6 di О substituted residues was also detected. It was shown, that residues of glucuronic acid are directly linked to the residues of galac turonic acid of the rhamnogalacturonan backbone.118 Pectic polysaccharides from the sugar beet pulp con tain residues of ferulic acid, which are linked to neutral side chains by ester linkage. Enzymatic hydrolysis of the beet pectin by Drizelase from fungus Basidiomycetes, which contains various exo and endo carbohydrases, including arabinase, cellulase, xylonase, galactanase and polygalac turonase, but does not show esterase activity against feru lic acid residues evidenced the presence of feruloil groups at galactose (45—50%) and arabinose (50—55%)119—122 residues. Ferulic acid is linked to O(2) position of 1,5 linked arabinose residues and to O(6) position of 1,4 linked galactose of side chains.119,123,124 The ferulic acid residues of arabinan chains readily form dehydromers in the presence of oxidases and oxidants.125 Treatment of sugar beet pectin with ammonium persulfate or peroxi dase increases its viscosity and enhances its gelation prop erties due to transformation of ferulate monomers to de hydromers.126 Pectic polysaccharides of olive fruits. Pectic polysac charides are significant components of fruits of olive Olea europea, their content can be as much as 40%.127 Though the pectin content varies depending on the type of applied extraction technique and environmental factors, their ba sic structure is linear1,4 -D galacturonan, accompanied by blocks of heavily branched RG I.128 Pectic polysaccharides from olive fruits contain a large quantity of arabinose. About 30% of 1,5 linked arabinose residues of the carbohydrate backbone are substituted at

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O(3) atom. Extraction of olive fruits with aqueous ЕDTA and KOH solutions pectic polysaccharides were obtained together with xylans, which contain up to 70% (see Ref. 127) of 3,5 di О substituted arabinose residues.

Among pectic polysaccharides, two types of galac tans were found, where most of galactose residues are substituted at positions O(3) and O(6).These galactans can be either incorporated in side chains RG I or exist as separate polysaccharides.129 Galactose residues are frequently connected to proteins, which have hydroxy proline.73 Solvents, which are used for extraction of pectic com pounds from olive fruits, do not solubilize them totally.

Even after the final extraction with 6 М alkali the press cake contains ~30% of the total mass of pectins, which are present in cell walls. Highly branched pectic polysaccha rides, which form the residue material, are, presumably, strongly bound to other components of the cell wall.

During the ripening, monosaccharide content of pectic polysaccharides changes, and the quantity of methyl es ters and acetyl groups is decreased.130 Sunflower pectin, which was obtained from heads and stems after removal of seeds, was found to be a low esteri fied (LM) pectin.131 It is extracted with aqueous solution of sodium hexametaphosphate with yield, which exceeds the yield of other pectins (apple, citrus) from traditional sources. This pectin contains from 77 to 85% of galactur onic acid residues, and 11% of those are methyl esteri fied.131 The content of acetyl groups is about 2.5%. Pectin forms viscous aqueous solutions and gels, their stability being comparable to standard commercial species.

Commercial pectins. Pectins are widely used as food additives due to their ability to form gels (code EU E440).

World annual pectin consumption is around 45 thousand ton with total market value up to 400 million euro.132 In food industry, pectins are known as thickeners are find application in manufacturing of jams and jellies, fruit juic es, desserts, as well as bakery products.5,6,129,133—135 Pec tins are also used as stabilizers of sour milk products and yoghurts. Though the majority of plant tissues contains pectins, manufacturing is almost entirely based on a limit ed number of sources.136 Currently, citrus peel and apple press cake are the principal materials for commercial pec tin production; meanwhile, other possibly valuable sourc es remain mostly untapped. Conditions of pectin industri al production are severe (high temperature, strong acids) and are optimized for HG fragments enrichment, as they account for gel forming properties of pectins. However, side chains are almost totally destroyed in these condi tions.4,137—140 Commercial pectins are, in general, classified accord ing to their degree of methyl esterification of galacturonic acid residues (DM), which is the main parameter account ing for their physical properties. The degree of methyl esterification is calculated as the number of moles of me

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thyl alcohol residues per 100 moles of galacturonic acid residues.139 Depending on the degree of methyl esterifica tion, pectins are divided into high (DM>50%) and low methyl estrified (DM<50%).

Pectins from sugar beet waste material are character ized with modest gel forming capacity due to low molecu lar mass and high percentage of acetyl groups. Treatment with acidified methanol removes acetyl groups and in creases methyl esterification dergee, but substantially de creases molecular mass and the number of galacturonic acid molecules.141 Nevertheless, acetylated pectins find an application due to their emulsifying properties.142 Gel forming ability of acetylated pectins is rather limited, be cause acetyl groups prevent the formation of ionic cross links between free carboxyl groups of galacturonic acid residues in the presence of cations of polyvalent metals.143 Low molecular mass and high content of neutral monosac charides also do not favor gelation.144,145 Nevertheless, one of the advantages of acetylated pectins is their ability to form hydrolysable and dehydratable gels.146 Today, amidated pectins are being used more and more, which are manufactured, as a rule, from high methyl es terified apple or citrus pectins by suspending of dried polysaccharide in an alcohol with the following treatment with ammonia. In this process, methyl esterified groups are randomly substituted for amido groups. When DM is 25—35%, degree of amidation is as much as 15—24%.

The peculiar feature of amidated pectin is its enhanced ability to form gels in the presence of calcium cations. In contrast to pectins and their modified derivatives, accept able daily dose of amidated pectins is limited and is equal to 25 mg per 1 kg of the body weight.147 In this way, though the main structural elements of pectic polysaccharides have been elucidated to date, and these polysaccharides are widely used in different branch es of industry due to their gel forming ability, many prob lems are waiting to be solved. In particular, it is not known so far, whether structural elements of pectic polysaccha rides are separate molecules or they exist in the form of covalently linked blocks within one pectin molecule. We hope, that complex application of modern analytical methods, such as enzyme immunoassay, NMR spectro scopy, atomic force microscopy and application of mono clonal antibodies will shed more light on the structure of pectic polysaccharides.

1.3. Biological functions of pectic polysaccharides in a plant cell Investigation of processes of biosynthesis is a com plicated task, and progress in this area is slow. At least 53 different enzymes148 are involved in the synthesis of pectins. The majority of these enzymes are glycosyl tranferases, but methyl and acetyltransferases are needed as well.148

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In order to elucidate mechanisms of enzyme action in 53 different processes, which are necessary for pectin syn thesis, corresponding genes have to be determined and cloned. No pectin biosynthetic enzyme has been obtained to date as a homogeneous compound, and, similarly, pec tin biosynthetic glycosyl , methyl , and acetyltransferases have not been cloned.9 However, recently, progress can be noted in the field of pectin biosynthesis research, and, in particular, in partial purification of a few enzymes and in determination of genes of pectin biosynthesis en zymes.149,150 Immunocytochemical analysis, which involves cell wall carbohydrate specific epitope antibodies,151—154 has shown, that HG and RG I are present both in cis , and medial cisternae of the Golgi apparatus. Presumably, syn thesis of HG and RG I is started in cis 155,156 and is con tinued in medial cisternae.152,154 Synthesis is catalyzed by galacturonosyltransferases2 and S adenosyl L methionine dependent methyltransferases.157 Esterification of HG, is, very likely, fulfilled in medial and trans cisternae of the Golgi apparatus,156 and the more intensive formation of branched areas of pectins takes place only in trans cister nae.154,156 Final arrangement of pectins is fulfilled in vesi cles of the Golgi apparatus, which are later transported to the plasmatic membrane and incorporated into the cell wall, most frequently in the form of high methyl esterified poly mer.158 Removal of methyl groups is catalyzed by pectin methylesterases, which are located in the cell wall.1,159 However, some types of cells, apparently secrete relatively low esterified pectins to the cell wall. For example, cells of melon callus160 and slimes, which are secreted by epider mal cells from clover roots155 contain non esterified HG in trans cisternae of the Golgi apparatus. Besides, non esterified HG were found in the plasmatic membrane of cell walls. Hence, it can be assumed that HG may also be incorporated into the cell wall in non esterified form and is not always synthesized in highly esterified form.161 Thus, significance of the degree of esterification of HG for the synthesis of pectins is still to be clarified.

Degree of methyl esterification is an important para meter, which influences the flexibility of the cell wall.162 The flexibility of the cell wall changes under the action of pectinmethylesterases, which are a group of enzymes, which catalyze demethylesterification of pectins to give free carboxyl groups.162,163 De esterification effects the interaction of pectins with other components of the cell wall, changes the structure of protopectic complex and of the cell wall as a whole.164,165 Besides, de esterified HG can form covalent bonds with RG I, RG II and xylo glucan, and, thus, influence the structure of the cell wall.2 Pectins account for the plants freezing tolerance by serving as a temperature buffer and contribute to plants cold acclimatization by preventing the ice spread and over cooling of the intracellular water on lowering of the ambi ent temperature.166 It was found, that the quantity of pec

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tin in rape seeds is elevated during the cold acclimatiza tion of the plants.167 Moreover, it was shown, that cold acclimatization promotes the activity of pectinmethyl esterases, and, as a result, de esterification facilitates the formation of semi rigid pectate gels158,168,169 in the cells, which consist of pectin molecules and Са2+ ions. This phenomenon can be connected with the reduction of pore size in the cell under cooling, which was observed in grape and apple cells.170 Though the transcription of pectinme thylesterase and its activity were registered within gene regulation during the cold stress,167,171 little is known about the mechanism of this process. During the develop ment of plants the reaction to the abiotic factors and the activity of these enzymes change,169,172—175 suggest ing that the mechanism of regulation of pectic polysac charide structure by pectinmethylesterases is rather com plicated. As far as pectins are one of the principal com ponents of plant cell walls, and pectinmethylesterases are the most abundant among the plant cell wall proteins, any processes, which cause changes in the cell wall, ini tiate alterations in expression and activity of pectin methylesterases.

Pectins maintain the texture of fruits and vegetables.

Presumably, due to the presence of homogalacturonan, pectins regulate porosity of the cell wall and the cell adhe sion and maintain the ionic transport and water bal ance.170,176,177 Metabolism of pectic polysaccharides plays a key role in ripening of the fruits, which to date it regarded as a well coordinated and genetically defined process of tissue dif ferentiation. While cellulose and hemicelluloses provide the rigidity of the cell wall, pectins connect other compo nents of the cell wall and form its texture; degradation of pectins results in decomposition of the protopectic com plex, and thus determines and accelerates softening of fruits.178,179 Decomposition of structural complexes of the cell wall includes coordinated and synergetic action of different enzymes, with one type of enzymes possibly mediating the other, and, as a result, the cell wall is mod ified.180 During ripening, pectins of the protopectin complex are solubilized, depolymerized, and de esteri fied.181,182 This process is controlled by specific en zymes.183 Depolymerization of the carbohydrate chain of galacturonan during ripening is caused by action of poly galacturonase.2,91,184—186 Degradation of carbohydrate chains of galacturonan is not obligatory; another type of reaction can be observed, which is stipulated by destruc tion of glycosidic bonds between the residues of galactose, arabinose, xylose, and mannose.91,187—189 Thus, during ripening of fruits of kiwi, tomatoes, apples, asian pear, avocado, strawberry, blackberry, watermelon, and plum, solubilization is accompanied by the decrease of the num ber of residues of both galacturonic acid and galactose in pectin carbohydrate chains.190—192 During the ripening of persimmon fruits, depolymerization of pectin carbohydrate

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chains with the decrease of the number of arabinose, ga lactose and uronic acids was registered, which did not correlate with activity of polygalacturonase.187 Decrease of galactose content was also reported for pectic polysac charides in tomatoes, peaches, melon Cucumis melo L. and apples, but it was not registered for pectins in plums Prunus spp., apricots, pears, blueberry Vaccinium angusti folium Ait. and raspberry Rubus idaeus L.193,194 It was found, that ripening of European pears and apricots is accompanied by the decrease of the content of xylose194 residues along with arabinose. Other researchers noted sub stantial decrease of the content of galactose and galactur onic acid residues during ripening of apricot fruits, with the concurrent increase of the content of arabinose resi dues.195 It was shown, that in cantaloupe fruits decrease of the content of galactose residues in pectin carbohydrate chains during ripening is caused by enhanced activity of  galactosidases.196 The presented papers contain contra dictory data; however, all of them evidence modification of pectins during the ripening of fruits.

Besides, pectic polysaccharides serve as protectors in a plant cell wall, which presents the first outer barrier to phytopathogens. Integrity of the plant cell wall has an effect on the susceptibility of plants to phytopathogens The majority of phytopathogenic fungi and bacteria se crete enzymes, which destroy cellulose, hemicellulose and pectins. Investigation of the degradation process of differ ent components of the cell wall at the early stages of the infection showed, that pectolytic enzymes, and among them polygalacturonases, pectatelyases, and pectinmethyl esterases, are the first enzymes, secreted by fungal and bacterial phytopathogens.174,197 Degree of methyl esterification of galacturonic acid residues in pectins accounts for the resistance of the plant cell wall to phytopathogens and has an effect on resistance of plants to deseases.177 Differences in the degree and char acter of methyl esterification define susceptibility of the cell wall of different varieties of carrots with similar pectin content to fungus Mycocentrospora аcerina.198 In pectins of near isogenic lines of wheat, susceptible to fungus Puccinia graminis, which is a causative agent of stem rust, block like distribution of methyl esterified residues of ga lacturonic acid, in contrast to resistant lines.199 High de gree of methyl esterification of pectins in different variet ies of potatoes correlates with their resistivity to Pecto bacterium carotovorum.200,201 It was shown that degree of methyl esterification is higher for varieties of beans, which are resistant to Colletotrichum lindemuthianum, than for the susceptible to its action nearest isogenic lines.202 Pectinmethylesterases play a crucial role in interac tions phytopathogens and plant cells. They de esterificate pectins, thus transforming them into the form, which is not resistant to the action of the pathogen, and make the plant cell available to other enzymes, which destroy the cell wall.

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Diverse biotechnological approaches, which are aimed at the increase of degree of methyl esterification of pectins in different plants, are concentrated mainly at downregu lation of pectinmethylesterases activity by intensification of in planta expression of corresponding inhibitors, as far as activity of pectinmethylesterase in the cell wall is regu lated both by pH and ionic strength and by pectinmethyl esterase inhibitors. Inhibititors of pectinmethylesterases were first found and characterized in kiwi cell walls.203,204 Later, they were unveiled in the cell walls of Arabidopsis thaliana, pepper, broccoli and wheat.205—209 Inhibitors, as a rule, slow down synthesis of pectinmethylesterases in the plant, but have no effect on the enzymes of the patho gen due to structural differences.210 Pectinmethylesteras es and corresponding inhibitors are tissue specific regula tory proteins, which belong to vast multigene families.177 It was shown, that increase of the degree of estrification of the residues of galacturonic acid in cell wall pectins of Arabidopsis thaliana reduces its susceptibility to Botrytis cinerea and Pectobacterium carotovorum.173 It was also found, that increase of the immune response in trans formed plants towards fungal and bacterial infections is connected with elevation of degree of esterification of a pectin, and does not depend on protective reactions switched on by infection. Polygalacturonase action in a plant cell results in accumulation of pectin degradation fragments, for example, oligogalacturonides, which are linear molecules containing from 2 to 20 1,4 linked resi dues of -D galacturonic acid.1,211 Degree and character of methyl esterification of a pectin have an effect on en zymes action. In order to modulate protective reac tions,212,213 interact with wall associated kinases,214 and activate the innate imunity,215,216 oligogalacturonides have to be de esterified. Biological role of oligogalacturonides it not limited to stimulation of protective reactions; they also provide the control over the cell growth and differen tiation.1 The mechanisms of action of oligogalacturonides can be diverse, as these molecules are charged and thus able to form non covalent bonds in cell walls. They can interfere the structures, which are ionically built of HG, loosen mechanical stress inside the cell wall, and thus initiate signal transduction cascades responsible for reac tion to mechanical stress.1 It was established, that xylogalacturonan biosynthesis genes activity is enhanced in response to the pathogen infection. This fact suggests that pectin galacturonan frag ments may be protected from the action of phytopathogen polygalacturonases by substitution of galacturonan with xylogalacturonan.217 Xylogalacturonans are resistant to action of polygalacturonases, which are synthesizes by both the phytopathogen and the plant itself. Xylogalacturonas es, which are able to destroy parts of xylogalacturonan, are synthesized by plants and pathogens less often, than endo polygalacturonases. In this way, the presence of substitut ed galacturonans can impact the development of the im

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mune response to phytopathogens. In this context it should be mentioned that the level of mRNA of the galactur onan deficient mutant gene is elevated in response to a number of phytopathogenic microorganisms, such as Botrytis cinerea, Phytophtora infestans и Pseudomonas syringae.217 Specific functions of xylogalacturonan have not been established so far; however, the presence of this polymer can change the physical properties of the pectin matrix.

Substitution of the residues of galacturonic acid with xy lose residues disables formation of calcium dependent in teractions between the HG chains in cell walls. It is still unclear how it can change the biological functions of the plant cell.217 It was demonstrated that RG II is involved in the interaction of plants and pathogens, though it is a minor component of the cell wall, by interfering with specific transmembrane transport of amino acids. Relatively long regions of galacturonan in the RG II molecule (which consist of seven or more galacturonic acid residues) are essential for fulfillment of biological functions of RG II in plants, and so do the residues of unusual monasaccha rides, such as KDO and apiose. It is possible that these monosaccharides can have synergetic effect on the pro cess of interaction of plants with phytopathogens and their combined presence is important for the fulfillment of biological functions of RG II.218 Besides, it is often antic ipated that the chemical content of plants is closely con nected with the environmental conditions, temperature, precipitation volume, type of soil, biotic parameters and even the state of atmosphere.219—223 Studies of leaves of true laurel Laurus nobilis L., which grows in two climatic regions in Tunisia and Algeria, showed that changes in their polysaccharide content in water stress regimen dif fer. Average concentration of cellulose in the laurel from Tunisia increases significantly at constant stress, while cellulose concentration in the leaves of a laurel from Alge ria does not change independent of the kinetics and inten sity of water stress. Concentration of hemicelluloses in laurel leaves from Algeria decreases in response to con stant stress, but it is elevated in response to cyclic stress, while concentration of hemicelluloses in the laurel leaves from Tunisia decreases in response to both procedures. In laurel from Algeria, concentration of pectic polysaccha rides increases in stress of medium intensity, and, in laurel from Tunisia, this effect arises only in response to high intensity srtess.224 It was established earlier that qualitative and quanti tative characteristics of pectins in plants changed depend ing on the vegetation period. The content of pectins in plant cells and the proportion of neutral monosaccharides and galacturonic acid in their carbohydrate chains changed during ontogenesis.225,226 In this way, the key role of pectic polysaccharides in plant development is established to date.

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2. Pectic polysaccharides as components of dietary fiber To date, the fields of application of pectins are numer ous. The most important one is the use as a nutritional component. Besides pectic polysaccharides are compo nents of fruits and vegetables, they are comprised in nutri tional ingredients and dietary supplements. Pectins ac count for the most of the dietary fiber (DF).

The interest to DF arose in 1950—1970 after a number of independent investigations demonstrated that inclu sion of DF in the ration had a positive effect on health.227—231 As a result of these studies, a belief emerged that diseases, such as atherosclerosis, obesity, appendicitis, constipa tion, colorectal cancer, diverticulitis, diabetes mellitus and cholecystitis are connected to the DF deficiency in the ration.232 Metabolic disorders, hypertonia, and other diseases of gastrointestinal tract, metabolic and car diovascular diseases are associated with the deficiency of DF.233,234 The term dietary fiber (DF) is close to cellulose and roughage, which are used in Russian and foreign litera ture.230,235 First this term referred to the residue, which was formed after the extraction of the food with diluted acid,236 and this term was also used to name the indigest ible plant food residue.237 For a long time, the precise meaning of this term has been widely discussed.238 In 1953 dietary fiber was referred to as the sum of cellulose, hemi celluloses and lignin.235 In 1975 it was understood as the rest of the plant cell, which is resistant to hydrolytic ac tion of human digestive enzymes (digestion) and consists of pectic polysaccharides, hemicelluloses, cellulose, lig nin, oligosaccharides, gums and slimes.230,238 Later the meaning was extended to all polysaccharides of the plant cell with the exception of starch.239 However, this inter pretation suggests, that DF include, mostly plant cell wall polymers, and it does not consider modified polysaccha rides (modified cellulose and starches), which are basic compounds for a number of food additives. Today, the majority of researchers interpret the term DF as the edible parts of plants and polysaccharides, which are comprised into the nutritional additives and are not digested with the secretions of human gastrointestinal tract with the total or partial fermentation in the large intestine.235 However, alternative interpretations of the term DF continue to appear.238 Chemical analysis showed that the majority of the com ponents of DF are non starch polysaccharides, which con sist of cellulose and non cellulose polysaccharides. The latter group includes hemicelluloses, pectins, storage polysaccharides, such as inulin and guar, and the plant gums and slimes. Non cellulose polysaccharides can be water soluble and water insoluble. Since pectic polysac charides in a plant cell can be present both in soluble (hydropectin) and insoluble (protopectin) forms, they are defined as soluble and insoluble components of DF.240

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2.1. Physiological activity of pectic polysaccharides Multiple results, which were obtained during studies of DF, were attributed to pectic polysaccharides as they are physiologically active components, which account for the major part of the polymer matrix of the primary cell wall of most of dicotyledons.170 It was established that a number of pectic polysaccha rides has a gastroprotective effect and is able to inhibit inflammation in the bowel.76,241â&#x20AC;&#x201D;244 It was found that citrus pectin enhances regeneration of the epithelium in the large intestine,245 and apple pectin diminishes bacteri al invasion and the endotoxin level in blood plasma of animals after the induced bowel inflammation.246 The an imal model of ulcerative colitis was used to demonstrate that, depending on structure, pectic polysaccharides are able to induce inflammation in the bowel wall of mam mals. This study also showed that galacturonan from any pectin has a pronounced anti inflammatory effect.247 Among different ways of pectin action on the gas trointestinal tract (GIT) the interaction with mucus has been most intensively studied. It is proved that pectins react with mucin, which is the major component of GIT, and form a gel network.248 A number of mechanisms has been suggested for interaction of pectins with mucus. It is probable that this process involves molecular intercala tion, which is accompanied by mechanical stirring, for mation of hydrogen bonds, and van der Waals interac tions, thus favoring reaction of mucin and pectin both on the surface and in the surface layer.249 Analysis of rheo logical data for mixtures of mucin and pectin demonstrat ed synergy of these two polymers and allowed to calculate the value of mucoadhesive interaction.250 Investigation of rheological properties within the study of interaction of galacturonides with GIT epithelium showed that pectins with low degree of esterification and with large number of linear galacturonide regions readily adhere to GIT epithe lium.248 Molecular structure of pectins plays a key role in stabilization of the formed pectin mucin gel. Pectins with low and high degree of esterification, as well as pectin derivatives with amido groups, demonstrate different gel forming ability. Highly charged pectins have enhanced bioadhesion and are able to interact with the mucus to form stable gels. Besides, highly esterified pectins also in teract with mucous glycoproteins with the following gel formation. In this way, pectic polysaccharides, which bear amido and ester groups enhance the protective properties of the mucus barrier, and can be used in cases of GIT injuries and infections.251 It is possible to change the sta bility of the formed gels and distribution of pectin in the pectin mucin complexes by altering the molecular para meters of pectin. This opens the way to regulation of pec tin mucin interaction with a view to adjustment of the properties of pectin to the requirements of drug delivery and clinical therapy.251

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During fermentation of pectins comprised in the DF in the large intestine bacteria generate volatile short chain fatty acids, which are the energy source for the cells of the columnar epithelium.193,252 Volatile short chain fatty acids promote absorption of sodium and water and stimulate proliferation of cells of the large intestine.245,253 Results of one of the first comparative in vitro studies of bifidogenic properties of pectins and oligogalacturonans show that pectic polysaccharides with low degree of me thyl esterification are more preferable substrates for proli feration of intestinal microflora than their highly methyl estrified analogs. Bifidobacteria Bifidobacterium angula tum, Bifidobacterium infantis and Bifidobacterium adoles centis do not grow in the medium with highly methyl es terified pectins. Bacteria Bacteroides thetaiotamicron and Clostridium ramosum and such useful microorganisms, as Bifidobacterium lactis Bb12, Lactobacillus plantarum and Lactobacillus pentosus grow in the medium with highly methyl estrified pectins.254 Effect of apples on the human intestinal microflora is associated with the presence of pectin.255 Incubation of the samples of faeces of healthy people in the liquid nutri ent medium with apple pectin results in elevated content of bifidobacteria and lactobacteria in comparison to the pectin free medium. After healthy volunteers (eight peo ple) ate apples (two apples a day for two weeks) the con centration of bifidobacteria in their faeces increased (p < 0.05 on the 7 day and p < 0.01 on the 14 day). The number of lactobacteria and streptococcus/enterococcus also increased. Meanwhile, the number of lecithinase pos itive clostridia (including Clostridium perfringens) is sig nificantly decreased, and the tendency to decrease was observed for enterobacteria and pseudomonas.255 Pectic oligo and polysaccharides induce apoptosis in cells of human colorectal adenocarcinoma.256 Vast data evidence that their complex side chains play an important role in anti cancer activity and other physiological properties.257 The ability of pectins to bind ions of heavy metals, radionuclides and toxic compounds from the organism has been shown258â&#x20AC;&#x201D;260, which is due to the presence of long galacturonan regions in their macromolecules, which act as ion exchange materials.

It is anticipated that pectins due to their ability to form viscous solutions and gels can include bile salts, fat and cholesterol in a gel matrix thus minimizing their absorp tion.261 This action of pectins can result in elevated faecal excretion of lipids, cholesterol and bile acids and decrease of the cholesterol level in blood.261â&#x20AC;&#x201D;266 This process de pends mainly on degree of methyl esterification of galac turonic acid residues, molecular mass and viscosity of pec tins.267 It was also shown that pectins produce hepato protective effect against induced liver toxicity in experi mental animals,268,269 which is provided by the ability of pectins to bind toxic compounds, which are accumulated in the development of toxic hepatitis in GIT.

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In this way, pectic polysaccharides (as DF, dietary additives, food ingredients), when eaten, influence not only the digestion, but also many other processes, which take place in the human body, wherein their structure is of key importance.

2.2. Structural changes of pectic polysaccharides during digestion Structure of pectic polysaccharides can undergo sub stantial modifications at the action of secretions of GIT. The pectin structure can be influenced by рН, ionic strength, activity of pectolytic enzymes. In the stomach, рН ranges form 1.5 to 2.0, and in the small intestine it is increased to 7.5—8.8. At low рН values hydrolysis of glyco sidic bonds in pectins can be observed. With the help of pectolytic enzymes, which are secreted by the intestinal microflora, hydrolytic and non hydrolytic decomposition ( elimination) of the carbohydrate backbone in pectin macromolecules takes place.129,270—273 The complete picture of structural transformations of pectic polysaccharides, which are one of the main compo nents of the plant food, in the process of digestion is un clear so far. This is due to the absence of adequate analyt ical techniques for elucidation of changes of monosaccha ride content and structural modifications of pectins dur ing digestion in GIT. Many researchers use the method of neutral detergent fiber (NDF),274 which helps evaluate quantitative changes in insoluble components of DF, whereas the fraction of soluble components of DF, such as soluble pectins, which are not bound to other cell wall components, is not measured. More informative analyti cal method was suggested by Englyst.275,276 It allows to evaluate the quantitative changes of soluble and insoluble components of DF and to determine the monosaccharide content of all non starch polysaccharides, and separately water soluble and water insoluble polysaccharides. How ever, as cellulose and hemicellulose are included in DF along with pectins, the problems arise, which concerns determination of monosaccharide content of separate components of insoluble DF. Different in vitro and in vivo models involving labora tory animals of volunteers were used for investigation of pectin digestion in GIT. In some experiments commercial pectins were used to elucidate their transformation during digestion, and in some cases degree of hydrolysis of pectic polysaccharides was evaluated directly in DF. Most experiments showed that pectins are not digested in the upper gastrointestinal tract, and are totally meta bolized by bacteria of the large intestine.277—284 It is as sumed that hydrolysis of pectic polysaccharides is fulfilled solely by enzymes, which are secretes by anaerobic intes tine flora (mostly of the large intestine) because no home enzymes were found inside the passageway of the human intestine, which are capable of catalyzing hydrolysis of

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pectins.232 It was shown that bacteria, which decompose carbohydrate bonds of pectic polysaccharides, populate the human large intestine. Although some of polysaccha ridases produced by bacteria from the human large intes tine are extracellular, the majority of the studied enzymes are bound to the bacterial cell wall.280 Of potentially large significance for the enteral pectin digestion is the fact that the majority of the microbial polysaccharide degrading enzymes are inducible.280 However, a number of experiments evidence that pec tins can be digested to some extent in the upper gastrointes tinal tract. In vivo experiments, which involved volunteers with ileostomas, which, on medical indications, under went removal of large intestine, showed, that up to 32% of pectic polysaccharides delivered with the plant food, is decomposed.281 In vivo experiments with commercial pec tin showed the degree of decomposition of pectins in the upper GIT to be 10—15%.283—285 Enteral digestion stud ies, which involved fermentation of commercial pectins with human faecal flora in vitro, demonstrated that pec tins are totally fermented in 6 h.286,287 In faeces of pa tients, which obtained 36 g of pectins (24 g of galacturon ic acid), no galacturonic acid was found.288 In vitro treat ment of apple pectin with faecal microflora from human faeces resulted in the decrease of the content of galactur onic acid by 37% in 6 h and by 90% in 12 h (see Ref. 289).

In beet pectin content of galacturonic acid residues de creased by 55% in 12 h of the treatment and only by 62% in 24 h. It is probable that the differences in the destruc tion of pectins are due to the presence of methoxyl and acetyl residues, as well as the ferulic acid residues.290 It was established that low esterified pectins are depolymerized and fermented faster than highly esterified ones and in 24 h are totally hydrolyzed in vitro by human faecal flora.291 In this way, accumulated to date information on di gestion of pectins in GIT is somewhat conflicting. The use of commercial pectins in these studies does not provide reliable data about their transformations, as the condi tions of industrial extraction of pectins are optimized for the better yield, and it is achieved by use of extractants with high pH, which results in the loss of side chains of the branched regions. As a result carbohydrate chains of com mercial pectins contain mostly HG regions, while natural pectic polysaccharides have diverse structure provided by their side chains, which have an impact on functional properties and biological activity of pectins. Fine struc ture, which determines physiological effects of pectins, can be extremely heterogeneous. Data on the structural modifications of pectic polysaccharides during digestion in GIT are of crucial importance, as there is a close rela tionship between the structure and physiological activity.

It was shown that structural changes have an effect on physiological activity: for example, fermentation of pec tins, which reduces the number of galactose and arabinose residues in side chains, decreases their physiological ac

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tivity.292 It was found that immunomodulating activity of arabinogalactan is diminished after the processing in a medium, which is close to gastric secretion. HPLC anal ysis showed that treatment of arabinogalactan with aque ous HCl (pH 2.00) and gastric juice (рН 1.95) causes similar destruction, which is accompanied by appearance of arabinose and smaller quantities of galactose, xylose, and mannose in the hydrolysate.Treatment of arabino galactan with gastric juice (рН 7.76) changes in monosac charide content of the polysaccharide were not observed, hence evidencing the effect of the acidity on the destruc tion of glycosidic bonds.293 3. Gel forming properties of pectins as dependent upon their physico chemical properties Pectic polysaccharides show the majority of their function al properties in a plant cell1,170 and as physiologically active components of dietary fiber54,251,252,254,255,258–266,268,269 in gel form. Ability of pectins to form gels was the first property, which was discovered during the studies of this type of plant polysaccharides, and it rendered their wide application in food and pharmaceutical indus tries.4,137,294,295 The process of gelation of pectins and their physico chemical properties are examined in detail using galacturonan as an example.

Conformational analysis of the galacturonan part of a pectin macromolecule seems to be the easiest. Use of X ray scattering, circular dichroism, and NMR spectro scopy made it clear, that galacturonan is an extended and flexible molecule that can exist in spiral conformations 21 and/or 31 (see Refs. 296 299) depending on the degree of hydration and the nature of counter ion in the solid/gel state. Results of computer modeling are in agreement with these data and indicate that conformational flexibility of galacturonan can be more expressed than it was anticipat ed. Along with spiral comformations 21 and 31, laevo rotating 32 and dextro rotating 41 spiral conformations300 are possible. Both the experimental and theoretical ap proaches were used for investigation of the process of complex formation of galacturonan with cations of poly valent metals.

Mechanism of pectin complex formation in the pres ence of metal ions is described within the approved model, which was suggested for metal complexes of anionic polysaccharides coordinated to s , p and d metals.301,302 According to this model deprotonated carboxyl groups of two acid residues of one part of a polysaccharide chain by means of ionic bonds form a negatively charged hydro philic cavity, which fits the size of a divalent metal cation.

Thus formed hydrophilic cavity is stabilized by van der Waals forces, hydrogen bonds and electrostatic interac tions.302—305 This is followed by cross linking of two par allel polysaccharide chains, which are in a spiral confor mation 21, to form egg box dimers. This model was

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suggested for complexes of  L guluronate with calcium ions, and later approved for a system calcium— D ga lacturonate.106,301,306 Alginate and pectinate molecules can be regarded as analogs because of structural similarities of their carbo hydrate chains, which are 1,4 -L guluronate and 1,4 D galacturonate, and, correspondingly, they bind with cal cium ions in a similar way.307,308 However, the latest stud ies indicate a number of differences of guluronate and galacturonate in binding with calcium ions. Computer modeling showed that the most favorable position of the galacturonate chains in initial dimers after the forma tion of ionic bonds is the antiparallel arrangement of chains in spiral conformations 31 and 21, which are stabilized, in addition to electrostatic interaction, with hydrogen bonds.309 The result of the antiparallel arrangement is the shifted egg box structure, in which the galacturonate chains are shifted by на 1.7 Å relative to each other; this favors more efficient binding of molecules due to additional in termolecular hydrogen bonds, van der Waals and electro static interactions310 and facilitates formation of cavities, which fit the size of the calcium ion. As a result, the chain length of galacturonate, which is sufficient for formation of stable binding site is shorter, than that of guluronate.309 Differenly from formation of alginate gels, where cal cium ions coordinate with various carbohydrate chains to form monocomplexes, egg box dimers and multimer struc tures,311 calcium ion binding during gel formation with low methyl esterified pectins includes mostly the first two stages. The following association of the previously formed egg box dimers into tetramers, hexamers, etc., can take place, but thus formed multimers do not have a specific structure, and, probably, are regulated by electrostatic in teractions alone. This behavior may be caused by structur al features of pectins. Random distribution of ester and amido groups alongside the pectin carbohydrate chain re sults in multiple defects during formation of this type of structure.312 Pectinate and alginate gels are characterized by different distribution of binding sites. Investigation of pectin gels in the presence of Са2+ using small angle X ray scattering (SAXS), analysis of mechanical properties, and the swelling capacity analysis showed that in the presence of Са2+ in pectin gels single randomly distributed binding sites are basically formed, in contrast to alginate gels, which are characterized by rod like binding sites with block distribution.313 Ability of pectins to bind cations of divalent metals is of primary importance both for the fulfillment of their functions in plant cell wall and for their application in various fields of economy. It is known, that in plant pec tins interact not with Са2+ ions alone, but also with Mg2+, Fe2+ and other ions.314 Ability of pectins,315 pectin con taining plant materials,316—320 gels321 and gel particles on the basis of pectins322,323 to absorb and desorb ions of metals is being studied with the view to their application

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as biosorbents for removal of heavy metals and radio nu clides from animal and human organisms,324,325 and puri fication of waste water, which contains ion of toxic metals, such as lead, copper, cadmium, and zink.326—328 The pos sibility was demonstrated to use pectins as flocculating or suspending agents in the presence of Fe3+ ions for prepa ration of stable and easily redisperged pharmaceutical sus pensions for a liquid system of drug delivery. Cations favor pectin adsorption at the surface of a suspended particle by lowering the negative charge of pectin molecules and by formation of cross links between the particles.329 Ability of pectins to selectively bind cations of metals can be influenced by the radius of the hydrated ion. It was found, that selectivity of citrus (DM 54%, DA 0%) and beet (DM 58%, DA 14%) pectins in cation binding is decreased in the following series: Cu2+ ~ Pb2+ >> Zn2+ > > Cd2+ ~ Ni2+ > Ca2+ (see Ref. 330). It was shown, that the strength of gels on the basis of citrus pectin (DM 30%), which were prepared with the use of solutions with similar cation concentration is increased in the following series of bound cations: La3+ < Ca2+ << Al3+ << Cu2+ (see Ref. 331). Besides, the number and the type of distribution of methoxyl and acetyl groups in the pectin carbohydrate chain also effect the configuration of the formed complex es. With the use of 13C CP MAS NMR spectroscopy it was established that non esterified calcium galacturonate has a spiral conformation 21, lead galacturonates adopt only the conformation 31, independent of degree of methyl es terification, partially acetylated galacturonates can adopt a broad range of conformations, which differ significantly depending on the bound cation.332 One of the factors responsible for the strength of inter action of carboxyl groups with cations is the number of methyl esterified residues of galacturonic acid, which are present in the carbohydrate chain of pectic polysaccha rides. Degree of methyl esterification defines the linear charge density of the macromolecule, which regulates the binding strength of pectins with cations. At high degree of esterification (DM > 50%), free negatively charged carb oxyl groups in pectic polysaccharides are distant from each other. When the degree of esterification is decreased, they are closer to each other, and, consequently, the charge of the macromolecule increases and facilitates stronger bind ing of pectins with cations. In the presence of 55% of sucrose or similar dehydrating agent at pH < 3.5 high methyl esterified pectins form gels due to formation of hydrophobic interactions and hydrogen bonds.333 In pec tic polysaccharides hydrophobic interactions between me thyl esterified carboxyls take place, mostly at decreased water activity, which occurs due to the presence of dis solved compounds. Hydrogen bonds are formed mostly at low рН values due to suppression of electrostatic repulsion between carbohydrate chains of pectic polysacccharides.334 It is the efficient gel formation that favors vast use of high methyl esterified pectins in food industry.4,136 Low methyl

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esterified pectins (DM < 50%) form gels in the presence of cations in the broad range of рН.296,305 Gel forming abili ty of low methyl esterified pectins in the presence of cat ions depends not only on the number of free carboxyl groups, but also on the type of their distribution along the carbohyrdate chain.129,335,336 According to plentiful ex perimental data the minimal quantity of sequentially con nected non esterified residues of galacturonic acid, which is necessary for stable cross linking of pectin carbohydrate chains and gel formation in the presence of calcium ions is 6 — 20 residues.335,337,338 Distribution of sequentially con nected non esterified residues of galacturonic acid along the carbohydrate chain also impacts the functional prop erties of pectin gels.339 Pectins, which have block distribu tion of non esterified carboxyl groups along the carbo hydrate chain, form gels more efficiently than molecules with similar DM, but with random distribution of non esterified carboxyl groups. Gel, which is formed by pectin with block distribution of non esterified residues of galac turonic acid, is stronger and more elastic.141,340,341 Yield strength measurements for specimen of different pectin based gels (DM 45%) indicate the considerable associa tion in the presence of calcium ions between pectin carbo hydrate chains with block distribution of non esterified carboxyl groups in contrast to pectins with random distri bution of these groups.298 Besides, pectins with carbo hydrate chains, which contain relatively short regions built of non esterified residues of galacturonic acid and distrib uted evenly along the whole carbohydrate chain, are able to form stronger gels, than pectins, where these residues form longer, but rare regions.342 The presence of acetyl groups favors the decrease of the degree of binding of calcium ions with pectin and pectic acid343,344 by inhibiting of the interaction of sec ondary hydroxyl groups with cation. It is known that acety lation of pectins results in inhibiting of coagulation and/or precipitation of pectins from solutions with the majority of tested cations of divalent metals (Ca2+, Mg2+, Sr2+, Ba2+, Zn2+, Mn2+, Co2+ и Ni2+), except Cu2+, Cd2+ and Pb2+ and polyvalent metals (Al3+, Fe3+, Cr3+ and Sn4+).345,346 Enzymatic deacetylation of beet pectin improves rheolog ical characteristics of gels, which are formed in the pres ence of calcium ions.347 Addition of sodium ions to pectin solutions results in decrease of electrostatic repultion between galacturonate carbohydrate chains due to formation of additional ionic bonds between carboxyl groups of galacturonic acid resi dues and sodium ions, thus significantly improving rheo logical parameters of the formed gel.348 After partial sub stitution (1—25%) of Na+ ions in sodium pectate for ions of s and d metals (Са2+ and Co2+, Cu2+, Fe2+) forma tion of water soluble types of complexes of pectins with metals was observed.349,350 Besides, pectins are able to bind different polycations, for example  L polylysine351 or polysaccharide chitosan, which contains amino groups.352

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Molecular mass of polysaccharide is among the fac tors, which have an effect on rheological characteristics of pectin gel in the presence of Ca2+ ions. Pectins with high molecular mass (over 100 kDa) are able to form strong gels. Depolymerization of a pectic polysaccharide reduces its gel forming ability, and the resulting gel is more fragile.347,353 In this way, structure of galacturonans, which form pectins, and position of ions, which are involved in cross linking, are of great importance for the arrangement of the gel structure, and, correspondingly, for exhibition of physico chemical properties of the pectin matrix.

The spatial organization of rhamnogalacturonan, structure and monosaccharide content of side chains, de gree of branching, degree of polymerization, and position of side chains in space determinate the contribution of branched areas of pectins in physico chemical properties of solutions and gels thereof. According to conformation al studies of pectic polysaccharides, the presence of a sin gle rhamnose residue in galacturonan should form a kink of the chain, differently from the relatively extended basic galacturonan chain. However, modeling has shown that introduction of more than two or three rhamnose residues into the carbohydrate chain of galacturonan returns the molecule to its extended state, which is close to that of the basic galacturonan. Predicted low energy conformation of the main carbohydrate chain of RG I, which comprises alternating residues of rhamnose and galacturonic acid was found to be one of the triple spirals.354 NMR analysis showed that side carbohydrate chains of RG I are rather flexible elements of the plant cell wall, which can be com pactly arranged near the carbohydrate backbone.354—356 Branched side chains are present in many natural polysaccharides, such as starch (amylopectin), galacto mannans, pectin, gum arabic. Presumably, side branches of carbohydrate chains substantially influence different physico chemical properties of polysaccharides, which are solubility, gelation, stability of the freezing — thawing process, film formation, by topological inhibition of inter molecular interactions. 357,358 Rheological theoretical approaches, which describe the effect of branching on rheological properties of polymers, are examined in detail using synthetic compounds and are often applied to describe the behavior of solutions of natural polysac charides.357,359,360 Rheological studies were conducted to evaluate the effect of branched side chains of pectins on physico chem ical properties of their solutions, which showed that be havior of branched macromolecules depends on the con centration of a polysaccharide in the solution. In diluted solutions, branched pectins, differently from linear ones, have smaller hydrodynamic volume, and their solutions are less viscous.361,362 Concentrated solutions of pectic polysaccharides, which have long and branched carbo hydrate side chains, are more viscous than solutions of

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compounds with similar molecular mass, but with negligi ble content of side chains.357,360 Circular dichroism anal ysis showed that conformation of pectin macromolecules does not depend on degree of branching of their carbo hydrate chains.363 Presumably, when concentration of so lutions is increased, side carbohydrate chains form addi tional intermolecular bonds, and, hence, rheological para meters are changed.357,360 Plentiful hypotheses were advanced, some of them con flicting with each other, which study the effect of branched areas of pectic polysaccharides on gelation, and few ex perimental works were reported, which focus on compari son of gelation properties of starting and modified pectins.

It is anticipated, that pectins with shorter side carbohydrate chains of RG I are more capable of gelation. Modified commercial apple pectin (Sigma), which was obtained by treatment with pectolytic enzymes, that have, i.a., endo glucanase, arabinase, galactanase and acetyl esterase ac tivity, forms gel with improved rheological parameters as compared to the starting polysaccharide. This pheno menon is attributed to the influence of steric factors, and, in particular, with the decrease of the length of carbo hydrate side chains built of neutral monosaccharide resi dues, and the increase of the negative charge of the modi fied pectin macromolecules.362 It was shown that treat ment of a pectin from carrot (Daucus сarota, Belgium) with high content of arabinose residues with a mixture of endo arabinase and arabinofuranosidase, which decrease the length of carbohydrate side chains in RG I, impaired rheological parameters of the gel, which if formed in the presence Са2+ ions. Authors anticipate that structural sta bility of pectic gels depends strongly on intermolecular interactions between long side chains consisting of ara binose residues, which form a tangle, hinder the move ments of the structural lattice, and improve rheological properties of gels.364 In this way, the contribution of structural features of the branched areas of pectins to physico chemical proper ties of their solutions and gels thereof still waites to de defined. Presumably, that regions of RG I enhance the rate of gel formation, facilitate formation of stable and strong gels and, at the same time, act as junction zone terminating structural elements, which restrict such events as turbidity, syneresis, and sedimentation during gela tion.129,339 As we see, the knowledge about the effect of structural organization of the entire pectin molecule on gel forma tion is not full. Formulation of the principles of formation of pectin gels and use of a number of pectins with defined structure will make it possible to apply them not only as gel forming agents for food and pharmaceutical indus tries, but also to obtain new biomaterials on their basis with pre defined physico chemical and functional prop erties, which will meet the contemporary requirements of biomedicine.295

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Conclusion

References

Pectic polysaccharides are complex plant polysaccha rides, which account for the functionally important part of primary cell walls. Pectic polysaccharides have extreme ly complicated and heterogeneous structure. Though sub stantial achievements have been made, especially in the field of elucidation of principal structural elements of pec tins, investigations of their physico chemical properties and biological activities, the problem of biosynthesis of pectic polysaccharides and definition of their fine struc ture still waits to be solved. We can anticipate that novel types of pectic polysaccharides will be described, which differ in the structure of side chains, biological functions and physiological activity. It is extremely important to elucidate the details of fine structure of pectins, which are character, structure and dimensions of side chains, and the nature of terminal monosaccharide residues, which, presumably, play a crucial role for biological and physio logical properties. The presence of protein components is not accidental and a profound study is needed to establish the relationship between polysaccharide chains and pro tein components and its function. Besides, pectic polysac charides which are essential components of the human ration, can effect the immunity, and this influence is not studied so far. It is known that pectic polysaccharides have immunostimulating action, but molecular mecha nism of their physiological action waits to be determined.

To achieve this aim, it is important to study the pathways of biotransformation of pectic molecules in the gastro intestinal tract. Still have to be clarified structural sites of pectins, which interact with regulating receptors in the gastrointestinal tract. The knowledge about the spa cial organization of the pectin molecule and its influence on gelation is limited to data on linear galacturonan.

Of particular interest is the data on the contribution of branched areas on the conformation of the macro molecule and gel forming ability of pectins, which can be used for formation of gels with predefined physico chem ical properties.

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This work was financially supported by the Rus sian Foundation for Basic Research (Projects № 12 04 00150 a, 13 04 92200) and Presidium of Russian Academy of sciences (Program "Molecular and cellular biology").

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Received January 20, 2014; in revised form August 6, 2014