Physico-chemical characterization of alkaline soluble polysaccharides from sugar beet pulp

Food Hydrocolloids 23 (2009) 1554–1562

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Physico-chemical characterization of alkaline soluble polysaccharides from sugar beet pulpq Marshall L. Fishman*, Hoa K. Chau, Peter H. Cooke, Madhav P. Yadav, Arland T. Hotchkiss Eastern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, 600 East Mermaid Lane, Wyndmoor, PA 19038, USA

a r t i c l e i n f o

a b s t r a c t

Article history: Received 25 June 2008 Accepted 24 October 2008

We studied the global (i.e. overall) structure of microwave-assisted alkaline soluble polysaccharides (ASP) isolated from fresh sugar beet pulp. The objective was to minimize the disassembly and possibly the degradation of these polysaccharides during extraction. Prior to ASP microwave-assisted extraction (MAE), pectin was removed and isolated by MAE. Two sub fractions, ASP I and ASP II were isolated. ASP I had about the same ratio of anhydrogalacturonate (AGA) to neutral sugar (NS) by percentage weight (%wt) whereas ASP II had about twice as much AGA to NS by %wt. Unlike the sugar beet pectin isolated, the degree of methyl esterification of AGA in both ASP fractions was very low. Arabinose was the most abundant neutral sugar followed by either rhamnose or galactose. For optimum sample 10/100/30, High Performance Size Exclusion Chromatography with molar mass and viscometric detection revealed that both ASP fractions are about 100,000 Da in Mw and about 10 and 16 nm in Rgz, respectively. ASP I and ASP II had weight average intrinsic viscosities of 0.31 and 0.33 dL/g respectively and their Mark–Houwink exponents indicated that they are relatively compact in shape. Molar Mass distributions of ASP appeared to be bimodal in shape. Atomic Force Microscopy (AFM) images of ASP I revealed a bimodal distribution of small and large compact asymmetric subunits when a 0.1 mg/mL air dried, aqueous solution was deposited on freshly cleaved mica and imaged. When the solution concentration was 25 mg/mL in ASP I, the subunits appeared to aggregate into skeletal structures whereas ASP II only formed compact asymmetric structures. Unlike AFM images of sugar beet pectin previously imaged, neither ASP I nor ASP II formed network structures at higher concentrations. Both ASP I and II were found to emulsify orange oil. Published by Elsevier Ltd.

Keywords: Arabinan–galactans HPSEC Viscosity Molar mass AFM

1. Introduction Each year it is estimated that the extraction of sugar from sugar beets in the U.S. produces about two million tons of sugar beet pulp (SBP) (Anonymous). This pulp has been used primarily as a low valued animal feed. The increased cost of energy to prepare this animal feed and increased world wide production of agricultural residues from sugar beets and other sources has diminished the demand for sugar beet pulp as a source of animal feed. Nonetheless, pulp is, based on dry weight, a rich source of carbohydrates in that it contains about 67% or more of potentially valuable polysaccharides (Buchholt, Christensen, Fallesen, Ralet, & Thibault, 2004; Oosterveld, Beldman, Schols, & Voragen, 1996; Sun & Hughes, 1999). Typically polysaccharides are obtained by sequentially

extracting sugar beet pulp to obtain three fractions. The first fraction extracted is pectin which may be extracted either by acid (Fishman, Chau, Cooke, & Hotchkiss, 2008), or by mild alkaline conditions (Guillon & Thibault, 1988). The second fraction solubilized under alkaline conditions, in this paper is labeled alkaline soluble polysaccharides (ASP). This fraction may at least be pectin associated in part (Sun & Hughes, 1999), and the third fraction (i.e. the extracted residue) is cellulose microfibrils (Dinand, Chanzi, & Vignon, 1999). The focus of this study is to obtain ASP (i.e. the second fraction), in order to assess its properties for value added applications. 2. Methods 2.1. Materials

q Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. * Corresponding author. Tel.: þ1 215 233 6450; fax: þ1 215 233 6406. E-mail address: marshall.fi[email protected] (M.L. Fishman). 0268-005X/$ – see front matter Published by Elsevier Ltd. doi:10.1016/j.foodhyd.2008.10.015

Partially dehydrated SBP with sugar removed from sugar beets, which were harvested about 60 days prior to sugar extraction, was a gift from American Crystal Sugar, Moorehead, MN. SBP was shipped frozen and stored at 20  C until prepared for extraction.

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The frozen sample was ground with a Comitrol 1700 (Urschel Laboratories, Valparaiso, IN) fitted with a microcut cutting head. In order to keep the pulp cool, ground dry ice was added to the pulp during grinding. Isolation of corn fiber gum, a potential emulsifier in food systems, has been described previously (Yadav, Johnston, Hotchkiss, & Hicks, 2007). Commercial gum arabic was used as received. 2.2. Microwave-assisted extraction (MAE) The use of microwaves to extract plant tissue has been described elsewhere with some modification (Fishman & Chau, 2000; Fishman, Chau, Hoagland, & Ayyad, 2000). Microwave heating was performed in a model Mars X microwave sample preparation system (CEM Corporation, Matthews, NC) equipped with valves and tubing which permitted the application of external pressure to each of the extraction vessels via nitrogen from a tank equipped with a pressure gauge. Samples were irradiated with 1200 W of microwave power at a frequency of 2450 MHz. Pectin, alkaline soluble polysaccharides I and II (ASP I, ASP II), and cellulose were obtained according to the procedure depicted in the flow chart in Fig. 1. For each experiment, vessels interconnected with tubing were placed in the sample holder, a rotating carousel. One vessel was equipped with temperature and pressure sensors which measured/controlled the temperature and pressure within the cell. After microwave heating, solubilized samples were allowed to cool in a water bath for ½ h at room temperature and treated as indicated in Fig. 1. Precipitated samples were dried and stored under vacuum until analyzed. 2.3. Chemical composition Anhydrogalacturonate content (%AGA) was determined by the Sulfamate/3-Phenylphenol Colorimetric Method (Fillisetti & Carpita, 1991; Yoo, Fishman, Savary, & Hotchkiss, 2003). Degree of methyl esterification (%DM) was determined by HPLC (Voragen, Schols, & Pilnik, 1986). Neutral sugar content (%NS) was determined with the Phenol–Sulfuric Acid Colorimetric Method (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956). Monosaccharide analysis was performed following methanolysis using High Performance

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Anion-Exchange Chromatography and Pulsed Amperometric Detection (HPAEC-PAD). A Dionex DX-500 system (Dionex Corporation, Sunnyvale, CA) was used which included a CarboPac PA-20 column operated at 0.5 mL/min. Neutral and acidic monosaccharides were separated in a single run using a mobile phase that was 14 mM NaOH isocratic for 13 min, then a 0–120 mM CH3COONa gradient in 100 mM NaOH for 30 min. The mobile phase returned to 14 mM NaOH for 40 min prior to the next injection. Other conditions were reported previously (Manderson et al., 2005). Percentage of alcohol insoluble pectin was determined gravimetrically (%PR). The protein content of pectin was estimated using standard methods for determining the nitrogen content of samples by use of a combustion instrument followed by thermal conductivity (AOAC Method 990.03, AACC Method 46–30). A Flash EA 1112 Elemental Analyzer (CE Elantech, Inc., Lakewood, NJ) calibrated with aspartic acid was used for the nitrogen determination. Percentage nitrogen was multiplied by 6.25 to obtain an estimation of protein (AOAC Method 22.052). 2.4. HPSEC with molar mass detection Dry sample (2 or 4 mg/mL) was dissolved in mobile phase (0.05 M NaNO3), centrifuged at 50,000 g for 10 min and filtered through a 0.22 or 0.45 mm Millex HV filter (Millipore Corp., Bedford, MA). The flow rate for the solvent delivery system, model 1100 series degasser, auto sampler and pump (Hewlett-Packard Corp.), was set at 0.7 mL/min. The injection volume was 200 mL. Samples were run in triplicate. The column set consisted of two PL Aquagel OH-60 and one OH-40 size exclusion columns (Polymer Laboratories, Amherst, MA) in series. The columns were in a water bath set at 35  C. Column effluent was detected with a Dawn DSP multi-angle laser light scattering photometer (MALLS) (Wyatt Technology, Santa Barbara, CA), in series with a model H502 C differential pressure viscometer (DPV) (Viscotek Corp., Houston, TX) and an Optilab DSP interferometer (RI) (Wyatt Technology). Electronic outputs from the 90 light scattering angle, DPV and RI were sent to one directory of a personal computer for processing with TRISEC software (Viscotek Corp.). Electronic outputs from all the scattering angles measured by the MALLS, DVP and RI were sent to a second directory for processing with ASTRAÔ software (Wyatt

Fig. 1. Flow diagram for the extraction of polysaccharides from sugar beet pulp.

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Technology). A dn/dc value of 0.148 was used for light scattering measurements on ASP I and ASP II. 2.5. Atomic force microscopy ASP I and ASP II samples, 10/100/30 (time/temperature/pressure), were dissolved in HPLC grade water and serially diluted to the desired concentration. Two microlitres of the solution were pipetted onto a freshly cleaved 10 mm diameter disk of mica and air dried. The mica was mounted in a Multimode Scanning Probe microscope with a Nanoscope IIIa controller, operated as an atomic force microscope in the Tapping Mode (Veeco Instruments, Santa Barbara, CA). The thin layer of pectin adhering to the mica surface was scanned with the AFM operating in the intermittent contact mode using etched silicon probes (TESP). Additional details concerning AFM measurements and image processing have been published previously (Fishman, Cooke, & Coffin, 2004)

Table 1 Weight percentage recovery of alkaline soluble polysaccharide (ASP). Samplea

ASP I

ASP II

Total

30/80/60 10/100/30 10/100/40 10/100/60 15/100/90 20/100/30 20/100/60 30/100/30 30/100/90 5/105/70 5/105/90 10/105/50 10/105/90 20/105/60 15/110/90 10/120/90 Average

10.9 4.2 12.3 16.0 4.4 9.9 8.8 13.9 6.4 4.4 8.3 12.8 3.3 6.2 5.5 14.6 8.8(4.2)b

22.8 21.6 15.4 13.8 17.1 15.4 22.8 19.4 24.8 16.6 12.2 12.8 15.0 21.8 19.2 10.2 17.5(4.4)

33.7 25.8 27.7 29.8 21.5 25.3 31.6 33.3 31.2 21.0 20.5 25.6 18.3 28.0 24.7 24.8 26.4(4.7)

a b

2.6. Analysis of ASP as an emulsifier Solutions, containing 6% (w/w) emulsifying agent (EA), 0.1% (w/ w) sodium benzoate (a preservative) and 0.3% (w/w) citric acid were prepared with stirring overnight to produce a homogeneous solution. Oil-in-water emulsions were prepared in triplicate by taking 0.104 g aliquot from the solution containing EA (i.e. 6.25 mg EA) and placing it in a vial. Then sufficient de-ionized water was added to give 2.375 g of the EA solution. Finally, 0.125 g of Valencia orange oil was added to give 2.5 g of an oil and water mixture containing emulsifier. No weighting agent was added to avoid the effect of such agent on the emulsification process. Weighting agents are added to foods containing oil flavor ingredients to increase their specific gravity and prolong the stability of the dispersion. Eliminating the use of such agents permits a measurement of the actual ability of a hydrocolloid to emulsify an oil in an aqueous environment. Then the oil and water mixture was vortexed and prehomogenized using a polytron bench top homogenizer equipped with a 12 mm diameter head (Brinkmann, Switzerland, PT 10/35) at 15,000 rpm for 30 s. The pre-homogenized emulsion was passed 3 times through a EmulsiFlex-B3 high-pressure homogenizer (Avestin Inc., Canada) at a homogenization pressure of 10,000 psi. The resulting emulsion concentrate was diluted to 78.125 g with a 10.0% (w/w) sucrose solution containing 0.1% (w/w) sodium benzoate and 0.3% (w/w) citric acid. The stability over time of the emulsion was measured by equating its turbidity to the 0 angle of scattered light of the suspended particles. The scattered light by the particles was obtained by measuring the absorbance of the emulsion at a wavelength of 650 nm using a UV-1700 Spectrophotometer (Shimadzu, Columbia, MA). Eq. (1) relates the turbidity, T, to the absorbance, A.

T ¼ 2:303A  D=l

(1)

where D ¼ dilution factor, and l ¼ path length of the cuvette in cm. The turbidity was determined immediately after preparing the dilute emulsion by measuring absorbance. The emulsion stability at each concentration was determined by emulsion breakage which was monitored by decrease in absorbance (loss of turbidity) against a 10.0% sugar solution containing 0.1% sodium benzoate and 0.3% citric acid over a two-week period. Further details concerning the preparation and measurement of emulsions have been published previously (Yadav et al., 2007).

temperature, pressure, ASP type, etc.). Multiple comparisons of treatment averages were performed using the Bonferroni LSD technique at the p ¼ 0.05 significance level (Miller, 1981). This statistical method allows one to determine if averages are significantly different at the 95% confidence level. 3. Results and discussion 3.1. Microwave-assisted extraction (MAE) In earlier work, we have shown that MAE under pressure produced pectin from citrus peels (Fishman, Chau, Hoagland, & Hotchkiss, 2006; Fishman, Chau, Kolpak, & Brady, 2001) and sugar beet pulp (Fishman et al., 2008) with higher molar mass, viscosity and significantly shorter heating times than pectin extracted with conventional heating. Because our aforementioned work on pectins demonstrated that MAE gave pectins with the least amount of degradation when compared to conventional methods of pectin extraction, we employed MAE in the removal of ASP from sugar beet pulp with the expectation that they too would undergo extraction with minimal degradation. Typically ASP are extracted for times in excess of 30 min whereas in this study they are extracted for 30 min or less and the sugar beet pulp has been depectinated under relatively mild conditions. Fig. 2 is a plot of pressure and temperature against time for optimal MAE of ASP. In

Table 2 Compositional analysis of alkaline soluble polysaccharides (ASP). Samplea

The data in Tables 1–6 has been analyzed by analysis of variance to determine the effects of the various treatments (time,

% AGA ASP I

SB pulpb 3/60/30c 60/100d 20/100/60e 30/100/90e 10/80/30e 10/90/30e 10/100/30e 10/110/30e a

% DM ASP II

34.4(4)f 48.3(2.3) 47.1(1) 29.5(3) 25.1(4) 25.0(3) 20.7(2) 41.4(0.05) 26.6(0.3) 21.6(0.2) 44.2(0.3) 27.7(0.1) 29.8(0.2) 23.2(0.2) 39.5(0.2)

ASP I

% NS ASP II

70.4(9) 113.7(13.0) 79.8(3) – – – – 3.9(1) 9.0(2) 11.8(1) 2.7(0.7) 6.4(0.8) 7.5(1) 11.5(0.6) 3.4(0.4)

ASP I

ASP II

37.8(4) 44.7(3.5) 29.3(0.2) – – – – 22.2(0.2) 22.8(0.04) 29.6(0.2) 24.1(0.1) 26.5(0.3) 13.9(0.1)) 25.7(0.1) 25.6(0.9)

Time (min.)/temp ( C)/pres. (psi). Unfractionated sugar beet pulp. c MAE sugar beet pectin. d Sugar beet pectin extracted in an open beaker. Conditions of pectin extraction 60 min, 80  C. e All fractions obtained by MAE. Conditions of pectin extraction, 3/60/30. f Standard deviation of triplicate analysis. b

2.7. Statistical analysis

Time (min.)/temp ( C)/pres. (psi). Standard deviation.

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Table 3 Percentage recovery of monosaccharides in alkaline soluble polysaccharides (ASP) and in acid extracted pectin.

Table 5 Weight average molar mass (Mw) and Mark–Houwink exponent (a) of alkaline soluble polysaccharides (ASP).

Samplea

Fraction

Ara

Gal

Rha

Sample

SB pulpb Commercialc 60/80d 3/60/30e 60/80d

Untreated Pectin Pectin Pectin ASP I ASP II ASP I ASP II ASP I ASP II ASP I ASP II ASP I ASP II

41.29 19.21 40.7 46.26 47.17 49.7 41.81 54.4 54.1 44.3 52.4 44.9 52.5 37.7

11.53 30.31 22.17 20.47 29.11 25.01 10.7 11.0 11.7 9.4 11.7 10.5 11.4 8.0

6.31 15.12 16.54 2.98 11.08 6.74 7.74 7.57 10.72 5.25 12.36 1.31 19.1 0.6 22.6 0.02 20.6 0.15 19.8 0.55 20.7 0.13 15.7 1.4 20.9 0.23 13.0 0.22

10/80/30f 10/90/30f 10/100/30f 10/110/30f

Glc

Xyl

Fuc

Gal A Gluc A

6.76 0.78 1.63 4.01 1.06 0.72 0.29 0.11 0.01 0.19 0.07 1.3 0.09 0.23

0.46 0.42 0.5 0.63 0.05 0.37 0.1 0.03 0.05 0.07 0.05 0.09 0.05 0.03

16.96 26.77 14.96 11.11 3.78 7.43 26.1 10.7 12.2 24.5 13.4 24.6 13.5 40.1

1.57 3.0 2.22 2.3 2.41 2.1 1.35 1.2 1.2 1.2 1.6 1.6 1.33 0.81

a

Time (min.)/temp ( C)/pres. (psi). Unfractionated sugar beet pulp. c Commercial sugar beet pectin. d Sugar beet pectin extracted in an open beaker. Conditions of pectin extraction 60 min, 80  C. e MAE sugar beet pectin. f All fractions obtained by MAE. Conditions of pectin extraction, 3/60/30. b

that experiment, the sample reached the desired temperature (ca. 100  C) and pressure (ca. 30 lb/in2) in a little more than 2 min. The total heating time for extraction was 10 min. By applying the pressure from an external source instead of generating it internally from the steam produced in a closed system, we were able to heat the sample at constant pressure. The %wt recovery of ASP under various extraction conditions of time, temperature and pressure are contained in Table 1. Total %wt recovery of ASPs ranged from 18.3 to 33.7%. Regression analysis on ASP II and on the total values using a linear model in time, temperature, and pressure showed significant linear effects for time. This model showed that ASP II and total values increased as the time increased (the correlation coefficients, r, were 0.715 and 0.765, respectively). Such values indicated a non-zero correlation at the 95% confidence level. There was no trend in recovery time for ASP I at the 95% confidence level, r ¼ 0.114. There were no significant effects due to pressure and temperature at the 95% confidence level for ASP I, ASP II and total values. The average recovery values for ASP I, ASP II and their total overall conditions were 8.8  4.2, 17.5  4.4 and 26.4  4.7 respectively. The %wt recovery of ASP II is about twice that of ASP I. 3.2. Chemical composition

30/80/60 10/100/30 10/100/40 10/100/60 15/100/90 20/100/30 20/100/60 30/100/30 30/100/90 5/105/70 5/105/90 10/105/50 10/105/90 20/105/60 15/110/90 10/120/90 Average a

Pectin

ASP I

ASP II

Sugar beet pulpb Commercialc 60/100d 10/80/30e 10/90/30ee 10/100/30e 10/110/30e

8.83(0.2)f 5.59(0.2) 6.29(0.03) 8.70(0.03) 8.70(0.03) 8.70(0.03) 8.70(0.03)

– – 3.82(0.01) 0.15(0.01) 2.65(0.01) 3.43(0.01) 1.69(0.04)

– – 6.69(0.07) 3.42(0.01) 3.75(0.03) 11.5(0.3) 5.18(0.1)

a b c d e f

Time (min.)/temp ( C)/pres. (psi). Unfractionated sugar beet pulp. Commercial sugar beet pectin. Extracted in an open beaker. Conditions of pectin extraction 60 min, 80  C. All fractions obtained by MAE. Conditions of pectin extraction, 3/60/30. Standard deviation of triplicate analysis.

a

MALLS

LS/V

99.2(5)a 94(9) 92.1(0.9) 92.2(0.3) 99.1(0.8) 106(1) 148(1) 109(3) 130(1) – 106(20) 106(3) 192(5) 130(2) 94.1(1) 83.3(1) 113(28)

90.9(3) 80(7) 87.9(0.1) 90.4(0.4) 92.8(0.3) 92.0(0.2) 188(3) 98.3(2) 158(5) – 92.5(0.5) 121(3) 299(11) 136(2) 91.8(1) 83.2(0.5) 132(66)

0.63(0.01) 0.59(0.04) 0.57(0.02) 0.57(0.02) 0.65(0.03) 0.56(0.01) 0.64(0.02) 0.52(0.03) 0.70(0.02) – 0.57(0.02) 0.63(0.01) 0.64(0.01) 0.67(0.02) 0.67(0.01) 0.66(0.01) 0.63(0.05)

Mw  103

a

MALLS

LS/V

150(7) 109(8) 113(4) 92.6(0.3) 137(3) 62.7(0.6) 160(10) 77.6(0.8) 94.6(2) 324(12) 189(6) 122(7) 252(4) 106(1) 126(2) 88.6(0.7) 137(68)

165(4) 126(21) 165(3) 123(2) 159(2) 65.9(0.8) 173(4) 91.5(1) 119(3) 252(6) 219(0.7) 142(3) 258(4) 125(1) 172(4) 123(3) 153(54)

0.60(0.02) 0.66(0.04) 0.59(0.01) 0.62(0.02) 0.67(0.01) 0.74(0.01) 0.56(0.02) 0.66(0.01) 0.63(0.01) 0.62(0.01) 0.53(0.03) 0.68(0.02) 0.54(0.02) 0.62(0.01) 0.56(0.01) 0.66(0.01) 0.63(0.06)

Standard deviation of triplicate analysis.

esterification (DM) and % neutral sugar(NS) were appreciably lower for ASP than for either pulp or MAE pectin (sample 3/60/30). As indicated by the percentage recovery of monosaccharides in Table 3, the largest amount of neutral sugar present is in the form of arabinose followed by either rhamnose or galactose. This is true for all samples examined other than commercial sugar beet pectin. In the case of commercial sugar beet pectins, unlike all other samples analyzed, the amount of galactose present is greater than arabinose. For MAE sugar beet pectin, the order of neutral monosaccharide recovery was Ara > Rha > Gal > Glc > Xyl > Fuc. Recently Kirby, MacDougall, and Morris (2008) found for the order of neutral sugar recovery Ara > Gal > Rha when they extracted pectin from freshly harvested sugar beet roots. In their study they found only trace amounts of Glc, Xyl, Fuc and Man but much larger amounts of uronic acid than found in the monosaccharide analysis performed in this study. Because monosaccharide analysis was optimized for the individual neutral sugars, the uronic acids were

Table 6 z-Average radius of gyration (Rgz) and weight average intrinsic viscosity [hw] of alkaline soluble polysaccharides (ASP). Samplea

Samplea

ASP II

Mw  103

Compositional analysis of ASP (Table 2) revealed that in all cases that % anhydrogalacturonate (AGA), % degree of methyl

Table 4 Percentage protein associated with pectin and alkaline soluble polysaccharides (ASP) fractions from sugar beet pulp.

ASP I

ASP I

ASP II [hw] (dL/g)

Rgz (nm)

30/80/60 10/100/30 10/100/40 10/100/60 15/100/90 20/100/30 20/100/60 30/100/30 30/100/90 5/105/70 5/105/90 10/105/50 10/105/90 20/105/60 15/110/90 10/120/90 Average a b

MALLS

LS/V

12.5(2)b 10.2(1) 10.1(2) 11.2(0.4) 12.3(0.9) 13.9(0.4) 20.4(0.4) 11.5(0.6) 21.3(0.4) – 15.3(0.2) 23.8(1) 23.1(1) 18.8(0.7) 13.3(2) 14.4(4) 16.1(4.6)

10.9(0.3) 8.4(0.3) 10.7(0.1) 11.0(0.3) 11.1(0.1) 11.6(0.1) 25.3(0.4) 11.4(0.2) 23.2(0.7) – 11.1(0.1) 17.4(0.2) 34.4(1) 19.7(0.4) 11.1(0.1) 10.6(0.1) 15.3(7.2)

0.38(0.01) 0.31(0.01) 0.37(0.01) 0.38(0.01) 0.37(0.01) 0.34(0.01) 0.43(0.01) 0.37(0.01) 0.53(0.01) – 0.38(0.01) 0.40(0.01) 0.40(0.01) 0.36(0.01) 0.36(0.01) 0.35(0.01) 0.42(0.13)

Time (min.)/temp ( C)/pres. (psi). Standard deviation of triplicate analysis.

[hw] (dL/g)

Rgz (nm) MALLS

LS/V

18.1(0.9) 15.9(2) 16.7(1) 14.0(1) 20.7(2) 21.0(4) 17.1(0.6) 16.2(1) 12.9(2) 23.1(0.6) 14.8(2) 23.2(3) 20.9(0.8) 15.3(0.4) 17.0(0.1) 16.3(0.6) 18.0(3.1)

20.9(0.3) 14.6(0.1) 20.9(0.3) 18.4(0.3) 19.8(0.1) 12.1(0.2) 19.6(0.3) 14.8(0.3) 17.6(0.1) 21.7(0.2) 20.4(0.4) 18.9(0.8) 21.5(0.2) 17.5(0.1) 20.5(0.4) 19.6(0.4) 18.8(2.6)

0.46(0.01) 0.33(0.03) 0.40(0.01) 0.34(0.01) 0.41(0.01) 0.31(0.01) 0.45(0.01) 0.32(0.01) 0.40(0.01) 0.43(0.01) 0.45(0.01) 0.41(0.03) 0.43(0.01) 0.41(0.01) 0.41(0.01) 0.36(0.01) 0.41(07)

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Fig. 2. Typical time/temperature/pressure curve for the extraction of ASP I from sugar beet pulp.

underestimated due to incomplete hydrolysis. Therefore we established the amount of uronic acids by a separate and fairly reliable colorimetric analysis (Fillisetti & Carpita, 1991; Yoo et al., 2003). Nonetheless, as expected, we found from monosaccharide analysis that the percentage of Gal A present was much greater than the percentage of Gluc A. Comparison of MAE sugar beet pectin with ASP I and II by analysis of variance revealed that the 3 fractions showed no difference in the relative amounts of neutral monosaccharides at the 95% confidence level. Percentage protein as determined by nitrogen analysis (see Table 4), revealed that generally, the relative order of protein present in the samples is pectin > ASP II > ASP I. An exception to that generality is the 10/100/30 sample in which ASP II had greater protein content than pectin. Possibly, at constant time and temperature, raising the pressure above 90 psi extracts an ASP II fraction which is richer in protein than those ASP II fractions extracted at lower pressures. Above 100 psi it appears that the protein starts to degrade.

2008). As indicated by molar mass values, radius of gyration and intrinsic viscosity measurements indicated that sugar beet pectins are larger and have higher molar masses than alkaline soluble polysaccharides. Superimposed light scattering (LS) at 90 , viscosity (DP) and refractive index (RI) chromatograms for ASP I and ASP II fractions obtained by MAE with 10 min heating time at 100  C and at a pressure of 30 psi plotted against retention volume are shown in Fig. 3A and B. Comparison of the chromatograms revealed that both fractions are bimodal in the region where macromolecules elute. The more complete overlap of chromatograms for ASP I as compared to ASP II, indicates that the ASP I has a more narrow molar mass distribution than ASP II. This observation is confirmed by the polydispersity values of Mw/Mn and Mz/Mn for ASP I in Table 7 which were 1.5 and 2.2 respectively, as compared with the values for ASP II, namely 3.1 and 11.9. These are the values for the whole chromatogram. The molar mass against elution time curves superimposed upon RI chromatograms in Fig. 4A and B also indicate that both fractions are bimolar as do the superimposed molar mass differential and integral distribution curves in Fig. 5A and B. Fig. 6A and B contain Mark–Houwink plots for ASP I and II respectively. We have divided both plots into three sections. The high molar mass end in which ASP I has an ‘‘a’’ value of 0.27 and ASP II has an ‘‘a’’ value of 0.26, a transition region in which the ‘‘a’’ values were 0.87 and 0.55 respectively and low molar mass region in which the ‘‘a’’ values are 0.86 and 0.89. Based on their M–H exponents, ASP in the high molar mass tend toward a spherical

3.3. Analysis of molecular properties by HPSEC with molar mass detection Table 5 contains the weight average molar mass, Mw, and the Mark–Houwink (M–H) exponent, ‘‘a’’, for ASP I and II and Table 6 contains the z-average radius of gyration (Rgz) and the weight average intrinsic viscosity ([hw]) for the two fractions. Mw is obtained by the MALLS and the LS/V methods for both fractions. No trends in either Mw or M–H exponents with time of heating in the MAE process are apparent. Differences in Mw by the two methods of determining Mw and for the two fractions are apparent for some specific heating conditions but overall differences between the MALLS and LS/V and between ASP I and II are not appreciably different due to sample variation as indicated by averaging overall samples. For ASP I, Mw values ranged between 83,000 Da and 299,000 Da and for ASP II between 62,000 Da and 324,000 Da whereas previously, Fishman et al. (2008) found that Mw ranged between 532,000 and 1.2 million Da for MAE pectin. The M–H exponent ranged between 0.52 and 0.70 for ASP I whereas the range of values was between 0.53 and 0.74 for ASP II. These values of M–H revealed that both fractions were relatively compact overall. Rgz values in Table 6 apparently show no clear trend with heating conditions. Overall average values in Table 6 show no appreciable differences between MALLS and LS/V; and ASP I and ASP II for Rgz and no appreciable differences in [hw] for ASP I and ASP II. We found relatively low values of Rgz and [hw], ranging from about 12 to 25 nm for Rgz and from [hw] about 0.31 to 0.53 dL/g. By way of contrast, sugar beet pectin values of Rgz ranged from 34 to 51 nm and [hw] values from about 3.0 to 4.2 dL/g (Fishman et al.,

Fig. 3. Superimposed 90 light scattering (LS), viscosity (DP) and refractive Index (RI) chromatograms. A.) ASP I. B.) ASP II. Sample 10/100/30.

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Table 7 Molecular properties of alkaline soluble polysaccharide fractions. Regiona Whole

e

Spherical Transg Asymh a b c d e f g h

Frac.b

% Rec.c

Mw  103

Mw/Mn

Mz/Mn

Rgz (nm)

Rhz (nm)

Rgz/Rhz

[hw] (dL/g)

a

ASP ASP ASP ASP ASP ASP ASP ASP

58(2) 45(1) 12(1) 5.0(0.2) 39(1) 20(1) 7.0(0.6) 20(1)

94(9)d 109(8) 205(31) 512(75) 74(5) 91(5) 24(2) 19(2)

1.5 2.8 1.4 1.2 1.1 1.2 1.0 1.1

2.2 10.9 2.6 1.5 1.2 1.5 1.0 1.2

10(1) 16(2) 13(0.5) 18(1) 7(1) 11(2) 3.9(0.1)f 3.5(0.1)f

9.5(0.3) 14(1) 14(0.6) 20(2) 7.4(0.2) 8.7(0.5) 3.5(0.2) 3.5(0.1)

1.1 1.1 0.93 0.92 0.98 1.2 1.1 1.0

0.31(0.01) 0.33(0.03) 0.40(0.01) 0.65(0.07) 0.30(0.01) 0.34(0.01) 0.13(0.01) 0.12(0.01)

0.59(0.04) 0.66(0.04) 0.27(0.03) 0.26(0.05) 0.87(0.2) 0.55(0.07) 0.86(0.06) 0.89(0.03)

I II I II I II I II

Region of the chromatogram. Fraction extracted. Percentage recovered after extraction. Standard deviation of triplicate analysis. Properties of entire sample 10/100/30 (heating time, minutes/temperature,  C, pressure, psi). Rgz obtained from LS/VIS data. Transition. Asymmetric.

shape whereas those in the low molar mass region tend toward a more asymmetric shape. Another, possibility is that molecules in the high molar mass region are extremely compact which may account for the very low value of the M–H exponent. Presumably the transition region contains a mixture of both kinds of molecules with the asymmetric shapes being greater in number. The M–H exponents for ASP fractions at the ends of the distribution are comparable to one another but somewhat larger than M–H exponents obtained previously for sugar beet pectin which we extracted

prior to extracting ASP (Fishman et al., 2008). The sugar beet pectin was extracted for 3 min at 60  C and 30 psi. In that case the high molar mass fraction had an ‘‘a’’ value of 0.08 and a low molar mass ‘‘a’’ value of 0.650. Because the transition region for sugar beet pectin in the M–H plot had a relative minimum and maximum, no ‘‘a’’ value was calculated. 3.4. Analysis of molecular properties by AFM Table 8 contains molecular dimensions for 50 of the largest molecules at four concentrations for ASP I dissolved in water. The data in Table 8 reveals that ASP I becomes more aggregated with increasing concentration in that all dimensions increase with increasing concentrations. Unlike the height dimension, the length

Fig. 4. HPSEC molar mass calibration curve superimposed on refractive index (RI) chromatogram. A.) ASP I. B.) ASP II. Sample 10/100/30.

Fig. 5. Molar mass distribution analysis. A.) ASP I. B.) ASP II. Sample 10/100/30.

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Fig. 6. Mark–Houwink Plot. A.) ASP I. B.) ASP II. Sample 10/100/30.

and width dimensions should be considered apparent dimensions due to the radius of curvature of the probe tip causing the image to be broadened (Fishman, Cooke, Chau, Coffin, & Hotchkiss, 2007). This phenomenon is referred to as tip broadening. In Fishman et al. (2007) it was calculated that tip broadening added about 16 nm to the width of a rod-like pectin molecule. Therefore we might assume that the values for length and width in Table 8 are over estimated by at value comparable to 16 nm. The values in parentheses are plus or minus one sigma of the molecular dimension distribution about the mean which is given outside of the parentheses. At 0.25 mg/mL, heights ranged from about 0.78 nm to 0.42 nm with the average height about 0.6 nm indicating that most molecules were only about one molecule high. The ratio of length to width indicates that on average the molecules are somewhat elongated. Fig. 7A contains ASP I molecules of sample 10/100/30 imaged from solution by AFM at a concentration of 0.125 mg/mL. As indicated by the inset height bars in Fig. 7, the molecules which rise above the surface are brighter in color. The molecules appear compact and somewhat asymmetric in shape. Remarkably, as shown in Fig. 7B, at the concentration of 25 mg/mL many of the smaller compact molecules

have aggregated into linear, circular or combined linear–circular skeletal structures (see Fig. 7B). Nevertheless, many of the smaller compact molecules remain visible. Data comparable to that found in Table 8 for ASP I but for the 50 largest ASP II molecules is contained in Table 9. Table 9 reveals a fairly large percentage increase in

Table 8 Molecular dimensions of fifty largest ASP I molecules determined by AFM.

Table 9 Molecular dimensions of fifty largest ASP II molecules determined by AFM.

Concentration (mg/mL)

Height (nm)

Length (nm)

Width (nm)

Concentration (mg/mL)

Height (nm)

Length (nm)

Width (nm)

0.125 1.25 12.5 25.0

0.60(0.18) 1.0(0.3) 1.3(0.5) 1.5(0.6)

56(22) 180(120) 243(235) 429(370)

33(16) 66(27) 111(93) 176(69)

1.25 6.25 12.5 25.0

0.71(0.26) 2.8(1.1) 1.8(0.9} 2.9(2.1)

73(25) 130(70) 120(41) 170(86)

32(17) 73(19) 51(21) 68(26)

Fig. 7. AFM images of ASP I deposited from water. Sample 10/100/30. A.) solution concentration 0.125 mg/mL. Scale bar is 250 nm; inset height scale is 0–5 nm. B.) Solution concentration 25 mg/mL. Scale bar is 250 nm; inset height scale is 0–10 nm.

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the mean molecular dimensions when the ASP II concentration is increased from 1.25 to 6.25 mg/mL. There appears to be no increase in dimensions above 6.25 mg/mL. In Fig. 8, are images of ASP II sample 10/100/30 deposited from solution at the concentration of 25 mg/mL. Fig. 8 is comprised mostly of compact molecules with heights ranging from 0.8 to 5 nm around a mean of 2.9 nm. Comparison of mean values in Table 9 with those in Table 8 reveals that at all concentrations, ASP II is greater in height than ASP I but smaller in length and width. Thus, one may conclude from AFM images that ASP I aggregates tend to be larger but less dense than ASP II. 3.5. Analysis of ASP as an emulsifier Studies have shown that sugar beet pectin can be used as an emulsifier in foods (Leroux, Langendorff, Schick, Vaishnav, & Mazoyer, 2003; Williams et al., 2005). This work has prompted us to initiate preliminary studies on the emulsification properties of ASP I and II. We measured the turbidity of emulsified orange oil as described in Section 2.6. The turbidity was given by Eq. (1) and was taken as proportional to the combined effect of the size and concentration of orange oil containing particles emulsified. In Fig. 9, we have plotted the persistence of turbidity of emulsified orange oil mixed with water in the presence of several emulsifiers. The turbidimetric technique for studying emulsion stability is a well documented and accepted method (Pearce & Kinsella, 1978) and it correlates well with particle size measurement. In our experiments turbidity is measured from the bottom of the diluted emulsion, which represents the most stable part. Thus, the higher turbidity is an indication of greater emulsion stability. The control contained no emulsifier. ASP I and II both emulsified the orange oil, ASP II giving the highest long term turbidity of the two. After about 8 days, only corn fiber gum gave more turbidity than ASP II. Furthermore, ASP I was equal to Gum Arabic in long term turbidity whereas ASP II was higher. Moreover, the 10/100/30 ASP II sample had a higher protein content than either pectin or 10/100/30 ASP I sample (see Table 4) which may be taken as further evidence, as suggested by Leroux

Fig. 9. Stability of orange oil-in-water emulsions. Oil to emulsifier ratio was 20:1. ASP I and II are sample 10/100/30.

et al. (2003) that protein plays an important role in the emulsification properties of certain polysaccharides 4. Conclusions HPSEC with on-line molar mass detection in conjunction with AFM are powerful tools for the structural characterization of ASP. By both methods we found ASP to largely be comprised of compact particles at low concentrations. Whereas ASP I aggregated into skeletal structures with increasing concentration, ASP II only aggregated into larger compact particles. HPSEC and AFM clearly showed that pectin (Fishman et al., 2008), ASP I and II (this study) have different structural characteristics. Preliminary emulsion studies revealed that ASP I and ASP II show promise as emulsifying agents. Possible applications for alkaline soluble polysaccharides include uses such as adhesives, thickeners, stabilizers, film formers and emulsifiers. Acknowledgement We thank Andre´ White for technical assistance in determining compositional analysis of ASP, Robyn Moten and Michael Kurantz for assistance in determining protein composition, John Phillips for assistance in performing statistical analysis and William Damert for helpful discussions concerning probe tip broadening of AFM images. Appendix Supplementary data associated with this article can be found in the online version at doi:10.1016/j.foodhyd.2008.10.015.

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Fig. 8. AFM images of ASP II deposited from water. Sample 10/100/30. Solution concentration 25 mg/mL. Scale bar is 250 nm; inset height scale is 0–10 nm.

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