Structural, rheological and functional properties of galactose-rich pectic polysaccharide fraction from leek

Journal Pre-proof Structural, rheological and functional properties of galactose-rich pectic polysaccharide fraction from leek Manol Ognyanov, Connie Remoroza, Henk A. Schols, Yordan N. Georgiev, Nadezhda Tr. Petkova, Magdalena Krystyjan

PII:

S0144-8617(19)31217-2

DOI:

https://doi.org/10.1016/j.carbpol.2019.115549

Reference:

CARP 115549

To appear in:

Carbohydrate Polymers

Received Date:

2 September 2019

Revised Date:

14 October 2019

Accepted Date:

27 October 2019

Please cite this article as: Ognyanov M, Remoroza C, Schols HA, Georgiev YN, Petkova NT, Krystyjan M, Structural, rheological and functional properties of galactose-rich pectic polysaccharide fraction from leek, Carbohydrate Polymers (2019), doi: https://doi.org/10.1016/j.carbpol.2019.115549

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Structural, rheological and functional properties of galactose-rich pectic polysaccharide fraction from leek Manol Ognyanova,*, Connie Remorozab, Henk A. Scholsb, Yordan N. Georgieva, Nadezhda Tr. Petkovac, Magdalena Krystyjand a

Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences,

Wageningen University & Research, Laboratory of Food Chemistry, Bornse Weilanden 9, 6708

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b

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Laboratory of Biologically Active Substances, 139 Ruski Blvd., 4000 Plovdiv, Bulgaria

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WG Wageningen, The Netherlands

University of Food Technologies, Technological Faculty, Department of Organic Chemistry and

University of Agriculture in Kraków, Faculty of Food Technology, Department of

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d

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Inorganic Chemistry, 26 Maritza Blvd., 4002 Plovdiv, Bulgaria

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Carbohydrates Technology, ul. Balicka 122, 30-149 Kraków, Poland

Manol Ognyanova* ([email protected]; [email protected]) Connie Remorozab ([email protected]) Henk A. Scholsb ([email protected])

Yordan N. Georgieva ([email protected])

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Nadezhda Tr. Petkovac ([email protected]) Magdalena Krystyjand ([email protected])

*

Corresponding author. Tel./Fax: +359 32 642759; E-mail: [email protected]; [email protected] M. Ognyanov, PhD 1

Highlights An acid-extracted pectin from leek consisted of a high Mw population (10-100 kDa).

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The pectin is mainly built up by HG segments with different blocks of ester distributions.

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RG-I segments were substituted with galactan side chains with varying length.

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The pectin exhibited higher water holding capacity than oil-holding capacity.

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Interaction between pectic segments may control the rheological properties.

Abstract

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An acid-extracted polysaccharide from alchohol-insoluble solids of leek was obtained. The sugar

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composition indicated that galactose and galacturonic acid were the major sugars, followed by small amounts of rhamnose and arabinose. The fraction contained a relatively high methylesterified homogalacturonan next to rhamnogalacturonan type I decorated with galactose-rich side chains. The fraction consisted of three high Mw populations, covering the range of 10–100 kDa. Enzymatic fingerprinting was performed with HG/RG-I degrading enzymes to elucidate the structure. The oligomers were analysed using LC-HILIC-MS, HPAEC, and MALDI-TOF MS. 2

The data revealed the presence of GalA sequences, having different patterns of methylesterification, RG-I composed of unbranched segments and segments heavily substituted with β(1→4)-linked galactan chains of varying length. The rheological study showed the shearthinning, weak thixotropic, anti-thixotropic, and non-Newtonian behavior of the polysaccharide. The pectin exhibited higher water holding capacity than oil-holding capacity and the fraction did

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form stable foams at high concentration.

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Key words: leek, pectic polysaccharides, enzymatic fingerprinting, HILIC-MS, rheology 1. Introduction

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Leek is botanically known as Allium ampeloprasum var. porrum L. and it is a member of

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the Alliaceae family in which also onion and garlic are included (Swamy & Gowda, 2006). Leek has been widely cultivated as a vegetable, but members of the family play a role as culinary

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herbs, spices, ornamental, and aromatic plants, as well (Swamy & Gowda, 2006). Leek is commonly known for its specific sharp odour and acrid taste. The whole plant or separate parts

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(leaves and long white stems) are cooked or freshly consumed as salads. The consumption of leeks is related to the observation of various beneficial health effects on the human body, such as antioxidant, antimicrobial, anticancer activity, hepatoprotective, antidiabetic, anti-inflammatory and gastroprotective effects (Lim, 2015). All these activities are mainly attributed to various

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chemical components present, such as dietary fibers, sulfur-containing compounds, saponins, vitamins, macro- and micro-elements and polyphenols (Fattorusso, Lanzotti, Magno, & Taglialatela-Scafati, 1998; Bernaert et al., 2012; Koca & Tasci, 2016). Most of the studies have been focused on the low-molecular weight compounds, although investigations related to the chemical composition provide us with incomplete and insufficient information about 3

carbohydrate present (Breu, 1996). Non-structural carbohydrates, including sugars, together with fructooligosaccharides and inulin have been found in leeks (Van Loo, Coussement, De Leenheer, Hoebregs, & Smits, 1995). Detailed studies entirely devoted to structural cell wall carbohydrates, comprising cellulose, hemicellulose and pectin were difficult to find, although the mentioned polysaccharides are very important active components responsible for the biological activity and functional properties of foods. Considerable attention must be paid to the pectic polysaccharides

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since its contribution to the biological activity and physiological functions have been previously

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shown (Yamada & Kiyohara, 2007).

In general, pectic substances are acidic polysaccharides, that play an important role in the

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primary cell wall and middle lamella. Pectic polysaccharides are composed of homogalacturonan

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(HG) and rhamnogalacturonan-I (RG-I) regions and a highly complex and conservative rhamnogalacturonan-II (RG-II) fragment. HG consists of a linear molecule composed of D-

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galacturonic acid (GalpA) units joined by α-(1→4)-glycosidic bond (Albersheim, Darvill, O’Neill, Schols, & Voragen, 1996). The GalpA residues can be partially methyl-esterified at C-6

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and acetyl-esterified at O-2 and/or O-3. RG-I is a side-chain-containing polysaccharide, whose backbone consists of the disaccharide [→4-α-D-GalpA-(1→2)-α-L-Rhap-(1→]n repeating units (Albersheim et al., 1996). The side chains in the RG-I region are mainly composed of α-(1→5)linked L-arabinofuranosyl residues, β-(1→4)-linked galactopyranosyl residues, and/or two types

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of arabinogalactan (AG I and II) of varying length and composition (Ridley, O'Neill, & Mohnen, 2001). RG-II has a HG backbone of eight GalpA residues substituted by four oligomeric side chains (defined as A–D), consisting of different sugars, including GlcpA, rhamnose, and monosaccharides rarely observed in other polymers, such as apiose, 2-O-methyl-fucose, 2-Omethyl-xylose, aceric acid (3-C-carboxy-5-deoxy-L-xylofuranose), 3-deoxy-D-manno-24

octulosonic acid (Kdo), and 3-deoxy-D-lyxo-2-heptulosaric acid (Dha) (Buffetto et al., 2014). The structure of pectic substances is extremely complex and depends on factors such as raw material origin and extraction conditions used (Voragen, Beldman, & Schols, 2001). Several reports have been previously published on the subject of the pectic polysaccharides from leek (Schols & Voragen, 1994; Kratchanova et al., 2008; Kratchanova, Nikolova, Pavlova, Yanakieva, & Kussovski, 2010; Nikolova et al., 2013; Ognyanov, Nikolova,

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Yanakieva, Kussovski, & Kratchanova, 2013). Nikolova et al. (2013) investigated the effect of

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polysaccharides isolated from fresh leek on the production of reactive oxygen and nitrogen

species by phagocytes. Polysaccharides markedly activated RAW 264.7 macrophages for reactive

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nitrogen species production in a concentration-dependent manner. Further analysis revealed that

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the effect was due to stimulation of the expression of NO synthase of murine macrophages. Kratchanova et al. (2010) demonstrated the ability of the polysaccharides to activate serum

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complement and enhance killing activity of the macrophages obtained from the pectin-treated mice. Research carried out by Ognyanov et al. (2013) indicated that galactose-containing

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polysaccharide from leek contributed to the activation of the complement system. Summarizing, these studies were mainly focused on the isolation and a first preliminary characterization of leek pectin, and evaluation of its biological activities. Consequently, the obtained polysaccharides were not fully structurally characterized and some chemical features were still insufficiently

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investigated. Information concerning pectin fragments, especially HG, some chemical characteristics (DA, etc.), methylester and acetyl group distribution in leek pectin as well as the evaluation of physicochemical and rheological properties are still missing. It could be recalled that the best-known uses of pectin are mainly in the food industry as gelling, thickening, and stabilizing ingredients (Rolin, 2002). Because of this, the study of some physicochemical 5

characteristics and rheological properties are of great importance and are required for a further successful development and understanding of the applications. The structural data would therefore reinforce the hypothesis of the structure-functional properties relationship and potential of the polysaccharide from non-traditional sources to be explored as an functional ingredient. In order to investigate this hypothesis, the structural characteristics of a galactose-rich polysaccharide fraction from leek were systematically

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elucidated by enzymatic fingerprinting (HG- and RG-I degrading enzymes) in a combination

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with several chromatographic methods (HILIC MS/ELSD, HPSEC, HPAEC) for analysis of oligomers. To further test the hypothesis, we studied rheological characteristics, foaming,

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emulsifying properties, water-holding, and oil-holding capacity, as well.

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2. Materials and Methods

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2.1. Preparation of alcohol-insoluble solid and polysaccharide isolation The leek was bought from the local market (Plovdiv, Bulgaria) and stored at -18ºC until use.

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Alcohol-insoluble solids (AIS) material was prepared from the white part (bulb) and stem as described by Kratchanova et al. (2008). Polysaccharides were extracted according to a procedure as described before (Kratchanova et al., 2010). Briefly, leek AIS was extracted with distilled water and the insoluble residue was further treated with 0.5% aqueous hydrochloric acid solution

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(final pH 2.0), yielding an acid-extracted polysaccharide fraction (AELkP). The fraction so obtained was further used in this study. 2.2. Degree of methyl esterification (DM) and acetylation (DA) AELkP (1 mg/ml) was saponified in 0.1M NaOH and after neutralization, the methanol content was determined using a combined enzymatic/colorimetric method (Klavons & Bennett, 1986). 6

The acetyl content was determined by using an acetic acid test kit (Megazyme (Bray, Wicklow, Ireland)) according to the instructions of the manufacturer. The degrees of methylation and acetylation were expressed as moles of methyl esters or acetyl groups per 100 moles of galacturonic acid, respectively. 2.3. Constituent monosaccharide composition analysis

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The AIS material was analyzed for its neutral sugar composition by gas chromatography (Englyst

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& Cummings, 1984), using inositol as an internal standard. The samples were treated with 72% (w/w) H2SO4 for 1 h at 30ºC, followed by hydrolysis with 1M H2SO4 for 3 h at 100ºC. Prior to

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analysis by gas chromatography, the released neutral sugars were converted to volatile alditol acetates (Blakeney, Harris, Henry, & Stone, 1983). The monosaccharide composition analysis

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(including GalA and GlcA) of AELkP was carried out by HPAEC-PAD after methanolysis with

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anhydrous 2M HCl in methanol for 16 h at 80ºC, followed by hydrolysis with 2M TFA for 1 h at 121ºC according to De Ruiter, Schols, Voragen, & Rombouts (1992).

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2.4. Uronic acid content

The uronic acid content of AIS material was estimated as described by Ahmed & Labavitch (1978). In brief, the AIS sample was firstly pre-hydrolysed with 12M H2SO4 for 1 h at 30ºC,

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followed by dilution to 1M H2SO4 and hydrolysis for 3 h at 100ºC. An aliquot of hydrolysate was taken for automated m-hydroxy-diphenyl analysis using a continuous flow analyzer Skalar San++ system (Skalar Analytical BV, Breda, Netherlands) (Thibault, 1979). Absorption was measured at 530 nm and galacturonic acid (12.5–100.0 μg/ml) was used for a calibration curve construction. 2.5. Qualitative test for 3-Deoxy-D-manno-2-octulosonic acid (Kdo) 7

The qualitative estimation of polysaccharide-linked Kdo was performed by the periodate thiobarbituric acid colorimetric assay (Karkhanis, Zeltner, Jackson, & Carlo, 1978). A modified method of York, Darvill, McNeil, & Albersheim (1985) was used. Initially, the sample (2 mg) was hydrolyzed for 30 min at 95ºC in 1 ml of 0.1M H2SO4. The hydrolysate was cooled into an ice water bath and then it was centrifuged for 10 min at 4ºC (18,228 ×g). Further, sodium metaperiodate solution (40 mM in 62.5 mM H2SO4, 0.25 ml) was added to the supernatant (0.5

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ml), and the mixture was allowed to stand for 20 min at room temperature. The oxidation was

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stopped by adding sodium sulfite solution (2.0% in 0.5M HCl, 0.3 ml). The sample was vortexmixed till the disappearance of brown color and after that freshly prepared aqueous 2-

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thiobarbituric acid solution (0.6%, 0.5 ml) was added. The mixture was incubated in a boiling

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water bath for 15 min and immediately after that DMSO (1 ml) was added to each test tube. After cooling to room temperature, the absorbance of the pink color was measured at 548 nm against a

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reagent blank prepared with distilled water instead of sample hydrolysate. Lavender polysaccharide chPS-L2 fraction was used as a positive control (Georgiev et al., 2017).

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2.6. Yariv test

The presence of arabinogalactan (type II) protein (AGP) was detected by the single radial gel diffusion test with β-D-glucosyl Yariv reagent (Biosupplies, Australia) as described by Van Holst

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& Clarke (1985).

2.7. Protein and L-Hydroxyproline content Protein content was estimated with the Bradford assay using Coomassie® Brilliant blue G-250 dye (Amresco®) and bovine serum albumin as a standard (Bradford, 1976). Hydroxyproline was assayed following the method of Kivirikko & Liesmaa (1958) after hydrolysis of the sample (5 8

mg) at 110ºC with 6M HCl (1 ml) in sealed tubes for 16 h. cis-4-L-Hydroxyproline was used as a standard. The reagent preparation and methodology were performed as described by York, Darvill, McNeil, Stevenson, & Albersheim (1986). 2.8. Total phenolic content Total phenols were determined with the Folin-Ciocalteu reagent using ferulic acid as a standard

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(Singleton & Rossi, 1965).

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2.7. Controlled enzymatic hydrolysis

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Leek pectin (5 mg/ml) was dissolved in 0.05 M sodium acetate buffer (pH 5.2) and incubated for 24 h with purified and well characterized HG- and RG-I-degrading enzymes. The enzymes used

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in the current study were Aspergillus aculeatus endo-galactanase (EC 3.2.1.89) (van de Vis, Searle-van Leeuwen, Siliha, Kormelink, Voragen, 1991), Chrysosporium lucknowense (C1) exo-

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arabinase (EC 3.2.1.1) (Kühnel et al., 2010), endo-arabinanase (EC 3.2.1.99) (Beldman, Searlevan Leeuwen, De Ruiter, Siliha, & Voragen, 1993), and RG-hydrolase A enzyme (EC 3.2.1.B9)

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(Mutter, Renard, Beldman, Schols, & Voragen, 1998), pectin lyase I (EC 4.2.2.10) (Van Alebeek, Christensen, Schols, Mikkelsen, & Voragen, 2002) and endo-polygalacturonase II (EC 3.2.1.15) (Limberg et al., 2000). Enzyme doses were sufficient to degrade theoretically their corresponding

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substrates within 6 h into monomers. The pectin solution was firstly incubated for 6 h with RG-I enzymes, followed by addition of endo-PG-II and subsequent incubation for another 18 h. The hydrolysis was performed at 40 °C in a head-over-tail rotator (Eppendorf® Thermomixer) at 850 rpm. The enzymes were inactivated at 100 ºC for 5 min and after cooling the digest was centrifuged at 20,311 ×g, 5 ºC for 10 min. The supernatant obtained was further analyzed for molecular weight distribution and oligomeric products by high-performance size-exclusion 9

chromatography, high-performance anion-exchange chromatography, and hydrophilic interaction liquid chromatography coupled to MS and ELSD detectors. To get information about the structural features of RG segments in leek pectin, AELkP was saponified and extensively digested by endo-PG I-M2 from Asp. aculeatus (Megazyme International Ltd., Bray, Co. Wicklow, Ireland) for the removal of maximum amount of HG fragments. The undegraded substrate was recovered by alcohol precipitation as described

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(Ognyanov et al., 2013). The fraction, referred to as AELkP-EPG, was further treated with RG-

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degrading enzymes as described above and was analysed by MALDI-MS.

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2.8. High performance size-exclusion chromatography (HPSEC) analysis of molecular weight distribution

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Pectin samples (initial and digest) were analyzed using HPSEC as described elsewhere

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(Remoroza et al., 2012). The analysis was performed on a Dionex Ultimate 3000 system coupled with a Shodex RI-101 refractive index detector using one guard column (6 mm i.d. × 150 mm)

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and three TSK-Gel super AW columns connected in series (4000, 3000 and 2500; 6 mm i.d. × 150 mm). Ten microliter of sample (2.5 mg/ml) was injected and eluted at 55ºC with 0.2 M sodium nitrate at a flow rate of 0.6 ml/min. The system was calibrated with pectin standards in the range of 10–100 kDa (Remoroza, Broxterman, Gruppen, & Schols, 2014).

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2.9. High performance anion exchange chromatography (HPAEC) The galacturonic acid oligomers were monitored using an ICS5000 HPAEC system (Dionex Corp. Sunnyvale, CA, USA) with pulsed amperometric and UV detection and controlled by Chromeleon 7.0 software (Dionex Corp.). The sample was diluted with water (1:1) before analysis and 10 μl was injected on a CarboPac PA-1 guard column (2 mm i.d. × 25 mm) attached 10

to a CarboPac PA-1 anion-exchange column (2 mm i.d. × 250 mm) and eluted with two mobile phases A) 0.1 M NaOH and B) 1 M NaOAc in 0.1 M NaOH with a flow rate of 0.3 ml/min. For the separation of uronic acid mono- and oligomers the following gradient was used: 0–60 min 20–70% B, 60–65 min70–100% B, 65–70 min 100% B, 70–70.1 min 100–20% B. The column was re-equilibrated in 20% B for 15 min (Remoroza, Buchholt et al., 2014). Peaks were identified by comparing their retention times with those of the saturated (DP 1–5) and unsaturated (DP 2–6)

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GalA oligomers with different concentrations. Calculated response factors from the peaks area

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were used to quantify oligogalacturonides in the digest (Remoroza et al., 2012).

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2.10. Hydrophilic interaction liquid chromatography (HILIC) MS/ELSD

Digests, diluted to 1 mg/ml in 50% (v/v) acetonitrile were analyzed using UPLC–ELSD–MSn

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coupled to an evaporative light scattering detector (Agilent 1200 series, Gen Tech Scientific Inc.,

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NY, USA) and an ESI-IT-MSn-detector (LTQ Velos Pro ion trap MS, Thermo Scientific). Chromatographic separation was performed on an Acquity UPLC BEH amide column (1.7 μm,

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2.1 mm × 150 mm) in combination with a Van Guard pre-column (1.7 μm, 2.1 mm × 5 mm; Waters Corporation, Milford, MA, USA). The elution was performed at a flow rate of 0.5 ml/min at 35ºC. The injection volume was 5 μl (Remoroza, Buchholt et al., 2014). The amounts of oligomers were quantified by ELSD using GalA oligomer standards as previously described

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(Remoroza et al., 2012).

2.11. Matrix-assisted laser-desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS)

The matrix solution was prepared by dissolving 20 mg 2,5-dihydroxybenzoic acid in 1 ml acetonitrile:distilled water (1:1 v/v). One μL of this solution was placed on the sample plate 11

(MTP 384) together with 1 μL of the sample solution and dried under a constant flow of warm air. The sample plate was then placed in the instrument. MALDI-TOF-MS were recorded on an UltrafleXtreme™ workstation (Bruker Daltonics, Bremen, Germany) equipped with a nitrogen laser of 337 nm and operated by FlexControl 3.3 software (Bruker Daltonics). The mass spectrometer was operated in a positive linear mode. The delayed extraction time was 120 ns and the acceleration voltage 25 kV. Mass spectra were calibrated with an external standard containing

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2.12. Fourier transformed infrared (FT-IR) spectroscopy

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mixture of maltodextrins (300–3000 Da).

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The sample (4 mg) was mixed with spectroscopic grade KBr and then was pressed into a pellet. FT-IR spectrum was collected on a Nicolet Avatar 330 (Thermo Electron Corp., USA)

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spectrometer. The spectrum was recorded over a wavenumber range of 4000–400 cm−1 at 132

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scans with a spectral resolution of 4 cm−1. Analysis and processing of the obtained spectrum were performed using the Spekwin32 software (version 1.71.5).

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2.14. Functional properties 2.14.1. Swelling properties

The swelling properties of the leek pectin were evaluated as previously described by Robertson et

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al. (2000). In brief, the dried pectin (100 mg dry weight) was hydrated in 10 ml distilled water in a calibrated cylinder (1.5 cm diameter) at room temperature. After equilibration (18 h), the bed volume was recorded and expressed as volume/g original substrate dry weight. 2.14.2. Water- holding and oil-holding capacity

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The water-holding and oil-holding capacities of the AELkP were determined in duplicate as described (Holloway & Greig, 1984). The samples (100 mg) were placed into pre-weighed 50 ml polypropylene centrifuge tubes and 10 ml deionized water or sunflower oil was added. The tubes were tightly closed, and the contents were vigorously mixed. They were held for 24 h at 20ºC before centrifugation at 3000 ×g for 15 min; the excess water or oil was decanted, and the tubes were inverted for 1 h at 20ºC. The tubes were then weighed and dried at 105ºC to constant

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weight.

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2.14.3. Foaming properties

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The foaming properties of the isolated pectin were studied by a stirring/shaking method (CanoMedina et al., 2011). The series of different concentrations of pectin solutions were prepared (0.5,

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1.0, 1.5, and 2.0% w/w). All foam tests were performed in duplicate. Reproducibility of the

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results was typically expressed as mean ± 10%. The foam ability (FA, %) was determined as an aliquot of 15 ml pectin solution whipped in a graduated 50 ml cylinder by hand for 60 s. The

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foam ability was determined by volume increase (%) immediately after shaking and was calculated by

FA, % 

(V1  V2 ) 100 V1

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where V2 is the volume of pectin solution immediately after whipping, and V1 is the volume of solution before whipping. The foam stability (FS, %) is characterized by the volume of entrapped air, still remaining in the foam after a certain period of time, t > 0. The foam stability was defined as the volume of the foam that remained after 60 min at room temperature (20ºC) and was expressed as a percentage 13

of the initial foam volume. The test was performed as described by Ivanov, Petkova, & Denev, (2017). The foam stability was given by the parameter percentage volumetric foam stability FS ,%  (V1 foam  V0 foam )

where “V0 foam” is the volume of the formed foam; “V1 foam” is the volume of the foam change with the time (t). Foam stability over time was assessed by measuring the foam volume from 1 to

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60 min.

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2.14.4. Emulsifying properties

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The emulsion capacity (EC) and emulsion stability (ES) of leek pectin were investigated as previously described by Wang et al. (2018) with a slight modification. Two types of emulsions

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with 50 % and 30 % oil phase were prepared with different concentrations of leek pectin (0.5, 1,

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2, 2.5 and 5%, w/v). In brief, 7 ml aqueous pectin solutions were mixed with 3 ml sunflower oil at room temperature. The emulsions were prepared by homogenization using high-speed shear homogenizer (T25, IKA Co., Germany) at 10,000 rpm for 1 min. Then the mixture was

EC (%) 

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centrifuged at 3000 ×g for 20 min. The EC was calculated using the equation: Emulsion volume 100 Total volume

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For evaluation of ES the prepared 50/50 (o/w) and 30/70 (o/w) emulsions were heated at 80 ºC for 30 min. Then they were cooled down to room temperature and centrifuged at 3000 ×g for 20 min. The ES was calculated as follows: ES (%) 

Final emulsion volume 100 Total volume

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2.15. Rheological characterization 2.15.1. Sample preparation Leek pectin solutions were prepared by dissolving appropriate amount of pectin (0.5, 1, 2.5 and 5% w/v) in distilled water. The solutions were stirred for 24 h at 25ºC in order to get complete hydratation. The pH of solutions was below 3.

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Leek pectin solution with sucrose addition was prepared in the same way as leek pectin. After 24 h of mixing to pectin solution was added sucrose (65%, w/v) and mixed for another 1 h. Then the

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solution was mixed for 1 h at 90ºC.

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2.15.2. Rheological analyses

Flow curves of pectin solution was carried out according to Krystyjan, Khachatryan, Ciesielski,

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Buksa, Sikora (2017) procedure using an RS6000 (Gebrueder Haake GmbH, Karlsruhe, Germany) rheometer in CR mode with a CC26 Ti measuring system and 1.9 gap, with

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measurements determined at 25ºC. The shear rate was raised from 0 to 100 s-1, over a 5-min period and a subsequent decrease of shear rate from 100 to 0 s-1, also over 5 min. The flow curves

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  K  ( )n

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obtained were described by Ostwald-de Waele rheological model:

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where τ is shear stress (Pa), K is consistency coefficient (Pa·sn),  is shear rate (s-1), and n is flow

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behavior index (-).

The areas of thixo- and antithixotropy hysteresis loops were calculated according to the method described by Sikora et al. (2015) by the summing up of the areas of particular trapeziums formed between the curves “up” and “down”. 2.15.3. Statistics 15

The experimental data were subjected to an analysis of variance, at the confidence level of p=0.05, using Statistica v. 8.0 software (Statsoft, Inc., Tulsa, OK, USA). Fisher test was used for determination of statistically significant differences. 3. Results and discussion 3.1. Isolation and characterization of AIS and AELkP

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3.1.1 Yield and chemical characterization of AIS

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The current study is focused on the isolation and characterization of a pectic polysaccharide from leek by extraction with a hydrochloric acid-aqueous solution. As a first step, we prepared AIS

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from leek as a source of cell wall material. The AIS fraction represented 36.7% (w/w) of the

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dried vegetable or 3.5% w/w of the fresh material (Table 1). These values were slightly lower than in previous study (Kratchanova et al., 2010). Further analysis of the sugar composition,

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which has not been reported before, showed that the AIS consisted mainly of glucose (21.7% w/w; 43.5 mol%), uronic acids (13.5% w/w; 25.1 mol%) and galactose (8.6% w/w; 17.2 mol%)

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(Table 1). In addition, AIS is composed of lower amounts of xylose (2.7% w/w; 6.4 mol%), arabinose (1.4% w/w; 3.3 mol%), and mannose (1.4% w/w; 2.8 mol%). Rhamnose (0.5% w/w; 1.1 mol%) and fucose (0.3% w/w; 0.7 mol%) were found in minor amounts. The monosaccharide composition suggests that leek AIS consisted of different types of polysaccharides, including

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cellulose, pectin, and hemicelluloses. The total carbohydrates accounted for 50.1% of the AIS, while the remaining part included other cell wall constituents, such as protein, non-extracted polyphenols (tannins, lignin) and moisture. 3.1.2 Yield and chemical characterization of AELkP

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The sequential extraction of the AIS could provide information about the presence and extractability of the different polysaccharide populations. Therefore, the leek AIS was sequentially extracted starting with hot water. The water unextractable part was further extracted with a hot diluted hydrochloric acid solution to obtain an acid-solubilized polysaccharide fraction. The yield and results of the composition analysis are summarized in Table 1. The diluted hydrochloric acid extraction resulted in the solubilisation of 13.1% (w/w dw) of the AIS. In

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addition, the acid treatment solubilized 43.1% of the total uronic acids present in the AIS, and the

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obtained fraction was characterized as an acidic polysaccharide having 45.0% w/w uronic acids (Table 1). The residue from the cell wall extraction still contained 35% of the uronic acids

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present in the AIS keeping in mind the yield and uronic acid content of the water-soluble

3.2. Monosaccharide composition

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polysaccharide fraction (4.7% w/w dw, 62% w/w, 21.5% uronic acid recovery).

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The extracted polysaccharide was assessed using sugar composition analysis as results were summarized in Table 1. The total sugar content of the fraction was 96.6% (w/w) while neutral

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sugars (51.6%) slightly predominated compared to the uronic acids (45.0%). The analysis for separately determination of D-glucuronic acid (GlcpA) and galacturonic acid demonstrated that GalpA was the predominant hexuronic acid (43.0% w/w). The fraction contained small amounts of GlcpA (2.0% w/w) which might be incorporated within the RG-I segments, Hyp-

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arabinogalactan subunit or directly linked to the GalpA in pectin backbone (Tan et al., 2010). The investigated polysaccharide consists mostly of the commonly reported pectic neutral sugars. Galactose (48.5% w/w) was the major sugar, which represented 94.0% of the total neutral sugars. Impressively, water solubilized only 4.0% of the total galactose present in the AIS, while the AELkP fraction represented 74% thereof. Moreover, other sugars such as arabinose (0.7% w/w) 17

and rhamnose (2.4% w/w) were present only in small amounts. The presence of rhamnose and galacturonic acid, combined with a higher amount of galactose, is typical for pectins with a RG-I backbone substituted with galactans (Ridley et al., 2001). Galactans are usually decorated with a small amount of arabinose which indicates the presence of arabinogalactans (type I and/or type II) as side chains. In addition, the lower (0.07) ratio between rhamnose and GalpA, which is indicative for the contribution of RG-I blocks within the pectin backbone, showed that the pectic

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population contained only low amounts of RG-I segments. The RG-I segments had a relatively

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high ratio (Ara + Gal)/Rha (18.8) approximately indicating the average length of the side chains. The acid-extracted pectic polysaccharide could be characterized with a low number of long-side

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chains. Other sugars such as glucose, mannose, fucose, and xylose were not found suggesting the

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lack in extractability of hemicellulose. In order to investigate the presence of RG-II, in the obtained fraction, the thiobarbituric acid assay for Kdo presence was performed. As a result, it

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could be noticed that the fraction contained a low amount of thiobarbiturate-positive substances (+0.115 absorbance unit/2 mg) compared to the lavender chPS-L2-positive control (+0.342 unit/2

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mg). This indicated that Kdo was present in minor amounts in a polysaccharide-bound form, and only a low amount of RG-II segments constituted this pectin fraction. Additionally, a minor amount of polyphenols (0.2%) and some proteins (4.1%) were present as non-carbohydrate cell wall components. The presence of protein suggested that some part of galactosyl and arabinosyl

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residues could also originate from highly branched arabinogalactan protein type II complexes which were additionally confirmed with weak but positive Yariv reactivity and 4-Lhydroxyproline presence (Table 1). It is known that such components could be incorporated in the networks of other cell wall constituents (pectin, cellulose, etc.) or covalently linked to them (Du, Clarke, & Bacic, 1996). Generally, the monosaccharide composition suggested that the acid18

extracted pectin fraction was predominantly composed of HG as a main building block and a highly branched RG-I substituted by galactose-rich side chains (arabinogalactans and/or galactans). These results were in agreement with previous study (Redgwell & Selvendran, 1986). These authors have isolated from onion (Allium cepa L.) polysaccharide containing mainly uronic acid and Gal together with a small proportion of neutral sugars such as Rha and Ara.

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3.3. Degree of methyl esterification and acetylation

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The polymer fraction isolated from leek was characterized as a high-methyl-ester pectin with degree of esterification 65%. The degree of methyl esterification strongly determines the physical

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properties of pectin. The fraction contains also other non-sugar substituents such as acetyl groups. The acetyl content was found to be only 0.4% w/w, and the fraction was characterized as a low

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acetylated pectin (DA 2.5 mol%) which was comparable with that of commercial citrus pectin.

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The result for DA was expressed as acetyl content per D-GalpA unit assuming that only D-GalpA residues were acetylated. However, previous studies showed that not only GalA residues in HG

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region, but also L-Rhap and galacturonosyl units in the RG-I segments could be partially Oacetylated (Remoroza, Buchholt et al., 2014). It should be pointed out that an high acetyl content has rather negative effect on the gelling and rheological properties of pectic polysaccharides. Typically, apple, citrus, and sunflower pectins are characterized as a low-acetylated, while sugar

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beet, potato, and plum exhibited higher DA (15-35 mol%) (Voragen et al., 2001). 3.4. Fourier Transform Infrared (FT-IR) Spectroscopy The FT-IR spectrum of AELkP fraction is presented in Fig. S3. The typical bands for pectic polysaccharides were found, and most of the bands coincided with previous report (Kačuráková, Capek, Sasinková, Wellner, & Ebringerová, 2000). The broadened band that occurs in the region 19

from 3730 to 3100 cm−1 was assigned to the ν(O–H) stretching vibrations of free hydroxyl groups involved in intra- and intermolecular H-bonding. The band at 2939 cm−1 was attributed to ν(C–H) stretching of CH2 groups. The presence of well-separated sharp signal that appears at 1749 cm−1 was for C=O stretching vibration of methyl-esterified carbonyl groups and it is characteristic for pectin: ν(C=O)COOH (COOCH3) of GalpA. The bands at 1648 cm−1 and 1446 cm−1 were assigned to the asymmetric νas(O=C–O) and symmetric νs(O=C–O) stretching vibrations of the non-

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esterified carboxyl groups, respectively. The weak absorption peak at 1413 cm−1 is typical for

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νs(COO−) of salts of pectic acid. The signal observed at 1373 cm−1 was due to ν(C–C) and

scissoring δ(CH2) ring vibration and those at 1338 cm−1, 1238 cm−1 were induced by the δ(C–H)

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vibrations of pectic acid salt. Bands occuring at 1147 cm−1, 1101 cm−1, and 1018 cm−1 were

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associated with the glycosidic bond vibrations involving ν(C–O–C), ν(C–C)(C–O), and δ(O=CH) bending in the pyranose ring indicated the presence of the HG segments in the polymer

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fraction. According to Barros, Coimbra, Barros, Rutledge, & Delgadillo (1997) uronic acidcontaining polysaccharides show intensive bands at 1100 and 1018 cm−1. Further analysis of the

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spectrum suggested β-arabinogalactan and/or β-galactan presence, because of appearance of bands at 1074 cm−1, 1049 cm−1, and 890 cm−1 that coincided with previously published report (Kačuráková et al., 2000).

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3.5. Molecular weight distributions of initial and enzyme degraded AELkP The molecular weight distribution of the initial leek pectin was determined by HPSEC as shown in Fig. 1A. It can be seen that the fraction consisted of a high molecular weight population, although a broad molecular weight range (> 100 kDa and < 10 kDa) was covered. In this range, three populations were recognized which were fully eluted < 12.0 min. HPSEC was also used for 20

monitoring the degradation of AELkP by HG/RG-degrading enzymes. The elution pattern is shown in Fig. 1B. Considerable change in the elution pattern was especially observed in the higher molecular weight range of the degraded fraction (~100 – 70 kDa). Combined enzyme treatment resulted in the conversion of part of the high molecular weight material into lower molecular weight compounds explaining the shift in the retention time for part of the material. By this way, it was demonstrated that the enzymes were able to solubilize pectin sample to oligomers

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which were eluted at retention time between 12 min and 14.5 min. However, Fig. 1B shows also

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that a residual material with high molecular weight (~ 64-20 kDa; 9-11 min), resistant to the combined action of HG- and RG-degrading enzyme, still present. Probably, this resistant

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polymeric material could be ascribed to highly branched RG-I fragments and/or HG domain with

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specific methylester distribution.

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3.6. Separation and identification of oligomer products

3.6.1. HPAEC analysis of the leek pectin digests after enzymatic digestion

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HPSEC did not reveal any information about the structure of the released fragments. Therefore, the digest was analyzed with HPAEC for the separation and identification of low molecular

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weight oligomers.

Fig. 2 line C illustrates that as a result of enzyme treatment saturated monomers and oligomers with different DP (2-13) were dominantly present. It can be observed that saturated pentamers were co-eluted with unsaturated trimers, but other unsaturated oligomers were not accumulated. 21

Among the total released oligosaccharides, a higher amount of saturated octamers (8) and nonamers (9) (33.6%) followed by hepta- (7) and deca-GalpA oligomers (27.7%) were found. The detected oligomers were released from the HG region of the pectin fraction. These oligomers must be methyl-esterified in different extent as the Me-ester level and distribution pattern did not allow them to be further endo-PG-degraded. The sum of mono-, di-, and trimer was 12.3% of the totally released mono- and oligomers as proportion and ratio between them was 1.0:2.0:2.6 and

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10/(20+30) = 0.22, respectively. This suggested the presence of small blocks of non-esterified

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GalA residue (Daas, Voragen, & Schols, 2000; Remoroza, Broxterman et al., 2014). Further, quantification of the HPAEC peaks showed that endo-PG/PL degradation of the fraction released

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15% GalpA mono- and oligomers of the total GalpA amount originally presented in the polymer

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fraction. The lower recovery value indicated that only a relatively low part of the pectin could be degraded during digestion with endo-PG and PL and demonstrated that part of the AELkP

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fraction was organized as HG. Surprisingly, some part of the HG region was inaccessible for endo-PG and PL action, probably due to a specific structural organization and a quite special

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methyl-ester distribution. Unfortunately, information about methyl-ester content of the oligomers could not be obtained by HPAEC, since a higher pH was employed during chromatographic separation.

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3.6.2. HILIC analysis of the leek pectin digests after enzymatic digestion Because of the inability to obtain precise structural information, the diagnostic oligosaccharides were further characterized by HILIC analysis, which enables a good separation of most oligomers present in the digest (Remoroza et al., 2012). The HILIC-MS elution pattern is shown in Fig. 3.

22

Additionally, a summary of m/z value of all oligomers, retention time, and the structure for the identified oligosaccharides is shown in Table 2.

As can be observed from the HILIC elution profile, a great variety of saturated partly Meesterified and un-esterified GalpA oligomers were accumulated in the digest. Only, dimers (200),

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trimers (300), and part of the tetramers (400) were found to present without any methyl-ester groups. The HILIC chromatogram shows that the digest contained higher DP saturated oligomers

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which is consistent with the results of HPAEC elution pattern. Amongst the hydrolysis products,

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pentamers containing one or two methyl-esters were detected (510 and 520). Interestingly, endoPGII resistant oligosaccharides with higher DP having two methyl-esters were present, e.g. 620,

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720, 820, 920. The saturated oligomer of DP 11 carrying 3 methyl-ester groups (1130) was also found. Such saturated and randomly Me-substituted GalpA oligomers originated from the endo-

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PGII action on HG region. The acetylated GalpA residues with/without methyl-ester groups were not identified, suggesting the absence of acetylated ‘endo-PG degradable’ GalpA sequences in

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the HG backbone. Additionally, Fig. 3 shows the presence of a low amount of unsaturated GalpA tetramer containing three methyl-ester groups (u430) in the digest which was not recognased in the HPAEC elution pattern. Two structures were proposed to be uGalAMe-GalA-GalAMe-GalAMe

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and uGalAMe-GalAMe-GalAMe-GalA (Table 3) based on the MS2 fragmentation (Remoroza et al., 2012). This shows that pectin HG from leek contained not only unsubstituted domains but also ‘PL-degradable’ highly methyl-esterified GalpA sequences. Further analysis of the elution profile shown in Fig. 3 indicates that a variety of RG-I backbonerelated oligomers such as (Rha-GalpA)2, (Rha-GalpA)3 were present. The accumulation of such kind of oligosaccharides (DP 4-6), containing no side chain, is in agreement with the mode of 23

action of RG-hydrolase that cleaves GalA-(1→2)-Rha glycosidic linkage within un-branched RG-I segments (Mutter et al., 1998). These results suggested that some Rha units were not substituted. In a recent study, un-substituted oligomers with the same DP have been identified in pectin from Panax ginseng (Sun et al., 2019). In addition, RG-I oligomers composed of Rha units substituted at O-4 with single Gal residue, along with RG oligomers consisting of O-acetylated GalpA units, were found, e.g., (Rha-GalpA)2Gal, (Rha-GalpA)3Ac. As can be noted, acetyl

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groups were only found in RG-I oligosaccharides. This was in agreement with the findings of

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Remoroza, Broxterman et al. (2014) who have detected in the digest of sugar beet pectin not only acetylated HG oligomers, but also a RG-I oligomer having an acetyl group. The presence of Gal

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linked to Rha units demonstrated that the direct connection of galactose-containing side chains to

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the RG-I backbone of the AELkP exists. The presence of Ara residue, separately or in combination with Gal, was not identified in the HILIC elution pattern. Somewhat, this could be

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due to the considerable lower amounts of the monosaccharide in the fraction (0.9 mol%, Table 1). Also, the Ara/Rha ratio (1:3) indicated a very low number of neutral Ara residues attached to the

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RG-I backbone. Arabinose moieties might be not present in arabinanase-degradable form because of the presence of too short side chains and/or linked to other sugar residue (e.g. Gal). In the digest, as a result of endo-β-(1→4)-D-galactanase (A. aculeatus) treatment a mixture of Gal oligosaccharides with different DP (Gal2-5) was accumulated (Fig. 3). The enzyme hydrolyses the

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(1→4) glycosidic linkage between β-linked galactosyl units within galactan chains to monomeric galactose residues and galacto-oligosaccharides (Gal2-3) (van de Vis et al., 1991). It means that galactose-rich side chains contain linearly β-(1→4)-linked galactose units, e.g. [β-D-Gal-(1→4)β-D-Gal-(1→4)-β-D-Gal]n. 3.7. Methylester and acetyl group distribution in AELkP 24

Based on the different diagnostic oligomers present after an extensive HG/RG enzymatic digestion, it was hypothesized that the distribution of methyl and acetyl esterification differ over the leek pectin backbone and different types of blocks exist. The finding of unsaturated oligomers (u430 – HILIC; u3 – HPAEC) in the digest indicated the presence of areas with highly methylesterified GalpA units that were easily degraded by PL. Further, the presence of nonesterified saturated GalpA oligomers without methyl esters and acetyl ester groups (100, 200, 300)

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demonstrated the presence of a few ‘endo-PGII degradable’ blocks in the HG segments, as well.

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In addition, it could also be suggested existence of partly methyl-esterified blocks, represented by GalpA oligomers with DP 5–11 carrying 2–3 methylesters substituted in a random manner. Based

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on the identified oligosaccharide structures, blocks of GalpA sequences substituted with acetyl

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groups was not found to present in the HG backbone. However, the presence of the partially

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acetylated RG-I region in leek pectin was confirmed.

3.8. MALDI-TOF MS analysis of the leek pectin digests after enzymatic digestion

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In order to reveal more structural information about AELkP fraction, the oligomer products accumulated after treatment with HG/RG enzymes were analysed by MALDI-TOF-MS (Fig. S1). From the MS spectrum, a wide range of oligomers of declining DP from 4 – 14 GalpA units partly substituted with 1 to 5 methyl esters (GalA4Me1 m/z 1039 – GalA14Me5 m/z 2679, etc.)

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were recognized. The presence of un-substituted oligomer with DP 4 was also observed (GalA4, m/z 849). Such non- and low-Me-esterified GalpA oligomers originated from the endo-PGII action on HG backbone, which confirmed the results obtained by HILIC-MS analysis. More interesting MALDI-TOF-MS pattern was observed for RG-degraded AELkP-EPG (Fig. S2). Based on the mass spectrum, galactose-substituted RG-I-derived oligomers with DP of 4 or 8 25

were detected. Several peaks corresponding to oligosaccharides consisted of Gal side chains with declining (m/z 162) length were identified, e.g. two sugar residue (Rha-GalA)4Gal2, single residue (Rha-GalA)4Gal, and no side chain-containing octamer (Rha-GalA)4 (m/z 1597, 1435, 1273). Keeping in mind that glycosidic linkage between Rha-Gal could not be hydrolyzed by endo-galactanase (van de Vis et al., 1991), the presence of such oligomer indicated that some part of Rha residues in initial polymer were not branched. The unbranched RG-I backbone oligomers

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with DP 4 and 6 were reported (Remoroza et al., 2012; Sun et al., 2019), while (Rha-GalA)4

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corresponding to DP 8 was not observed earlier as a result of RG-hydrolase treatment. Oligomers composed of side chain with Gal4 and Gal3 units (m/z 1377, 1175) were also present, e.g. (Rha-

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GalA)2Gal4, (Rha-GalA)2Gal3. Because galactanase requires at least (≥3) three linear Gal residues

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for action (van de Vis et al., 1991), it was unlikely that four Gal units are attached to single Rha residue. Therefore, the four Gal units should be distributed between the two Rha residues in a

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different pattern which, incidentally, has been demonstrated (Gur’janov, Gorshkova, Kabel, Schols, van Dam, 2007). The detection of such oligosaccharides also confirmed directly

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connection of galactose-containing side chains to the RG-I backbone presence. Further, the presence of RG oligomer with oligomeric side chain composed of Ara residue in combination with Gal was identified (Rha-GalA)2GalAra2 (m/z 1111). In this structure, both Ara units might be linked to the Gal residue as in another published study (Zheng & Mort, 2008). However,

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single Ara moieties directly linked to Rha unit was not detected. Similar RG-I oligosaccharides with different substitution pattern have been already reported for flax stem (Gur’janov et al., 2007) and ginseng pectin fractions (Sun et al., 2019). Further analysis of the mass spectrum revealed the presence of neutral fragments with DP ranging from 4 to 8. The m/z value (689 to 1337) corresponded to the M + Na+ ions. The pattern of galacto-oligomers (Gal4-8 + Na+) was in 26

consistent with that estimated previously by Zheng et al. (2018) using MALDI-TOF MS technique for characterization of potato galactan oligosaccharides. Interestingly, endogalactanase displayed low activity towards Gal oligomers with higher DP. These oligosaccharides could be accumulated as a result of degradation of a very heavily substituted RG-I. The side chains must be composed of several branching points and/or different types of linkage between Gal residues. Summarizing, although leek pectin did contain low amounts of

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RG-I domain, the latter was composed of unbranched segments and regions with varying

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substitution pattern and Gal-containing side chains structure. The length of the side chains varies from 1, 2, and 3 Gal residue substitutions. Also, the ratio between Gal and Rha in initial polymer

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was 18.5 indicated the average lenght of the side chains. However, as above-mentioned, not all

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DP and Gal/Rha ratio may also be present.

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Rha units were substituted with Gal residues suggested that branched side chains with a higher

3.9. Rheological properties of leek pectin fraction

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Pectin is well-known for its gelling, thickening, emulsifying, and stabilizing properties, which enables it to be incorporated in food products (Rolin, 2002). Therefore, physico-chemical and rheological behaviour need to be additionally studied. The rheological properties of AELkP solutions was presented in Fig. 5 and Table 3,

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respectively. As it was confirmed by n value (n<1) all investigated samples were non-Newtonian liquids, but at low concentrations (0.5 and 1.0%) their rheological properties were close to the Newtonian fluids. This is due to the fact that in diluted pectin solutions homogeneously dispersed particles are too far apart to interact with each other. As mentioned already by Guimarães, Coelho Júnior, & Garcia Rojas (2009), the observed slight increase in the viscosity of these systems is 27

the result of the distortion of the liquid velocity pattern by an increased share of hydrated molecules. The shear-thinning nature of pectin solutions increased with their increasing concentration (Table 3). This behaviour has also been noticed for pectin isolated from tamarillo fruit (Nascimento, Simas-Tosin, Iacomini, Gorin, & Cordeiro, 2016). With the increasing of leek pectin concentration in solution, a statistically significant increase in the K-factor coefficient was noted (Table 3). In the case of 0.5, 1 and 2.5% concentrations the

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shear stress for the mentioned systems were at a very low level (Fig. 5A). Only at a concentration

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of 5% higher viscosity was observed. It means that at higher concentration intermolecular

interactions between the pectin molecules increased, as the distance between them decreased

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(Guimarăes et al., 2009). Pectin solutions were characterized by weak thixotropic and anti-

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thixotropic properties (Table 3) which provides ability to rebuild damaged structure due to the large number of hydrogen bonds. According to Oakenfull & Scott (1984) hydrogen bonding is

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the main interactions that keep pectin gel structure. However, it is insufficient to overcome the entropic barrier to gelation. These processes highly depend on the presence of co-solutes such as

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sucrose, glucose, fructose or polyols like glycerol (Tsoga, Richardson, & Morris, 2004), which stabilize junction zones by promoting hydrophobic interaction between methyl-ester groups (Oakenfull & Scott, 1984). Additionally, pH and temperature are factors that affect the gelation process. At low pH electrostatic repulsions along and between pectin chains are reduced, and

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non-dissociated carboxylic groups form inter- and intramolecular hydrogen bonds with secondary alcohol groups. da Silva, Gonçalves, & Rao (1995) confirmed that at intermediate temperature the hydrogen bonds and hydrophobic interactions are intensified. In our case of leek pectin solutions, the obtained systems were characterized by pH below 3, and the temperature of mixtures preparation was 25°C. Nevertheless, Evageliou, Richardson, & Morris (2000) claimed 28

that high stability of pectin gel structure could be obtained at 30°C despite of the pH difference. The mechanical spectra of leek pectin solutions (Fig. 5B) confirmed that they were stable in time, however, they exhibited the properties of concentrated solutions rather than gels. The presence of sucrose in the solution increased the viscoelastic properties of the solution, but still with the

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majority of the former.

It must be stated that many structural features such as the presence and distribution of methyl-

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and acetyl-ester groups, and consecutive galacturonic acid residues in the polysaccharide

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determine mechanical properties and mechanism of gelation (Buchholt, Christensen, Fallesen, Ralet, & Thibault, 2004). In general, a highly methyl-esterified pectins form a gel at acidic pH (<

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3.5) in the presence of large amounts of sugars through hydrophobic interactions and hydrogen bonding. On the other hand, the gelation of low-esterified pectins is mainly realized with Ca2+

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ions by forming ‘egg-box’ junction zones with free carboxylic groups from the independent pectin chains. However, another type of structure-depending gel-formation including RG-I

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backbone and neutral sugar side chains have been recently introduced and well-investigated (Sousa, Nielsen, Armagan, Larsen, & Sørensen, 2015; Mikshina et al., 2017). Some evidences indicated that galactan side chains contributed to network-interconnection and stabilization of

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backbone, but too short chains are not capable of forming junction zones (Mikshina et al., 2017). Thus, an explanation of the observed properties could be found in the chemical composition and molecular weight distribution. For instance, high methyl-esterified (≥ 80%) citrus pectins, wellknown for their ability to form gels, have galacturonic acid content over 75 mol%, while neutral sugars are minor components (Kaya, Sousa, Crépeau, Sørensen, & Ralet, 2014). In contrast, the 29

AELkP fraction was characterized with 65% degree of methyl esterification, 43.0 mol% galacturonic acid content, and galactose was the major sugar (51.8 mol%). This suggested the possible contribution of galactan side chains to the formation of compact structure together with HG-related gelation. However, polysaccharide, simultaneously composed of HG domain and RG-I with galactan side chains in nearly equal molar amounts, showed properties of concentrated solution rather than gel regardless of the appropriate conditions for gelation. This could be due to

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the peculiarity of the organization and distribution of the side-chains and HG segments, which

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may be involved in self-regulation (control) of association and network gel-formation.

Alternatively, gel-formation ability could be improved through modification or with preparation

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of mixed gels, i.e. composed of leek pectin and alginate.

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3.10. Functional properties

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3.10.1. WHC and OHC

The swelling properties of AELkP were 13 ml water/g sample and this values demonstrated

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ability of the polysaccharide fraction to absorb water. The obtained value was lower than that of citrus pectin (19 to 21 ml/g sample) (Wang et al., 2018). The WHC value was 12.47±0.90 g water/g sample which was higher than its OHC. WHC of leek pectin demonstrated values close to WHC of pectin from Fumaria officinalis in vitro cultures (Ognyanov et al., 2018). WHC is

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used to evaluate the stability, texture and sensory of sample and with its results leek pectin demonstrate good ability to hold water. Our results were lower than reported value for potato pectin (37.84 g water/g sample) (Yang, Mu, & Ma, 2018). However, oil holding capacity of AELkP (2.84±0.25 g oil/g) was in a good agreement with reported values for water-soluble polysaccharides from Rosa roxburghii Tratt fruits (3.29±0.38 g oil/g) and pectin from Fumaria 30

officinalis in vitro cultures (2.4 g oil/g), respectively (Wang et al., 2018; Ognyanov et al., 2018). Therefore, OHC of leek pectin could bring to potential use of this polysaccharide as mouthfeel enhancer in food design. 3.10.2. Foaming and emulsion properties The results for foaming ability and foaming stability of AELkP fraction are shown in Fig. 6A and

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Fig. 6B, respectively.

The results demonstrated that leek polysaccharide formed foams and emulsions at a concentration

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range from 0.5 to 5%. However, the 5% water solution of this pectin was viscous and hardly formed foams. The FA values increased with increasing the concentration of pectin solution. FA

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of 2.5 and 5% solutions demonstrated insignificant differences. At a low concentration (0.5%)

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leek pectin did not form stable foams. Only for 5 min the foaming stability decreased with more than 20%. The leek pectin concentration from 1 to 5% formed stable foams, as the most stable were foams formed with 2.5% leek pectin.

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For evaluation of the emulsifying properties of leek polysaccharide EC and ES were defined. ES is important factor determining the self life of commertial products and an indicator of the effectiveness of emulsifier to stabilize emulsion. The results were presented in Fig. 6C and Fig.

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6D. As shown, EC and ES values increased with the increase of concentration. Emulsion capacity of 5 % leek pectin in 50/50 o/w and 30/70 o/w emulsions reached 77-71%. The EC of 2% concentration of 30/70 o/w reached 37%, that was comparable with EC of the same emulsion type prepared with 2% pectins from Rosa roxburghii Tratt fruits (Wang et al., 2018), EC: 37 and

31

40%, respectively. Moreover, the formed 50/50 o/w emulsions with AELkP were more stable than 30/70 o/w emulsions. 4. Conclusion For the first time, we reported an in-depth structural characterization of leek polysaccharide fraction obtained by dilute hydrochloric acid extraction using enzymatic fingerprinting approach

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and modern analytical methods. The fraction consisted of HG composed of different blocks of nonesterified, partly methylesterified, and highly methylesterified GalA residues and low amount

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of a highly branched RG-I substituted by β-(1→4)-linked galactan side chains. The results

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revealed that polysaccharide had good foaming properties, oil-holding, and emulsifying capacity, although it exhibited the properties of concentrated solutions rather than gels, caused of unique

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and complexity of the structure. The rheological behavior may be controlled by interaction between polysaccharide fragments in which HG and RG-I/galactans seems to be mainly involved.

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The physicochemical characterisation of leek pectin could be useful for further investigation regarding its biological activity properties and potentially utilisation separately or in combination

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with other ingredients (polysaccharides, protein, etc.) in acidic emulsion-based foods including mayonnaise, dressings, dairy products, meat products as a fat replacer and as a health-promoting functional ingredient.

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Acknowledgment

This study was supported by Project BG051PO001/3.3-05 “Science and Business”, financed by the European Social Fund of the European Union and Bulgarian Ministry of Education and Science and partly by Project BG161PO003-1.1.05-0024-C0001 “Development of nutraceuticals

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with antioxidant and immune-stimulating action” of Operational Program “Competitiveness” of the European Union. References Ahmed, A. E. R., & Labavitch, J. M. (1978). A simplified method for accurate determination of cell wall uronide content. Journal of Food Biochemistry, 1, 361–365.

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Albersheim, P., Darvill, A. G., O’Neill, M. A., Schols, H. A., & Voragen, A. G. J. (1996). An hypothesis: the same six polysaccharides are components of the primary cell walls of all higher plants. In J. Visser, & A. G. J. Voragen (Eds.), Progress in Biotechnology, (pp. 47–55). Amsterdam: Elsevier Science.

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Barros, M., Coimbra, A. M., Barros, A., Rutledge, D., & Delgadillo, I. (1997). Analysis of uronic acid in pectic material by FT-IR spectroscopy. In P. Carmona, R. Navarro & A. Hernanz (Eds.), Spectroscopy of Biological Molecules: Modern Trends (pp. 275-276). Dordrecht: Kluwer Academic Publishers.

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Beldman, G., Searle-van Leeuwen, M. J. F., De Ruiter, G.A., Siliha, H. A., Voragen, A. G. J. (1993). Degradation of arabinans by arabinanases from Aspergillus aculeatus and Aspergillus niger. Carbohydrate Polymers, 20, 159-168.

lP

Bernaert, B., De Paepe, D., Bouten, C., De Clercq, H., Stewart, D., Van Bockstaele, E., De Loose, M., Van Droogenbroeck, B. (2012). Antioxidant capacity, total phenolic and ascorbate content as a function of the genetic diversity of leek (Allium ampeloprasum var. porrum). Food Chemistry, 134, 669–677.

ur na

Blakeney, A. B., Harris, P. J., Henry, R. J., & Stone, B. A. (1983). A simple and rapid preparation of alditol acetates for monosaccharide analysis. Carbohydrate Research, 113, 291– 299. Bradford, М. М. (1976). A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248-254.

Jo

Breu, W. (1996). Allium cepa L. (Onion) Part 1: Chemistry and analysis. Phytomedicine, 3, 293306. Buchholt, H. C., Christensen, T. M. I. E., Fallesen, B., Ralet, M.-C., Thibault, J.-F. (2004). Preparation and properties of enzymatically and chemically modified sugar beet pectins. Carbohydrate Polymers, 58, 149-161.

33

Buffetto, F., Ropartz, D., Zhang, X. J., Gilbert, H. J., Guillon, F., & Ralet, M.-C. (2014). Recovery and fine structure variability of RGII sub-domains in wine (Vitis vinifera merlot),” Annals of Botany, 114, 1327–1337. Cano-Medina, A., Jiménez-Islas, H., Dendooven, L., Herrera, R. P., González-Alatorre, G., & Escamilla-Silva, E. M. (2011). Emulsifying and foaming capacity and emulsion and foam stability of sesame protein concentrates. Food Research International, 44, 684–692. Daas, P. J. H., Voragen, A. G. J., Schols, H. A. (2000). Characterization of non-esterified galacturonic acid sequences in pectin with endopolygalacturonase. Carbohydrate Research, 326, 120–129.

ro

of

da Silva, J. A. L., Gonçalves, M. P., & Rao, M. A. (1995). Kinetics and thermal behaviour of the structure formation process in HMP/sucrose gelation. International Journal of Biological Macromolecules, 17, 25–32.

-p

De Ruiter, G., Schols, H. A., Voragen, A. G. J., & Rombouts, Fr. M. (1992). Carbohydrate analysis of water-soluble uronic acid-containing polysaccharides with high-performance anionexchange chromatography using methanolysis combined with TFA hydrolysis is superior to four other methods. Analytical Biochemistry, 207, 176-185.

re

Du, H., Clarke, A. E., Bacic, A. (1996). Arabinogalactan-proteins: a class of extracellular matrix proteoglycans involved in plant growth and development. Trends in Cell Biology, 6, 411-414.

lP

Englyst, H. N., & Cummings, J. H. (1984). Simplified method for the measurement of total nonstarch polysaccharides by gas-liquid chromatography of constituent sugars as alditol acetates. Analyst, 109, 937-942.

ur na

Evageliou, V., Richardson, R. K., & Morris, E. R. (2000). Effect of pH, sugar type and thermal annealing on high-methoxy pectin gels. Carbohydrate Polymers, 42, 245–259. Fattorusso, E, Lanzotti, V., Magno, S., & Taglialatela-Scafati, O. (1998). Sapogenins of Allium porrum L. Journal of Agricultural and Food Chemistry, 46, 4904-4908.

Jo

Georgiev, Y. G., Paulsen, B. S., Kiyohara, H., Ciz, M., Ognyanov, M. H., Vasicek, O., Rise. F., Denev, P. N., Yamada, H., Lojek, A., Kussovski, V., Barsett, H., Krastanov, A. I., Yanakieva, I. Z., Kratchanova, M. G. (2017). The common lavender (Lavandula angustifolia Mill.) pectic polysaccharides modulate phagocytic leukocytes and intestinal Peyer’s patch cells. Carbohydrate Polymers, 174, 948–959. Guimarães, G. C., Coelho Júnior, M. C., & Garcia Rojas, E. E. (2009). Density and kinematic viscosity of pectin aqueous solution. Journal of Chemical & Engineering Data, 54, 662–667. Gur’janov, O. P., Gorshkova, T. A., Kabel, M., Schols, H. A., van Dam, J. E. G. (2007). MALDITOF MS evidence for the linking of flax bast fibre galactan to rhamnogalacturonan backbone. Carbohydrate Polymers, 67, 86-96. 34

Holloway, W. D., & Greig, R. I. (1984). Water holding capacity of hemicelluloses from fruits, vegetables and wheat bran. Journal of Food Science, 49, 1632-1633. Ivanov, I., Petkova, N., & Denev, P. (2017). Physicochemical characterization of pectin from orange peels obtained after different extracting conditions. Industrial Technologies, 4, 21–25. Kačuráková, M., Capek, P., Sasinková, V., Wellner, N., & Ebringerová, E. (2000). FT-IR study of plant cell wall model compounds: pectic polysaccharides and hemicelluloses. Carbohydrate Polymers, 43, 195–203.

of

Karkhanis, Y. D., Zeltner, J. Y., Jackson, J. J., & Carlo, D. J. (1978). A new and improved microassay to determine 2-keto-3-deoxyoctonate in lipopolysaccharide of gram-negative bacteria. Analytical Biochemistry, 85, 595–601.

ro

Kaya, M., Sousa, A. G., Crépeau, M-J., Sørensen, S. O., & Ralet, M.-C. (2014). Characterization of citrus pectin samples extracted under different conditions: influence of acid type and pH of extraction. Annals of Botany, 114, 1319–1326.

-p

Kivirikko, K. I., & Liesmaa, M. (1959). A colorimetric method for determination of hydroxyproline in tissue hydrolysates. Scandinavian Journal of Clinical and Laboratory Investigation, 11, 128–133.

re

Klavons, J. A., & Bennett, R. D. (1986). Determination of methanol using alcoholoxidase and its application to methyl ester content of pectins. Journal of Agriculture and Food Chemistry, 34, 597–599.

lP

Koca, I., & Tasci, B. (2016). Mineral composition of leek. In A.F. Gokce (Ed.), Acta Horticulturae № 1143, VII International Symposium on Edible Alliaceae (pp. 147–152). Nigde: International Society for Horticultural Science.

ur na

Kratchanova, M., Gocheva, M., Pavlova, E., Yanakieva, I., Nedelcheva, D., Kussovski, V., & Slavov, A. (2008). Characteristics of pectic polysaccharides from leek obtained through consecutive extraction with various reaction agents. Bulgarian Chemical Communications, 40, 561–567.

Jo

Kratchanova, M., Nikolova, M., Pavlova, E., Yanakieva, I., & Kussovski, V. (2010). Composition and properties of biologically active pectic polysaccharides from leek (Allium porrum). Journal of the Science of Food and Agriculture, 90, 2046-2051. Krystyjan, M., Khachatryan, G., Ciesielski, W., Buksa, K., Sikora, M. (2017). Preparation and characteristics of mechanical and functional properties of starch /Plantago psyllium seeds mucilage films. Starch/Stärke, 69, 1700014, DOI 10.1002/star.201700014. Kühnel, S., Hinz, S.W.A., Pouvreau, L., Wery, J., Schols, H. A., Gruppen, H. (2010). Chrysosporium lucknowense arabinohydrolases effectively degrade sugar beet arabinan Bioresource Technology, 101, 21, 8300-8307. 35

Lim, T. K. (2015). Edible medicinal and non medicinal plants. (vol. 9). Dordrecht: Springer, (Allium ampeloprasum). Limberg, G., Körner, R., Buchholt, H. C., Christensen, T. M. I. E., Roepstorff, P., Mikkelsen, J. D. (2000). Quantification of the amount of galacturonic acid residues in blocksequences in pectin homogalacturonan by enzymatic fingerprinting with exo- and endo-polygalacturonase II from Aspergillus niger. Carbohydrate Research, 327, 321-332.

of

Mikshina, P. V., Makshakova, O. N., Petrova, A. A., Gaifullina, I. Z., Idiyatullin, B. Z., Gorshkova, T. A., Zuev, Y. F. (2017). Gelation of rhamnogalacturonan I is based on galactan side chain interaction and does not involve chemical modifications. Carbohydrate Polymers, 171, 143151.

ro

Mutter, M., Renard, C. M. G. C., Beldman, G., Schols, H. A., Voragen, A. G. J. (1998). Mode of action of RG-hydrolase and RG-lyase toward rhamnogalacturonan oligomers. Characterization of degradation products using RG-rhamnohydrolase and RG-galacturonohydrolase. Carbohydrate Research, 311, 155-164.

re

-p

Nascimento, G. E., Simas-Tosin, F. F., Iacomini, M., Gorin, P. A. J., & Cordeiro, L. M.C. (2016). Rheological behavior of high methoxyl pectin from the pulp oftamarillo fruit (Solanum betaceum). Carbohydrate Polymers, 139, 125–130.

lP

Nikolova, M., Ambrozova, G., Kratchanova, M., Denev, P., Kussovski, V., Ciz, M., & Lojek, A. (2013). Effects of pectic polysaccharides isolated from leek on the production of reactive oxygen and nitrogen species by phagocytes. Journal of Medicinal Food, 16, 711-718. Oakenfull, D., & Scott, A. (1984). Hydrophobic interaction in the gelation of high methoxyl pectins. Journal of Food Science, 49, 1093–1098.

ur na

Ognyanov, M., Nikolova, M., Yanakieva, I., Kussovski, V., & Kratchanova, M. (2013). Influence of composition on the biological activity of pectic polysaccharides from leek. Journal of BioScience and Biotechnology, 2, 13-20. Ognyanov , M., Georgiev, Y., Petkova , N., Ivanov , I., Vasileva , I., & Kratchanova, M. (2018). Isolation and characterization of pectic polysaccharide fraction from in vitro suspension culture of Fumaria officinalis L. International Journal of Polymer Science, 2018, Article ID 5705036.

Jo

Redgwell, R. J., & Selvendran, R. R. (1986). Structural features of cell-wall polysaccharides of onion Allium cepa. Carbohydrate Research, 157, 183–199. Remoroza, C., Cord-Landwehr, S., Leijdekkers, A. G. M., Moerschbacher, B. M., Schols, H. A., & Gruppen, H. (2012). Combined HILIC-ELSD/ESI-MSn enables the separation, identification and quantification of sugar beet pectin derived oligomers. Carbohydrate Polymers, 90, 41–48.

36

Remoroza, C., Buchholt, H.C., Gruppen, H., Schols, H. A. (2014). Descriptive parameters revealing substitution patterns in differently prepared sugar beet pectins. Carbohydrate Polymers, 101, 1205-1215. Remoroza, C., Broxterman, S., Gruppen, H., Schols, H. A. (2014). Two-step enzymatic fingerprinting of sugar beet pectin. Carbohydrate Polymers, 108, 338-347. Ridley, B. L., O'Neill, M. A. & Mohnen, D. (2001). Pectins: structure, biosynthesis, and oligogalacturonide-related signaling. Phytochemistry, 57, 929–967.

of

Robertson, J. A., de Monredon, F. D., Dysseler, P., Guillon, F., Amado, R., & Thibault, J.-F. (2000). Hydration properties of dietary fibre and resistant starch: a European collaborative study. LWT - Food Science and Technology, 33, 72–79.

ro

Rolin, C. (2002). Commercial pectin preparations. In G. B. Seymour, & J. P. Knox (Eds.), Pectins and their Manipulation (pp. 222–241). Oxford: CRC Press/Blackwell Publishing.

-p

Schols, H. A., & Voragen, A. G. J. (1994). Occurence of pectic hairy regions in various plant cell wall materials and their degradability by rhamnogalacturonase. Carbohydrate Research, 256, 8395.

re

Sikora, M., Adamczyk, G., Krystyjan, M., Dobosz, A., Tomasik, P., Berski, W., Lukasiewicz, M. & Izak, P. (2015). Thixotropic properties of normal potato starch depending on the degree of the granules pasting. Carbohydrate Polymers, 121, 254–264.

lP

Singleton, V., & Rossi, J. (1965). Colorimetry of total phenolic with phosphomolibdiphosphotungstic acid reagents. American Journal of Enology and Viticulture, 16, 144-158.

ur na

Sousa, A. G., Nielsen, H. L., Armagan, I., Larsen, J., Sørensen, S. O. (2015). The impact of rhamnogalacturonan-I side chain monosaccharides on the rheological properties of citrus pectin. Food Hydrocolloids, 47, 130-139. Swamy, K. R. M., & Gowda, R. V. (2006). Leek and shallot. In K. V. Peter (Ed), Handbook of herbs and spices (pp. 365–389). Abington Cambridge: Woodhead Publishing Limited.

Jo

Sun, L., Ropartz, D., Cui, L., Shi, H., Ralet, M.-C., Zhou, Y. (2019). Structural characterization of rhamnogalacturonan domains from Panax ginseng C. A. Meyer. Carbohydrate Polymers, 203, 119-127. Tan, L., Varnai, P., Lamport, D. T. A., Yuan, C., Xu, J., Qiu, F., & Kieliszewski, M. J. (2010). Plant O-hydroxyproline arabinogalactans are composed of repeating trigalactosyl subunits with short bifurcated side chains. Journal of Biological Chemistry, 285, 24575-24583. Thibault, J.-F. (1979). Automatisation du dosage des substances pectiques par la méthode au méta-hydroxydiphényl. Lebensmittel-Wissenschaft und Technologie, 12, 247-251. 37

Tsoga, A., Richardson, R. K., & Morris, E. R. (2004). Role of cosolutes in gelation of highmethoxy pectin. Part 1. Comparison of sugars and polyols. Food Hydrocolloids, 18, 907-919. Van Alebeek, G.-J. W. M., Christensen, T. M. I .E., Schols, H. A., Mikkelsen, J. D., & Voragen, A. G. J. (2002). Mode of action of pectin lyase A of Aspergillus niger on differently C6substituted oligogalacturonides. Journal of Biological Chemistry, 277, 25929-25936. Van de Vis, J. W., Searle-van Leeuwen, M. J. F., Siliha, H. A., Kormelink, F. J. M., Voragen, A. G. J. (1991). Purification and characterization of Endo-1,4-β-D-galactanases from Aspergillus niger and Aspergillus aculeatus: Use in combination with arabinanases from Aspergillus niger in enzymic conversion of potato arabinogalactan. Carbohydrate Polymers, 16, 167-187.

of

Van Holst, G. J., & Clarke, A. E. (1985). Quantification of arabinogalactan-protein in plant extracts by single radial gel diffusion. Analytical Biochemistry, 148, 446-450.

ro

Van Loo, J., Coussement, P., De Leenheer, L., Hoebregs, H., & Smits, G. (1995). On the presence of inulin and oligofructose as natural ingredients in the western diet. Critical Reviews in Food Science and Nutrition, 35, 525-552.

re

-p

Voragen, A. G. J., Beldman, G., & Schols, H. A. (2001). Chemistry and enzymology of pectins. In B. V. McLeary, & L. Prosky (Eds.), Advanced Dietary Fibre Technology (pp. 379–398). Oxford: Wiley-Blackwell Science.

lP

Wang, L., Zhang, B., Xiao, J., Huang, Q., Li, C., & Fu, X. (2018). Physicochemical, functional, and biological properties of water soluble polysaccharides from Rosa roxburghii Tratt fruit. Food Chemistry, 249, 127–135.

ur na

Yamada, H., & Kiyohara, H. (2007). Immunomodulating activity of plant polysaccharide structures. In J. P. Kamerling (Ed.), Comprehensive Glycoscience (pp. 663–694), Amsterdam: Elsevier. Yang, J.-S., Mu, T.-H., & Ma, M.-M. (2018). Extraction, structure, and emulsifying properties of pectin from potato pulp. Food Chemistry, 244, 197–205. York, W. S., Darvill, A. G., McNeil, M., & Albersheim, P. (1985). 3-deoxy-D-manno-2octulosonic acid (KDO) is a component of rhamnogalacturonan II, a pectic polysaccharide in the primary cell walls of plants. Carbohydrate Research, 138, 109–126.

Jo

York, W. S., Darvill, A. G., McNeil, M., Stevenson, T. T., & Albersheim, P. (1986). Isolation and characterization of plant cell walls and cell wall components. Methods in Enzymology, 118, 3-40. Zheng, Y., & Mort, A. (2008). Isolation and structural characterization of a novel oligosaccharide from the rhamnogalacturonan of Gossypium hirsutum L. Carbohydrate Research, 343, 10411049.

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Zheng, Y., Li, L., Feng, Z., Wang, H., Mayo, K. H., Zhou, Y., Tai, G. (2018). Preparation of individual galactan oligomers, their prebiotic effects, and use in estimating galactan chain length in pectin-derived polysaccharides. Carbohydrate Polymers, 199, 526-533.

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Figure 1. HPSEC elution pattern of AELkP fraction before (A) and after (B) combined enzyme

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digestion. Pectin standards (10–100 kDa) were used to estimate the molecular weights.

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Figure 2. HPAEC elution pattern of AELkP fraction after HG/RG enzyme digestion. Peak annotation: 1-10 stands for DP 1-10 saturated GalpA mono- and oligomers; u2-u6 stands for DP 2-6 unsaturated GalpA oligomers; AELkP after enzyme digestion (C), distribution of both types of standards, respectively for saturated (B) and unsaturated (A) oligomers.

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Figure 3. HILIC-MS profile of partly methylesterified, saturated and unsaturated pectin oligomers derived from AELkP fraction after HG and RG-I enzyme digestion. Peak annotation: u430, DP 4, 3 O-methylester, 0 O-acetyl group; (u-unsaturated).

42

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Figure 5. Flow curves (A) and mechanical spectra of AELkP and AELkP solutions with addition of sucrose (B).

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Figure 6. Foaming ability (A) and foaming stability (B) of AELkP fraction at different concentrations. Emulsifying properties of different concentrations AELkP in (C) 50/50 o/w emulsions and (D) 30/70 o/w emulsions.

44

Table 1. Yield and chemical characterization of AIS and AELkP fraction* AELkP

3.5a

36.7b 0.5a

Protein

4.0

4.1

Hyp

n.d.

0.05

Total phenols

n.d.

0.2

Total sugars

50.1

96.6

Neutral sugars

14.7b

26.2c

51.6b 55.5c

Rhamnose

0.5

1.1

2.4

Galactose

8.6

Arabinose

1.4

Glucose

21.7

Xylose Kdo

ur na

AG-II (Yariv test) Uronic acids

ro 2.8

17.2

48.5

51.8

3.3

0.7

0.9

43.5

0.0

1.4

2.8

0.0

0.3

0.7

0.0

6.4

0.0

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Fucose

1.2e

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Mannose

13.1b

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Yield

-p

AIS

2.7

n.d.

+

n.d.

+

13.5

25.1c

45.0

44.6c

GalA

n.d.

43.0

42.6

GlcA

n.d.

2.0

2.0

Degree of methylation n.d.

65

d

Degree of acetylation

2.5d

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n.d.

*

Values are the average of two replicates; aValues express as g/100 g fw; bValues express as g/100 g dw; c Values represent the monosaccharide composition in mol%; dMoles methanol or acetyl per 100 moles of galacturonic acid; eHyp content as part of total protein content; n.d. not determined.

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Table 2. Retention time, m/z values and structures of oligomers released by HG- and RG-I enzymes after digestion of AELkP fraction determined by HILIC-ELSD-MSn. RT (min)

m/z

Structure

5.65

387.2

(Gal)2

Gal-Gal

9.07

549.2

(Gal)3

Gal-Gal-Gal

745.2

30

10.70

u4

uGalAMe-GalA-GalAMe-GalAMe uGalAMe-GalAMe-GalAMe-GalA

711.3

(Gal)4

Gal-Gal-Gal-Gal

00

14.90

369.1

2

15.60

873.4

(Gal)5

Gal-Gal-Gal-Gal-Gal

16.21

657.1

(Rha-GalA)2

Rha-GalA-Rha-GalA

17.58

511.1

(Rha-GalA)2Gal

RhaGal-GalA-Rha-GalA

18.80

1007.2

(Rha-GalA)3Ac

Rha-GalAAc-Rha-GalA-Rha-GalA

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GalA-GalA

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13.4

Rha-GalA-Rha-GalAAc-Rha-GalA 545.1

300

20.72

735.2

4

10

21.34

965.2

(Rha-GalA)3

925.3

5

GalA-GalA-GalAMe-GalA

20

re

21.76

GalA-GalA-GalA

Rha-GalA-Rha-GalA-Rha-GalA GalA-GalAMe-GalA-GalAMe-GalA

lP

19.89

GalA-GalAMe-GalAMe-GalA-GalA

400

24.03

911.2

5

10

25.33

1101.2

620

28.22 30.63 32.82

GalA-GalA-GalA-GalA GalA-GalA-GalA-GalAMe-GalA GalA-GalA-GalAMe-GalA-GalAMe-GalA GalA-GalA-GalAMe-GalAMe-GalA-GalA

1277.3

720

GalA-GalA-GalA-GalAMe-GalA-GalAMe-GalA

1453.3

820

GalA-GalA-GalA-GalAMe-GalA-GalAMe-GalA-GalA

1629.3

20

1995.4

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34.37

ur na

721.1

24.03

9

11

30

GalA-GalA-GalA-GalAMe-GalA-GalAMe-GalA-GalA-GalA GalA-GalA-GalAMe-GalA-GalA-GalAMe-GalA-GalA-GalA GalA-GalAMe-GalA-GalAMe-GalA-GalA-GalAMe-GalAGalA-GalA-GalA GalA-GalA-GalA-GalAMe-GalA-GalAMe-GalA-GalAMeGalA-GalA-GalA GalA-GalA-GalA-GalAMe-GalA-GalA-GalAMe-GalAGalAMe-GalA-GalA

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Table 3. Rheological properties of AELkP solutions at different concentrations. Model of Ostwald de-Waele

Thixotropy (Pa/s)

Concentration (%) n (-)

0.5

0.003±0.000d

0.923±0.004d 0.9989±0.0002

0.090±0.013b 0.030±0.000c

0.120±0.014c

1

0.004±0.001c

0.951±0.004c 0.9990±0.0001

0.065±0.007b 0.078±0.006c

0.143±0.086c

2.5

0.027±0.000b

0.868±0.001b 0.9999±0.0000

1.495±0.035a 0.200±0.057b

1.685±0.175b

5

0.253±0.001a

0.757±0.002a 1.0000±0.0000

1.535±0.021a 1.540±0.000a

3.075±0.021a

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K (Pa·sn)

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R2

Antithixotropy Total (A + T) (Pa/s) (Pa/s)

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Parameters in columns denoted with the same letters (a, b, etc.) do not differ statistically at the level of confidence p=0.05.

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