Characterization of a water soluble, hyperbranched arabinogalactan from yacon (Smallanthus sonchifolius) roots

Accepted Manuscript Characterization of a water soluble, hyperbranched arabinogalactan from yacon (Smallanthus sonchifolius) roots Alejandra Castro, Francisco Vilaplana, Lars Nilsson PII: DOI: Reference:

S0308-8146(16)32027-1 http://dx.doi.org/10.1016/j.foodchem.2016.12.019 FOCH 20311

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

17 October 2016 7 December 2016 8 December 2016

Please cite this article as: Castro, A., Vilaplana, F., Nilsson, L., Characterization of a water soluble, hyperbranched arabinogalactan from yacon (Smallanthus sonchifolius) roots, Food Chemistry (2016), doi: http://dx.doi.org/ 10.1016/j.foodchem.2016.12.019

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Characterization of a water soluble, hyperbranched arabinogalactan from yacon (Smallanthus sonchifolius) roots Alejandra Castro a ,b ,d, Francisco Vilaplanac, Lars Nilsson a* a

Department of Food Technology, Engineering and Nutrition, Lund University, P.O. Box 124, SE-222 01 Lund,

Sweden. b

Center for Food and Natural Products, San Simón University, Cochabamba, Bolivia.

c

Division of Glycoscience, School of Biotechnology, KTH Royal Institute of Technology, AlbaNova University

Centre, SE-106 91, Stockholm, Sweden d

Current affiliation: SOLVE Research & Consultancy AB, Medicon Village, Scheelevägen 2, SE-22381 Lund,

Sweden * corresponding author: [email protected]

Abstract Yacon (Smallanthus sonchifolius Poepp. & Endl.) roots are largely grown in Andean countries and have attracted recent interest due to their antioxidant and prebiotic effects. Yacon is typically consumed as a fruit due to its sweet taste and juiciness. The macromolecular properties of an aqueous extract of yacon are investigated using asymmetric flow field-flow fractionation (AF4) coupled to UV, multiangle light scattering (MALS) and differential refractive index (dRI) detection. The method allows for determination of molar mass and size over the size distribution. Three major populations were found of which one strongly dominates in concentration. Through collection of fractions from AF4, carbohydrate composition and glycosidic linkage analysis for the dominating population was performed. The results show that the dominating population consists of a highly branched arabinogalactan (type 2) with a molar mass of approximately 1-2·105 g/mol, a hydrodynamic radius of approximately 6-10 nm and a relatively high apparent density (approx. 70-150 kg/m3).

Key words: AF4, asymmetric flow field-flow fractionation, arabinogalactan, yacon, Smallanthus sonchifolius

1. Introduction Yacon (Smallanthus sonchifolius Poepp. & Endl.) is a plant originally from Andean countries in South America. Yacon is considered novel in Europe and has not been introduced on the European markets yet. Nevertheless, yacon roots have been studied with regard to their antioxidant composition and content using yacon varieties from Bolivia, Ecuador and cultivated in Czech Republic, (Fernandez, Viehmannova, Lachman & Milella, 2006; Lachman, Fernández, Viehmannová, Sulc & Cepková, 2007; Ojansivu, Ferreira & Salminen, 2011). Inulin-type fructans are one of the major fiber components in yacon roots and they are considered water ‘soluble’ and can act as a dietary fiber in the human diet. The content of fructans in yacon roots varies between varieties and landraces in relation to their ploidy in terms of their tuberous root contents of short-chain fructooligosaccharides and long-chain fructooligosaccharides (Fernandez et al., 2013). (Campos, Betalleluz-Pallardel, Chirinos, Aguilar-Galvez, Noratto & Pedreschi, 2012) analyzed 35 yacon varieties cultivated in Peru, and found that the content of fructans varied between 6 and 65 g /100 g dry matter (DM). Sixteen of the 35 varieties had a content of fructans between 6-30 g /100 g DM, 10 varieties had a content of fructans between 31-40 g/100 g DM, and 9 varieties had a content of fructans between 41-65 g /100 g DM. Inulin-type fructans from yacon roots have been reported to have a degree of polymerization (DP) of 3-10 (Ohyama et al., 1990) and due to the low DP they are often referred to as fructooligosaccharides (FOS). Yacon FOS has been identified as a novel source of prebiotics 2

and have been shown to be efficiently utilized by both Lactobacilli and Bifidobacterium (Pedreschi, Campos, Noratto, Chirinos & Cisneros-Zevallos, 2003). Inulin-type fructans in aqueous solution has already been shown to aggregate at low concentration (approx. 0.07 mg/mL), which is related to the rather limited solubility, giving rise to colloidal particles with diameters in the range of approx. 20-200 nm as determined by dynamic light scattering and transmission electron microscopy (Dan, Ghosh & Moulik, 2009). The formation of inulintype fructan particles has been reported to occur even at low DP (i.e. DP=10). It has been shown that the presence of inulin particles play an important role for rheology and gel formation in aqueous systems and, thus, it is of interest to investigate whether a solution of yacon root extract contain such inulin or FOS particles (Bot, Erle, Vreeker & Agterof, 2004; Glibowski & Pikus, 2011). Asymmetric flow field-flow fractionation (AF4) (Wahlund & Giddings, 1987) is a versatile separation technique suitable for the separation and characterization of food macromolecules and colloidal particles over a wide size range (Nilsson, 2013). AF4 coupled to detectors such as multiangle light scattering (MALS) and differential refractive index (dRI) detectors enables the determination of molar mass (M) and root-mean-square radius (rrms) over a population while hydrodynamic radii (rh) can be determined directly from AF4 retention times. AF4 is a “chromatography-like” method although it is not a chromatographic method as instead of a column for separation an open channel, void of a stationary phase, is utilized. For the interested reader the principles of AF4 can be found elsewhere (Wahlund, 2000; Wahlund & Nilsson, 2012). In this paper we study the macromolecular and colloidal composition as well as properties in the aqueous extract of yacon pulp obtained from yacon grown in the tropical regions of Bolivia. The characterization is achieved using viscosity measurements, AF4-UV-MALS-dRI, cryogenic electron transmission microscopy (cryo-TEM) and fructose determination in the

aqueous extract. Furthermore, carbohydrate composition and glycosidic linkage analysis is performed on fractions obtained from AF4.

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2. Material and Methods 2.1 Sample preparation Yacon (Smallanthus sonchifolius) roots were purchased from Colomi, a locality in Cochabamba, Bolivia. The dry matter content in the roots was determined gravimetrically to 8%. The roots were washed and peeled under distilled water and cut into 1×1×1 cm cubes and immersed in 5 g/L ascorbic acid solution for 1 min, to avoid oxidation, and then drained. The cubes were milled in a knife mill (Grindomix® GM200 Retsch, Germany) in batches of 300 g at 7000 rpm for 45 seconds in order to obtain yacon paste. Potassium sorbate (1.5 g), sodium benzoate (0.8 g) and ascorbic acid (3 g) were added to one liter of yacon paste as preservatives. The yacon paste was stored in plastic containers with a capacity of 1 kg at a temperature of -20 ºC, and thawed at room temperature before analysis. The paste was separated into pellet and supernatant by centrifuging in 50 mL centrifuge tubes at 3000×g for 20 min at 20 °C after which the supernatant was analyzed.

2.2 Determination of inulin-type fructan content The content of inulin-type fructans in the supernatant was determined according to the method described by Steegmans et al. (Steegmans, Iliaens & Hoebregs, 2004), which consists of extracting the sample with boiling water and hydrolyzing with sucrase and fructanase. The amount of glucose and fructose were measured in the initial extract and in both hydrolyzates using a kit for glucose and fructose determination (Boehringer kit E0139106, R-Biopharm, Germany). The amount of inulin-type fructans was calculated based on fructose measurements, subtracting free fructose and contributions from sucrose. The results were expressed as g inulin-type fructans /100 g sample (wet basis). The degree of polymerization of fructans was estimated by dividing the amount of fructose with the amount of glucose, assuming that each fructan chain has a glucose unit end.

2.3 Viscosity of the supernatants and the volume fraction of particles The viscosity of the supernatant was measured with a stress controlled rheometer (Stress Tech, Reologica AB, Sweden) equipped with a bob and cup concentric cylinder (outer diameter:inner diameter = 27:25 mm), applying an increasing stress in 50 intervals from 0.01 to 10 Pa at a temperature of 20 °C controlled by a water bath.

2.4 Cryo Transmission Electron Microscopy (Cryo-TEM) Specimens for electron microscopy were prepared in a controlled environment vitrification system (CEVS) to ensure stable temperature and to avoid evaporation and concentration effects. The sample (5 µL of the aqueous phase) was put on a lacey carbon film supported by a copper grid and was gently blotted with a filter paper. The sample was quenched in liquid ethane at -180°C and stored under liquid nitrogen until measured. The microscope was a CM120 cryo-TEM (Philips, Eindhoven, Netherlands) operated at 120 kV and equipped with a CT-3500 cryo holder (Oxford Instruments, High Wycombe, UK) and a post-column energy filter (GIF100, Gatan, Pleasanton, CA, USA). Images were recorded with a CCD camera under low electron dose conditions.

2.5 Asymmetrical flow field flow fractionation (AF4) The AF4 instrument was an Eclipse 3+ Separation System (Wyatt Technology Europe, Dernbach, Germany). It was connected to a Dawn Heleos II multiangle light scattering (MALS) detector (Wyatt Technology) and an Optilab T-rEX differential refractive index (dRI) detector (Wyatt Technology) both operating at a wavelength of 658 nm. An Agilent 1100 series isocratic pump (Agilent Technologies, Waldbronn, Germany) with an in-line vacuum degasser and an Agilent 1100 series autosampler delivered the carrier flow and

6

handled sample injection onto the AF4 channel. Between the pump and the channel was placed a filter-holder with a 100 nm pore-size Millipore polyvinylidene fluoride membrane (Merck Chemicals and Life Science, Solna, Sweden) to ensure that particle free carrier entered the channel. The AF4 channel was a Wyatt short channel (Wyatt Technology) having a tip-to-tip length of 17.4 cm and a nominal thickness of 350 µm. The actual thickness was determined to be 289 µm by calibration against bovine serum albumin (BSA) described in literature (Håkansson, Magnusson, Bergenståhl & Nilsson, 2012) using a hydrodymanic diameter of 6.6 nm for BSA (Li & Hansen, 2000). The ultrafiltration membrane forming the accumulation wall was made of regenerated cellulose with a cut-off of 10 kDa (MicrodynNadir GmbH, Wiesbaden, Germany). The sample injection onto the channel was performed at a flow rate of 0.20 mL/min during 3 min. The sample volume injected onto the channel was 180 µL. The injected amount was optimized in order to ensure no overloading in the channel by confirming that retention times were independent of the injected amount. A 5 min focusing/relaxation step (including injection) was performed prior to elution with the focusing flow rate being identical to the initial cross-flow rate during elution (1.5 mL/min). In order to avoid excessive retention and long elution times a cross-flow rate which decays exponentially with time (half-life=6 min) was used according to equation 1


  =
 0



  /

(1)

where Qc is the volumetric crossflow Q0 is the initial volumetric crossflow, t1/2 is the half-life of the decay and t is the time. In the present case Q0=2 mL/min and t1/2=6 min. The resulting flow profile is shown in Figure 1. After elution, the channel was flushed without any crossflow for 5 min before the next analysis. Detector flow rate (Qout) was constant at 1.0 mL/min

throughout the separation. The carrier liquid was 10 mM NaNO3 (Merck, Darmstadt, Germany) and 0.02% (w/v) NaN3 (BDH, Poole, UK) dissolved in pure water prepared with a Milli-Q system (Millipore Corp.). Fraction collection from AF4 was performed using a Frac-100 fraction collector (Pharmacia, Uppsala, Sweden). The collected fractions were pooled based on 10 consecutive injections. Processing of light scattering data was performed with the Astra software, version 5.3.4.14 (Wyatt Technology). The molar mass (M) and the root-mean-square radius (rrms) were obtained by the Berry method (Andersson, Wittgren & Wahlund, 2003; Berry, 1966) performing a first order fit to data obtained at 44.0-126.0° scattering angle. The lowest scattering angles, 32.0° and 38.0°, were not included, as the data obtained was imprecise. A dn/dc value of 0.146 mL/g was used and the second virial coefficient was assumed to be negligible. The hydrodynamic radius (rh) was obtained from the Stokes-Einstein equation (Einstein, 1905)

rh ,i =

k bT 6πηDi

(2)

where kb is the Boltzmann constant, T is the temperature, η is the dynamic viscosity of the solvent and Di is the diffusion coefficient. In turn, the diffusion coefficient was obtained AF4 retention times the procedure reported previously (Håkansson et al., 2012). The apparent densities were obtained from the molar mass and either rrms or rh. As rh gives only an approximate description of the volume the density obtained should be considered as an apparent property. The apparent density based on rh, ρh,i, for component i of the sample is calculated from

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ρ h, i =

Mi V (rh ) i ⋅ N A

(3)

where M is the molar mass, V (rh) is the volume of a sphere with radius rh. The apparent density based on rrms, ρrms, i, for component i of the sample is calculated from

ρ rms , i =

Mi ⋅α V (rrms ) i ⋅ N A

(4)

where M is the molar mass, V (r) is the volume of a sphere with radius r and α is given by equation 5.

α=

Vsphere (rrms ) Vsphere (r )

3 rrms = 3 = r

(

3/ 5 ⋅ r r3

)

3

3

 3 2 =  5

(5)

2.6 Carbohydrate composition and glycosidic linkage analysis Neutral carbohydrate composition analysis of the polysaccharide fraction was performed by acid hydrolysis, derivatization of the released monosaccharides to their alditol acetates and final quantification by gas chromatography – electron impact mass spectrometry (GC-EI/MS) (Blakeney, Harris, Henry & Stone, 1983). In brief, the polysaccharides were hydrolyzed with 2 M trifluoroacetic acid (TFA) at 121°C for 3 h and further derivatized into alditol acetates by reduction with NaBH4 and acetylation with acetic anhydride and pyridine. Quantification of the alditol acetates was performed using a HP-6890 GC coupled to a HP-5973 electron-impact mass spectrometer (Agilent Technologies, Santa Clara, CA) on a SP-2380 capillary column (30 m x 0.25 mm ID, Agilent Technologies) with a temperature ramp of 1 °C/min from 160 to 210°C. Neutral sugar standards (fucose, rhamnose, arabinose, xylose, galactose, glucose, mannose) were used for quantification of the response factors.

Acidic monosaccharides (uronic acids) were identified and quantified by the same hydrolysis procedure (2 M TFA at 121°C for 3 h) and direct analysis of the dried and re-dissolved sugars by high-pH anion exchange chromatography with pulsed amperometric detection (HPAECPAD). Separation and quantification of the uronic acids (GalA and GlcA) was performed on an ICS3000 system (Dionex, Sunnyvale, CA) by injection of 10 µL samples onto a Dionex Carbopac PA1 column at 30 °C at a flow rate of 1 mL/min. A 30 min gradient from 30 mM sodium hydroxide to 30 mM sodium hydroxide + 400 mM sodium acetate was applied for the separation. Glycosidic linkage analysis was performed by methylation under alkaline conditions (excess of NaOH) in dimethyl sulfoxide (DMSO) using the conditions reported by (Ciucanu & Kerek, 1984). The partially methylated polysaccharides were hydrolyzed with 2 M TFA at 121°C for 3 h and further derivatized into permethylated alditol acetates (PMAAs) by reduction with NaBH4 and acetylation with acetic anhydride and pyridine. The PMAAs were identified and quantified by GC-EI/MS on a SP-2380 capillary column (30 m x 0.25 mm ID, Agilent Technologies) with a temperature ramp of 1 °C/min from 160 to 210°C. The PMAAs were assigned to the different glycosidic linkages by comparison of the retention times and the fragmentation spectra by EI-MS to reference polysaccharides. The molar abundances of each linkage were corrected using the relative molar-response factors provided by Sweet et al. (Sweet, Shapiro & Albersheim, 1975). The analyses were performed in triplicate.

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3. Results and Discussion

3.1.Viscosity of the supernatant and inulin-type fructan content The supernatant obtained after centrifugation behaved like a Newtonian fluid. The dynamic viscosity was about twice the dynamic viscosity of water i.e. 2.23 mPa s. The concentration of fructans in the supernatant was determined to 3.2±0.5 g per 100 g supernatant (wet basis) performed in triplicate. The estimated average DP of fructans in yacon obtained from the chemical determination of fructose and glucose was about 5, which is similar to values reported for yacon in literature (i.e. DP 3-10) (Ohyama et al., 1990).

3.2.Asymmetrical flow field flow fractionation (AF4) The results obtained from the asymmetrical flow field flow fractionation (AF4), revealed the presence of three populations of particles in the supernatants (named as peak 1, 2, and 3), which are shown in Fig. 2 and 3. The figures show the detector responses from MALS and dRI and it can be clearly seen that population 2 dominates the sample concentration-wise (i.e. very high dRI signal compared to populations 1 and 3) and has a molar mass ranging from approximately 1-2·105 g/mol (Figure 2) and a molar mass weight-average (Mw) of 1.4·105 g/mol. Population 1 has a pronounced UV-peak (results not shown), suggesting that population 1 could be of a proteinaceous, or more likely, a polyphenolic nature. Population 2 showed no UV-absorption suggesting that this population most likely is of carbohydrate nature. The late eluting population 3 is only present in low amounts but has a very high MALS-signal due to its large size. The M for this population is huge and ranges between approximately 108-1010 g/mol (Figure 2) with Mw=4.7·109 g/mol. Population 3 also has a pronounced shoulder at elution times > 30 min. Figure 3 shows the same fractograms as Figure 2 but with the radii as overlay. Population 2 is too small for determination of rrms with

the MALS-detector, i.e. it gives rise to isotropic light scattering, and hence the radii obtained for this population is the rh (from AF4 retention times). The rh for population 2 approximately ranges between 6-10 nm with an z-average of 8 nm. Population 3 has an rrms that ranges approximately between 50-400 nm. The uppermost part of this range should be considered apparent as the limit of the light scattering models is most likely being reached somewhere in that range (i.e. the size of the analytes are approaching the wavelength of the MALS-detector). However, the quality of the light scattering fit is not impaired (result not shown). The larger size of population 3 complicates any chemical information obtained from UV adsorption as such relatively large species would also scatter UV light. The rh could only be obtained for part of population 3 (Figure 3) as it is not possible to calculate rh from AF4 elution times longer than approximately 24 min under the utilized separation conditions. In turn, the limitation is related to the high retention level (RL) (Wahlund et al., 2012) of polutation 3 i.e. an elution time of 24 min corresponds to RL=86. Such high RL should be avoided in AF4 experiments as excessive retention can cause anomalities in the elution behaviour (Litzén, Walter, Krischollek & Wahlund, 1993; Wahlund, 2013). This could also be the reason for the levelling off of the rrms vs. elution time for population 3 at high retention (Figure 3). The remedy of this limitation is to modify the separation conditions and, thus, the RL. However, as the main focus of the present paper is the investigation of population 2, no further optimization of the separation method was performed. Figure 4 shows the differential molar mass distributions for populations 2 and 3. Furthermore, the figure shows the conformational properties, obtained from the AF4-MALS-dRI i.e. the apparent densities and rrms/rh, over the molar mass distribution. The reason for presenting different apparent densities for the two populations is that only one apparent density is possible to calculate for the respective populations (due to the limitations discussed above for

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determination of rrms and rh). This means that the densities are not directly comparable with each other but it should in any case be remembered the apparent density, as the name suggests, is an apparent property. For population 2 the apparent density decreases over the molar mass distribution. For population 3 the lower M species display a very strong scaling of the apparent density in relation to increasing M. The lowest M species appear to be rather dense (approx. 500 kg/m3) and as M increases the apparent density decreases drastically to approx. 30 kg/m3. The high values for the lower M species are somewhat scattered which originates from a noisy dRI-signal in this range due to the low concentration of these populations. This means that the values of the apparent density, in this range, are somewhat uncertain. The strong scaling in apparent density with increasing M suggests a fractal scaling for the species in both populations. (Wyss, Tervoort & Gauckler, 2005) The rrms/rh can only be obtained for a narrow M-range (approx. 2-4·108 g/mol. The values of rrms/rh are quite low (0.3-0.4) and correspond to a conformation which suggest a micro-gelled structure (Schmidt, Nerger & Burchard, 1979) and similar results have previously been found for aggregated cereal β-glucan (Håkansson et al., 2012) and glycogen (Fernandez, Rojas & Nilsson, 2011).

3.3. Identification of populations obtained from AF4 fraction collection In order to identify the species present in population 2 and 3, further analyses were performed. Fractions were collected from the AF4 and analyzed for this purpose. As the initial hypothesis was that the species in the sample could be particles of aggregated FOS, fructan analysis was performed but no fructan was found in either population. Thus, the FOS found in the sample were not aggregated to any large extent and most likely lost, due to their low molar mass (approx. 900 g/mol) through the accumulation wall in the AF4 separation channel. It had been reported that inulin particles can dissociate in aqueous environments at 20°C (Glibowski et al., 2011). Thus, FOS particles, could have dissociated upon dilution by the carrier liquid

during AF4 separation. However, cryo-TEM micrographs of the sample before AF4 separation confirmed the absence of particles (results not shown) and, thus, most likely the FOS were lost through the accumulation. Population 3 is most likely of carbohydrate nature (i.e. no UV-absorbance was observed) and probably contains pectin as the supernatant contains 0.03 wt% pectin based on dry matter (Castro, Cespedes, Carballo, Bergenståhl & Tornberg, 2013). However, compositional analysis from collected fractions was not possible due to the low concentration of this population. An initial qualitative scanning of the carbohydrate composition (both neutral sugars and uronic acids) of fraction 2 was performed by acid hydrolysis and subsequent determination of the released sugars using HPAEC-PAD by comparison with monosaccharide standards. The main detected monosaccharides were galactose (Gal) and arabinose (Ara), with a minor presence of glucose (Glc). No uronic acids (neither galacturonic acid nor glucuronic acid) were detected in the hydrolysate (results not shown), which indicate that the collected polysaccharide is a neutral polysaccharide consisting of arabinogalactan with no acidic (galacturonan and rhamnogalacturonan) domains. Further quantification of the monosaccharide and glycosidic linkage composition (Table 1) was performed by the determination of the alditol acetates (AAs) and partially-methylated alditol acetates (PMAAs), respectively, using gas chromatography – mass spectrometry (GC-MS). The results from the methylation glycosidic linkage analysis confirm the structure of the polysaccharide fraction as a highly-branched arabinogalactan (type 2) (Pettolino, Walsh, Fincher & Bacic, 2012), consisting of a backbone of (1→3)-Galp with 31% (1, 3→6)-Galp branching points. These branching points may result in further elongated (1→6)-Galp side chains, (1→2)-Araf, (1→5)-Araf short-chains, and/or terminal t-Araf units, similar to a brush comb copolymer. Arabinogalactans can be found as components of the plant cell walls, normally attached to a protein core. The glycosylated part of arabinogalactan proteins (AGPs)

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adds up to over 90% of the molecular mass (Serpe & Nothnagel, 1999), with arabinose and galactose being the main components and minor amounts of neutral (rhamnose, fucose, xylose) and acidic (glucuronic and galacturonic acid) sugars (Showalter, 2001). The structure of the arabinogalactan segment is extremely complex, and varies not only amongst plant species but also amongst tissues within the same plant (Seifert & Roberts, 2007). The structure found in this paper is similar to already reported AGPs in radish roots (Tsumuraya, Ogura, Hashimoto, Mukoyama & Yamamoto, 1988) and wheat flour (Tryfona et al., 2010). Interestingly, although we expected that the arabinogalactan populations could be closely associated to proteins, in our case no UV absorption was observed for population 2.

4. Conclusions

The aqueous phase of a yacon extract was analyzed with AF4-UV-MALS-dRI and three major populations were found. The population that by far dominates, based on concentration, was identified to consist of a highly-branched arabinogalactan (type 2) with a molar mass of approximately 1-2·105 g/mol and a hydrodynamic radius of approximately 6-10 nm. A low concentration population containing ultra-high molar mass species (108-1010 g/mol) was also found in the AF4-UV-MALS-dRI result. This fraction could not be identified due to its low concentration but could consist of highly aggregated pectin. The results demonstrate the strength of AF4 for separation of natural mixtures of biomacromolecules that span over several orders of magnitude in molar mass in a single run. The results also demonstrate the strength of AF4 as a preparative method for further analysis of collected fractions.

Acknowledgements

The authors wish to thank the Swedish International Development Agency (SIDA) for financial support.

Tables Table 1. Monosaccharide (% mol) and glycosidic linkage composition (%mol) of the fraction

2 based on the GC-MS analysis of the hydrolysed alditol acetates and partially O-methylated alditol acetates. The standard deviation of the analyses is presented in brackets.

Figure Captions Figure 1: The decaying crossflow (Qc) profile utilized in the AF4 separations.

Figure 2: AF4 fractogram of the supernatant from extraction of yacon. dRI-signal (blue

trace), MALS 90° scattering signal (black trace) and the molar mass (Mw) (red symbols) vs. AF4 elution time. 1-3 indicate the 3 major populations found and t0 is the void time.

Figure 3: AF4 fractogram of the supernatant from extraction of yacon. dRI-signal (blue

trace), MALS 90° scattering signal (black trace), the hydrodynamic radius (rh) (red symbols) and root-mean-square radius (rrms) (black symbols) vs. AF4 elution time. 1-3 indicate the 3 major populations found and t0 is the void time.

Figure 4: Differential molar mass distribution for population 2 and 3 (black trace). The

apparent density based on rh (ρh, blue symbols), the apparent density based on rrms (ρrms, red symbols) and rrms/rh (green symbols).

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Steegmans, M., Iliaens, I., & Hoebregs, H. (2004). Enzymatic, Spectrophotometric Determination of Glucose, Fructose, Sucrose, and Inulin/Oligofructose in Foods. Journal of AOAC International, 87, 1200-1207. Sweet, D. P., Shapiro, R. H., & Albersheim, P. (1975). Quatitative-analysis by various GLC response-factor theories fro partially methylated and partially ethylated alditol acetates. Carbohydrate Research, 40, 217-225. Tryfona, T., Liang, H.-C., Kotake, T., Kaneko, S., Marsh, J., Ichinose, H., Lovegrove, A., Tsumuraya, Y., Shewry, P. R., Stephens, E., & Dupree, P. (2010). Carbohydrate structural analysis of wheat flour arabinogalactan protein. Carbohydrate Research, 345, 2648-2656. Tsumuraya, Y., Ogura, K., Hashimoto, Y., Mukoyama, H., & Yamamoto, S. (1988). Arabinogalactan-proteins from primary and mature roots of radish (Raphanus Sativus L). Plant Physiology, 86, 155-160. Wahlund, K.-G. (2013). Flow field-flow fractionation: Critical overview. Journal of Chromatography A, 1287, 97-112. Wahlund, K. G. (2000). Asymmetrical flow field-flow fractionation. In: M. E. Schimpf, K. Caldwell, & J. C. Giddings (Eds.), Field-flow fractionation handbook (pp. 279-294). New York: John Wiley & Sons Inc. Wahlund, K. G., & Giddings, J. C. (1987). Properties of an asymmetrical flow field-flow fractionation channel having one permeable wall. Analytical Chemistry, 59, 1332-1339. Wahlund, K. G., & Nilsson, L. (2012). Flow FFF - Basics and key applications. In: S. K. R. Williams, & K. Caldwell (Eds.), Field-flow fractionation in biopolymer analysis (pp. 1-21). Wien: Springer Verlag. Wyss, H. M., Tervoort, E. V., & Gauckler, L. J. (2005). Mechanics and microstructures of concentrated particle gels. Journal of the American Ceramic Society, 88, 2337-2348.

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Partially O-methylated alditol acetates Araf

Linkage Type

Total Ara

Relative abundance (%mol) 41.5 (1.7)

2,3,5-Me3-Araf

Araf-(1→

32.1 (1.0)

3,5-Me2-Araf

→2)-Araf-(1→

5.7 (0.3)

2,5-Me2-Araf

→3)-Araf-(1→

0.8 (0.3)

2,3-Me2-Araf

→5)-Araf-(1→

2.6 (0.2)

3-Me-Araf

→2,5)-Araf-(1→

0.3 (0.1)

Galp

Total Gal

55.3 (2.5)

2,3,4,6-Me4-Galp

Galp-(1→

2.6 (0.0)

2,4,6-Me3-Galp 2,3,6- Me3-Galp 2,3,4-Me3-Galp

→3)-Galp-(1→ →4)-Galp-(1→ →6)-Galp-(1→

5.1 (0.5) 0.8 (0.0) 15.3 (0.8)

2,4-Me2-Galp

→3,6)-Galp-(1→

31.4 (1.3)

Glcp

Total Glc

3.2 (0.8)

• • •

26

Investigation of water soluble macromolecular species form yacon (Smallanthus sonchifolius Poepp. & Endl.). Identification and characterization of chemical structure of a novel arabinogalactan. Determination of arabinogalactan molar mass and size with AF4-MALS-dRI.