Extraction and Characterization of Sugar Beet ... - ACS Publications

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Extraction and Characterization of Sugar Beet Polysaccharides Marshall L. Fishman,* Peter H. Cooke,† and Arland T. Hotchkiss Jr. Crop Science and Engineering Research Unit and Microbial Biophysics and Residue Chemistry and Core Technology Research Unit, Eastern Regional Research Center, Agricultural Research Center, U.S. Department of Agriculture, 600 East Mermaid Lane, Wyndmoor, Pennsylvania 19038 *[email protected] †Present address: Electron Microscopy Laboratory, New Mexico State University, PO Box 30001, MSC 3EML, Las Cruces, NM 88003

Sugar Beet Pulp (SBP), contains 65 to 80% (dry weight) of potentially valuable polysaccharides. We separated SBP into three fractions. The first fraction, extracted under acid conditions, was labeled pectin, the second was comprised of two sub fractions solubilized under alkaline conditions and was labeled alkaline soluble polysaccharides (ASP) and part of the remaining fraction was solubilized by derivatizing with carboxy methyl groups (DWCM). We have studied the global structure of these fractions after microwave-assisted extraction (MAE) from fresh sugar beet pulp. MAE was employed to minimize the disassembly and possibly the degradation of these polysaccharides during extraction. Fractions were characterized by their carbohydrate composition, by HPSEC with on-line molar mass and viscosity detection and by imaging with Atomic Force Microscopy (AFM). AFM revealed that pectin formed integrated networks comprised of spheres and strands. ASP aggregated but did not form networks.

Introduction In the U.S., each year it is estimated that the extraction of sugar from sugar beets produces about two million tons of sugar beet pulp (SBP) (1). SBP has been © 2010 American Chemical Society In Green Polymer Chemistry: Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


used primarily as a low valued animal feed. Curently, demand and utilization of sugar beet pulp for animal feed is high. Nonetheless, on a dry weight basis, SBP is a rich source of carbohydrates in that it contains 65 to 80% of potentially high valued polysaccharides (2). Typically, polysaccharides are obtained by sequentially separating SBP into three fractions. The first fraction, extracted under acid conditions, has been labeled pectin. Pectins are polysaccharides in which (1→4) linked α-D-galacturonates and its methyl ester predominate (3). In addition to these “smooth” homogalaturonan (HG) regions, also common to all pectins are “hairy” regions which are comprised of rhamnogalacturonan (RG) units (3). HG units may contain OH protons substituted with acetyl groups or may be substituted with xylose. The (1→2) linked α –L- rhamnose units in the RG region may be substituted with galactan, arabinan or arabinogalactan side chains in the 4 position. Pectin from sugar beets often differs from pectin from other sources in that it tends to have a higher degree of acetylation and a higher neutral sugar content (4). Furthermore, unlike pectins from many other sources, sugar beet pectin contains feruloyl groups (4). Moreover, unlike pectin from citrus peels or apple pomace, sugar beet pectin does not gel when high concentrations of sugar and acidic conditions are present. The poor gelling properties of sugar beet pectin have been attributed to the presence of acetyl groups and relatively low molar mass (5, 6). It also has been suggested that the relatively high amounts of neutral sugar side chains in sugar beet pectin prevent it from gelling (7). There have been numerous studies on the structural characteristics and physico-chemical properties of pectin from sugar beet pulp (8). Also there have been many studies involving the structural characteristics and physico-chemical properties of enzymatically and chemically modified sugar beet pectins from pulp (9). Most of these studies were concerned with the fine structure of sugar beet pectin and how it might affect its functional properties. Few if any of these studies were directed toward understanding the global structure of sugar beet pectin. Recently, sugar beet pectin extracted by acid from cell walls isolated from fresh sugar beet roots and characterized by atomic force microscopy (AFM) revealed linear and branched molecules attached to globular molecules (10). The linear molecules were attributed to polysaccharides whereas the globular particles were attributed to proteins. The second fraction, solubilized under alkaline conditions, is labeled alkaline soluble polysaccharides (ASP). This fraction may be associated in part with pectin (11). The third fraction (i.e. the extracted residue) contains cellulose microfibrils (12). The insoluble fraction was partially characterized after solubilizing by introducing carboxy methyl groups. The objectives of the study was to minimize the disassembly and possibly the degradation of these polysaccharides during extraction and to obtain all three fractions from the same matrix in order to assess their properties for value added applications. Therefore, we chose extraction conditions which minimized the disassembly and possibly the degradation of the extracted polysaccharides.

72 In Green Polymer Chemistry: Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


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

Experimental Materials Partially dewatered SBP with sugar removed was a gift from American Crystal Sugar, Moorhead, MN. SBP was shipped frozen and stored at -20 °C until prepared for extraction. The frozen sample was dried and ground to approximately 20 mesh. Microwave Assisted Extraction (MAE) The flow diagram for the extraction of the various polysaccharides is illustrated in Figure 1. Details of the extractions of pectin (13) and alkali soluble polysaccharides (ASP) (14) have been described elsewhere. Briefly, microwave heating was performed in a CEM Corporation, model Mars X microwave sample preparation system 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 watts of microwave power at a frequency of 2450 MHz. Carboxymethylation (CM) of the SBP Residue and Determination of Degree of Substitution (DS) Derivitization of the insoluble fraction was done by the method of Heinze and Pfeiffer (15). One gram of cellulose was slurried in iso-propanol, sodium hydroxide solution (various concentrations) was added to the mixture and stirred 73 In Green Polymer Chemistry: Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


for an hour. Then sodium monochloroacetate was added to the mixture which was heated for 2 to 5 hours at 55°C. The resultant product was then isolated and dried. FTIR spectra of CMC were used to calculate its degree of substitution (DS) according to the method and parameters of Pushpamalar et al. (16). FTIR spectra were collected with a Thermo Electron Nexus 670 FTIR. Two hundred fifty mg of the CMP was dissolved in 5 mL of water with stirring overnight. The resulting solution was centrifuged at 50,000g in a Sorvall RC-5B centrifuge. Ten drops of the supernatant were placed on a CaF2 plate, allowed to air dry overnight, and then vacuum dried for thirty minutes. The films were scanned from 1200 cm-1 to 3700 cm-1 with a resolution of 4 cm-1. Each sample underwent an average of 64 scans. The degree of substitution was calculated from the methine absorbance at 2920 cm-1 and the carboxyl absorbance at 1605 cm-1. Chromatography Dry sample (2 or 4 mg/ml) was dissolved in mobile phase (0.05 M NaNO3), centrifuged at 50,000 g for 10 minutes and filtered through a 0.22 or 0.45 micrometer Millex HV filter (Millipore Corp., Bedford, MA). The flow rate for the solvent delivery system, model 1100 series degasser, auto sampler and pump (Agilent Corp.), was set at 0.7 mL/min. The injection volume was 200 µL. 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 degree 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 ASTRA™ software (Wyatt Technology). Atomic Force Microscopy (AFM) AFM of pectin (13) and ASP (14) have been described previously. Typically, dry sample was dissolved in HPLC grade water and serially diluted to the desired concentration. Two µLs of the solution was 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). The instrument was operated in soft tapping mode. Images were analyzed by software version 5.12 rev. B which is described in the Command Reference Manual supplied by the manufacturer. 74 In Green Polymer Chemistry: Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

Table 1. Compositional Analysis of Sugar Beet Pectin Extracted at 60 °C and for 3 Minutes Pressure lb/in2

% PR


Commercial

%AGA

%NS

%DM

57(1)a

39(2)

83(9)

%DA

Freshb

33

38

37

54

28

25

13.0

52(3)

41(1)

99(6)

59(4)

30

11.3

48(2)

45(4)

114(13)

68(5)

75

9.2

60(3)

29(3)

77(4)

44(2)

Averagec

11(2)

54(6)

38(8)

96(18)

57(12)

a

Standard deviation of triplicate analysis. commercial or fresh sample

b

Data from reference (7).

c

Does not include

Compositional Analysis Determination of anhydrogalacturonate content (%AGA), degree of methyl esterification (%DM), acetylation (%DA) neutral sugar content (%NS) and monosaccharide analysis have been described previously (14). Percentage of alcohol insoluble pectin was determined gravimetrically (%PR).

Results and Discussion Characterization of Pectin Compositional Analysis Table 1 contains percentages of pectin recovered (%PR), anhydrogalacturanate (%AGA), neutral sugars (%NS), degree of methylesterification (%DM) and degree of acetylation (%DA) for microwave extractions at 60 °C . Heating time was 3 minutes and pressures ranged from 25 to 75 lbs/in2. At 25 lb/in2, values of percentage PR, AGA, NS, DM and DA were comparable to those found for commercial sugar beet pectin. By way of comparison (7) for fresh sugar beets extracted at pH 1, 75 °C for 30 minutes with HCl, %PR was 33, %AGA was 38, %NS was 37, %DM was 54, and %DA was 28. The lower values of %AGA, %DM and %DA and the higher value of %PR over those found for the 3 minute, 25 lb/in2 extraction may indicate more pectin degradation occurred in the fresh sample extracted by conventional heating for 30 minutes.

Analysis of Molar Mass, Radius of Gyration and Intrinsic Viscosity Pectin heated for 3 minutes gave weight average molar masses (Mw) that ranged from 517,000-1.2 million Daltons, radii of gyration (Rgz) from about 35-40 nm, weight average intrinsic viscosity (ηw) from about 3.00-4.30 dL/g. 75 In Green Polymer Chemistry: Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


Table 2. Molecular Properties of Pectin from Sugar Beet Pulp Heated for 3 Minutes

a

Temp/Press. ( °C, lb/in2)a

Mw × 10-3

Rgz (nm)

Commercial

640(14)

40.4(2)

3.27(.03)

60/19

956(5)

40.1(.3)

4.23(0.05)

60/23

978(3)

35.2(.5)

3.81(.06)

60/25

1020(70)

37.3(1)

3.49(.08)

60/30

1200(6)

40.3(.3)

3.97(.02)

60/50

622(3)

37.6(1)

2.83(0.03)

60/60

517(7)

34.3(.4)

2.58(.05)

60/75

753(8)

40.8(2)

4.24(.07)

70/30

781(5)

36.3(.5)

3.45(.03)

[ηw] (dL/g)

Sample concentration, 2 mg/ml

Figure 2. Mark-Houwink plot for sugar beet pectin. Heating time 3 minutes. Temperature 60 °C. Pressure 30 lb/in2. We have plotted log intrinsic viscosity against log molar mass in Figure 2, the Mark Houwink (M-H) plot. The plot has been divided into three regions as marked by the vertical lines with the values of “a”, the slope or Mark-Houwink exponent, indicated for each end section of the plot. The molecular properties for these three regions are given in Table 3. The high molar mass region had an M-H exponent of 0.076 which indicates an extremely compact molecule. This region comprised about 7.8% of the sample. The low molar mass region comprised about 34 % of the sample and had an M-H exponent of 0.65 which indicated a much less compact molecule than the high molar mass fraction. 76 In Green Polymer Chemistry: Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

Table 3. Molecular Properties of Sugar Beet Pectin Fractionsa % recovery

Mw×10-4

[η]w (dl/g)

Rgz (nm)

ab

7.8(0.6)d

372(154)

3.8(0.1)

54(1.0)

0.076(0.02)

2. Trans. Region

60(1)

132(37)

4.1(0.1)

39(1)

---------

3. Random Coilc

34(1)

394(8)

3.7(0.1)

38(1)

0.650(0.06)

Fraction 1. Spherec

(3/60/30) (time/temperature/pressure). b Mark-Houwink exponent. “a”. d Standard deviation (triplicate analysis).


a

c

Determined from

Figure 3. AFM image of sugar beet pectin network deposited from water at a concentration of 12.5 µg/mL. Scale bar is 100 nm, inset height scale, 0-2.5 nm. (see color insert) AFM To further elucidate its structure, images of sugar beet pectin were obtained by AFM. Figure 3 is an image of a sugar beet pectin network obtained by depositing a 2 µL drop of pectin dissolved in water at a concentration of 12.5 µg/mL and air drying it. The image revealed a number of spherical or odd shaped particles surrounded by strands. Many of the particles appear to be in contact with multiple strands thus appearing to be embedded in an open network structure. Dilution of the sugar beet pectin in water to 6.25 µg/mL produced the image in Figure 4. In that Figure the network has dissociated into particles attached to expanded strands. The strands appear to be comprised of rods, kinked rods, and segmented 77 In Green Polymer Chemistry: Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


rods. Many of these appear to be aggregated end to end and/or side by side. Some of the strands have formed closed loops. The images in Figure 3 appear similar to peach pectin networks dissociated by dissolution in 5 mM NaCl and imaged by electron microscopy (17, 18). Nonetheless, spherical or odd shaped particles were not observed in the peach pectin images.

Figure 4. AFM image of sugar beet pectin network deposited from water at a concentration of 6.25 µg/mL. Scale bar is 100 nm, inset height scale, 0-5 nm. (see color insert)

Characterization of Alkaline Soluble Polysaccharides (ASP) % Weight Recovery and Composition Analysis The % wt recovery of ASP after 10 minutes of heating time in the microwave at various temperatures and pressures are contained in Table 4. Total % wt recovery of ASP ranged from 16.3 to 29.8%. Heating at 100 °C and 60 lb/in2 gave the highest ASP I recovery and a somewhat smaller recovery of ASP II. Nonetheless the quantity of ASP II recovered was sufficient for further characterization studies. Composition analysis of ASP (Table 5) revealed that in all cases that % anhydrogalacturonate (AGA), % degree of methyl esterification (DM) and % neutral sugar (NS) were appreciably lower than thiose values for either pulp or MAE pectin (sample 3/60/30 in Table 1).



Table 4. Weight percentage recovery of alkaline soluble polysaccharide (ASP) heated for 10 Minutes

a

ASP I

ASP II

Total

100/30

4.2

21.6

25.8

100/40

12.3

15.4

27.7

100/60

16.0

13.8

29.8

105/50

12.8

12.8

25.6

105/90

3.3

15.0

16.3

120/90

14.6

10.2

24.8

Average

11(5)a

15(4)

25(5)

Standard Deviation

Table 5. Compositon Analysis of Alkaline Soluble Polysaccharides % AGA Samplea

ASP II

ASP I

% NS

ASP II

ASP I

ASP II

pulpb

34.4(4)e

70.4(9)

37.8(4)

3/60/30c,d

48.3(2.3)

113.7(13.0)

44.7(3.5)

SB

a

ASP I

% DM

10/80/30d

41.4(0.05)

26.6(0.3)

3.9(1)

9.0(2)

22.2(0.2)

22.8(0.04)

10/90/30d

21.6(0.2)

44.2(0.3)

11.8(1)

2.7(0.7)

29.6(0.2)

24.1(0.1)

10/100/30d

27.7(0.1)

29.8(0.2)

6.4(0.8)

7.5(1)

26.5(0.3)

13.9(0.1))

10/110/30d

23.2(0.2)

39.5(0.2)

11.5(0.6)

3.4(0.4)

25.7(0.1)

25.6(0.9)

c

d

Time(min.)/temp(°C)/pres.(lb/in2).

MAE.

e

b

Unfractionated; Standard deviation of triplicate analysis.

Pectin.

Obtained by

Neutral Sugar Analysis As indicated by their molar percentages in Table 6, the largest amount of neutral sugar present is in the form of arabinose followed by either rhamnose or galactose. For MAE sugar beet pectin, the order of neutral sugars detected was Ara>Gal>Rha>Glc>Xyl>Fuc. Recently reported research (10) on sugar beet pectin found that the order of neutral sugar detection was similar (Ara>Gal>Rha) when freshly harvested sugar beet roots were extracted. In that study only trace amounts of Glc, Xyl, Fuc and Man were found . In contrast, we detected the molar percentages of neutral sugars to be in the order Ara>Rha>Gal for ASP I and ASP II fractions extracted from sugar beet. We note that the ratio of uronate to neutral sugars in Table 6 is appreciably lower than expected for pectin. Possibly, this is a result of the hydrolysis being optimized for neutral sugar recovery. It is well known that the neutral sugars glycosidic linlages in sugar beet pectin are 79 In Green Polymer Chemistry: Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

more readily hydrolyzed than (1→4)-α –D-galacturonate linkages. The presence of galacturonic acid in the ASP fractions posibly indicates that residual pectic polysaccharides may have been extracted with the ASP’s or that they are covalently linked to the ASP’s.


Table 6. Percentage Recovery of Neutral Sugar in Alkaline Soluble Polysaccharides (ASP) and in Acid Extracted Pectin Samplea

Fraction

Ara

Gal

Rha

Glc

Xyl

Fuc

GalA

GlcA

SB pulpb

Control

41.29

11.53

6.31

15.12

6.76

0.46

16.96

1.57

3/60/30c,d

Pectin

46.26

20.47

7.74

7.57

4.01

0.63

11.11

2.3

10/80/30d

ASP I

41.81

10.7

19.1

0.6

0.29

0.1

26.1

1.35

ASP II

54.4

11.0

22.6

0.02

0.11

0.03

10.7

1.2

ASP I

54.1

11.7

20.6

0.15

0.01

0.05

12.2

1.2

ASP II

44.3

9.4

19.8

0.55

0.19

0.07

24.5

1.2

ASP I

52.4

11.7

20.7

0.13

0.07

0.05

13.4

1.6

ASP II

44.9

10.5

15.7

1.4

1.3

0.09

24.6

1.6

ASP I

52.5

11.4

20.9

0.23

0.09

0.05

13.5

1.33

ASP II

37.7

8.0

13.0

0.22

0.23

0.03

40.1

0.81

10/90/30d

10/100/30d

10/110/30d

Time(min.)/temp(°C)/pressure (lb/in2). MAE. a

b

Unfractionated.

c

Pectin.

d

Obtained by

Table 7. Molecular Properties of ASP Heated for 10 Minutes ASP I

Sample

a

ASP II

Temp./ Press °C lb/in2

Mw x 10-3

Rgz (nm)

[ηw] (dL/g)

Mw x !0-3

Rgz (nm)

[ηw] (dL/g)

100/30

94(9)a

10.2(1)

0.31(0.01)

109(8)

15.9(2)

0.33(0.03)

100/40

92.1(0.9)

10.1(2)

0.37(0.01)

113(4)

16.7(1)

0.40(0.01)

100/60

92.2(0.3)

11.2(.4)

0.38(0.01)

92.6(0.3)

14.0(1)

0.34(0.01)

105/50

106(3)

23.8(1)

0.40(0.01)

122(7)

23.2(3)

0.41(0.03)

105/90

192(5)

23.1(1)

0.40(0.01)

252(4)

20.9(0.8)

0.43(0.01)

120/90

83.3(1)

14.4(4)

0.35(0.01)

88.6(0.7)

16.3(0.6)

0.36(0.01)

Average

109(40)

15.(6)

0.37(0.03)

130(61)

18(3)

0.38(0.04)

Standard deviation of triplicate analysis.



Figure 5. Mark-Houwink Plot. A.) ASP I. B.) ASP II. Sample 10/100/30.

Analysis of Molar Mass, Radius of Gyration and Intrinsic Viscosity Table 7 contains the weight average molar mass, Mw, the z-average radius of gyration (Rgz) and the weight average intrinsic viscosity ([ηw]) for ASP I and II. For ASP I, Mw values ranged between 83,000 Da and 192,000 Da and for ASP II between 92,000 Da and 252,000 Da. For ASP I, values ranged from about 10 to 24 nm for Rgz and about 0.31 to 0.40 dL/g for [ηw]. For ASP II, values ranged from about 14 to 23 nm for Rgz and about 0.33 to 0.43 dL/g for [ηw]. By way of contrast, sugar beet pectin values of Mw, Rgz, and [ηw] indicated that sugar beet pectins are larger and have higher molar masses than alkaline soluble polysaccharides (see Table 2).


Table 8. Molecular properties of alkaline soluble polysaccharide fractions Regiona

Frac.b

% Rec.c

Mw x 10-3

Rgz nm

ηw dL/g

a

Wholee

ASP I

58 (2)

94(9)d

10(1)

0.31(.01)

.59(.04)

ASP II

45(1)

109(8)

16(2)

0.33(.03)

.66(.04)

ASP I

12(1)

205(31)

13(.5)

0.40(.01)

.27(.03)

ASP II

5.0(.2)

512(75)

18(1)

0.65(.07)

.26(.05)

ASP I

39(1)

74(5)

7(1)

0.30(.01)

.87(.2)

ASP II

20(1)

91(5)

11(2)

0.34(.01)

.55(.07)

24(2)

3.9(.1)f

0.13(.01)

.86(.06)

19(2)

3.5(.1)f

0.12(.01)

.89(.03)

Spherical

Transf


Asymg

ASP I ASP II

a

7.0(.6) 20(1) b

c

Region of the chromatogram. Fraction extracted. Percentage recovered after extraction. d Standard deviation of triplicate analysis. e Properties of entire sample 10/100/30 (heating time, minutes/temperature, °C, pressure, lb/in2.) f Transition. g Asymmetric.

Figures 5a and 5b are M-H 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 (See Table 8.). Based on their M-H exponents, ASP in the high molar mass region tend toward a compact, possibly spherical, shape whereas those in the low molar mass region tend toward a more asymmetric shape. 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 the two 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 (see Figure 2). 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.

AFM Figure 6A contains ASP I molecules of sample 10/100/30 imaged from solution by AFM at a concentration of 0.125 µg/mL. As indicated by the inset height bars in the Figure, the higher the molecules rise above the surface, the brighter is their color. Size measurements at four concentrations on the fifty largest molecules revealed that the particles were asymmetric in shape and grew in size as their concentration increased (14). Remarkably, as shown in Figure 6B, at the concentration of 25 µg/mL many of the smaller compact molecules have aggregated into linear, circular or combined linear-circular skeletal structures. Nevertheless, many of the smaller compact molecules remain visible. 82 In Green Polymer Chemistry: Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


Figure 6. AFM images of ASP I deposited from water. Sample 10/100/30. A.) solution concentration 0.125 µg/mL. Scale bar is 250 nm; inset height scale is 0-5 nm. B.) Solution concentration 25 µg/mL. Scale bar is 250 nm; inset height scale is 0-10 nm (see color insert) Figure 7 contains images of ASP II, extracted from sample 10/100/30 and deposited from solution at the concentration of 25 µg/mL. Unlike AFM images of sugar beet pectin (see Figure 3), network structures of ASP I and ASP II were not observed at 25 µg/mL. Figure 7 is comprised mostly of compact molecules with 83 In Green Polymer Chemistry: Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


heights ranging from 0.8 to 5 nm and with a mean of about 2.9 nm, surrounded by fine strands (14).

Figure 7. AFM images of ASP II deposited from water. Sample 10/100/30. Solution concentration 25 µg/mL. Scale bar is 250 nm; inset height scale is 0-10 nm. (see color insert) Partial Characterization of Carboxy Methyl Cellulose Extracted from the Residue The insoluble residue which remained after removal of pectin, ASPI and II was partially solubilized by derivatizing with carboxy methyl groups (CWMG). Depending on reaction conditions, degree of carboxymethylation ranged from 0.59 to 1.38. High Performance Size Exclusion Chromatography with molar mass and viscometric detection revealed that solubilized material had Mw values ranging from about 84,000 to 206,000 Daltons, Rgz values ranging from 32 to 47 nm and [ηw] values ranging from 1.92 to 4.66 dL/g.

Conclusion We have demonstrated that MAE of sugar beet pulp under moderate pressure and temperature could extract under acid conditions high molar mass, moderate viscosity pectin and alkaline soluble polysaccharides in minutes rather than hours as is required by conventional heating. HPSEC with online light scattering and viscosity detection revealed that both acid and alkaline soluble polysaccharides are bimodal distributions of high molar mass compact particles and lower molar mass less compact particles. AFM images of air dried solutions of sugar beet pectin corroborated this conclusion and further revealed that in the case of pectin 84 In Green Polymer Chemistry: Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

strands and spherical particles were integrated into networks. In the case of ASP, only the compact spherical particles were visible when imaged by AFM at low concentrations. ASP I aggregated into skeletal structures with increasing concentration, ASP II only aggregated into larger compact particles. HPSEC and AFM in combination clearly showed the structural similarities and differences between pectin, ASP I and II. Initial results on their molecular properties indicate that sugar beet pulp has the potential to be a sustainable source of carboxymethyl cellulose.


Acknowledgments We thank André White for his technical assistance. We thank Hoa K. Chau and David R. Coffin for their editorial and technical assistance. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.

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