Effect of Macromolecules on Sugar Processing: Comparison of Cane and Beet Macromolecules

Transcription

1 Effect of Macromolecules on Sugar Processing: Comparison of Cane and Beet Macromolecules Mary An Godshall, John R. Vercellotti and Ron Triche Sugar Processing Research Institute, Inc. New Orleans, LA ABSTRACT The major macromolecules in sugar processing include colorant and polysaccharides, along with minor amounts of protein, soluble lignin, colloidal silicates and possibly calcium complexes. These high molecular weight components negatively affect sugar processing and have been implicated in the inclusion of color in crystals, formation of color on storage, processing problems, and final product quality issues, such as turbidity and acid beverage floc. It has long been accepted that the high molecular weight components are the most difficult to remove during refining. However, it is also of interest to note that the transfer of color (from syrup to crystal) is much higher in cane sugar processing than in beet sugar processing. White beet sugar, with color of IC, can be boiled from IC color syrup, but only from about 200 IC cane syrup. In chromatographically separated beet molasses extract, the color of the syrup can be as high as IC and still produce a IC sugar. What is the reason for this? This paper will discuss recent studies comparing beet sugar colorant and cane sugar colorant. The results indicate that beet and cane colorant are fundamentally different: Beet colorant tends to be produced during processing, mainly from alkaline degradation of invert, while cane colorant enters the process in the cane juice as plant pigments associated with polysaccharide, and changes very little in process, due to the milder conditions associated with cane processing. INTRODUCTION Courtois (2001) stated in a recent presentation, that sugar is an affair of macromolecules, referring to the importance of high molecular weight molecules in sugar processing and in color transfer from syrup to sugar crystals. Beet sugar colorant has been studied for over a century (Broughton, et al., 1987), with emphasis on color precursors, the role of enzymes on phenolic compounds (Gross and Coombs, 1976; Winstrom-Olsen, 1981; Winstrom-Olsen, et al., 1979; Maurandi, 1988; Rhinefeld, et al., 1984), the development of color on storage (Shore, et al., 1984; Richmond, 1990; Godshall, et al., 1991; 1998a), and the distribution of color within the crystal (Shore, et al., 1984; Mantovani, et al., 1986). In recent years, a great deal of interest has developed in comparing the nature of cane and beet colorants to understand the differences between the two types in transfer of color into crystals (Godshall and Baunsgaard, 2000). New techniques, such as fluorescence spectroscopy, have been employed to study beet colorants (Baunsgaard, et al., 2000; 2001). It is generally accepted in the literature that high molecular weight compounds have a greater tendency to go preferentially into the sugar crystal and thus impact refined sugar quality (Tu, et al., 1977; Godshall, et al., 1987; Lindeman and O Shea, 2001). Reports in the literature also indicate that cane sugar colorants have a considerably higher molecular weight than beet sugar colorant. The molecular weight range of beet sugar colorants has been reported as 5000 to 40,000 Da (Broughton,

2 et al., 1986), whereas cane sugar colorants are in the 30,000 to 1,000,000 Da range (Godshall, et al., 1988). The role of polysaccharides in beet and cane processing has been of interest over the years. The major polysaccharide in beet processing is pectin, present between 1-2% on beets. Extraction of juice is optimized as to temperature and alkalinity to prevent excessive pectin release into the juice, as it will cause severe problems in purification and filtration. Vogel and Schiweck showed that pectins are degraded or almost completely removed during juice purification (1988). A range of other polysaccharides has been reported in beet juice, such as galactan and araban, but these are not well defined and probably represent soluble hemicellulose (Clarke, et al., 1991). The major polysaccharides in cane processing include starch and indigenous sugarcane polysaccharide (ISP), both native to the cane plant, along with dextran, a microbial polysaccharide that is common and sometimes abundant during cane processing. ISP is an acidic arabinogalactan polysaccharide, which can be classified as soluble cell wall hemicellulose. A recent study showed the distribution of these polysaccharides in raw sugar (Godshall, et al., 2001). Godshall and Baunsgaard (2000) noted that polysaccharides in beet processing tend to be lower over-all than in cane, and may account in part for the lower color transfer rate of beet colorant into crystal compared to cane colorant. Cane polysaccharides have been shown to be associated with polyphenolic acids and are involved in color formation in cane juice (Godshall, et al., 1998b; 2001). Fares, et al., (2001) reported that the presence of monomers of galacturonic acid in factory juice in beet processing increased color formation. Godshall showed the presence of two high molecular weight compounds in beet white sugar, one, about 20,000 Da, having a yellow color and polysaccharide nature (Godshall, 1992). MATERIALS AND METHODS The high molecular weight material (HMW) was obtained by dialysis in regenerated cellulose acetate membranes with a 12,000 Da cut-off. The HMW obtained by this manner was then subjected to various tests, including gel permeation chromatography (PC), hydrolysis and determination of monosaccharides as alditol acetates by gas chromatography (GC). GPC was accomplished with five GPC columns (TSK Gel PWXL, 6000, 5000, 4000, 3000 and 2500 angstrom pore size with precolumn) in series, equilibrated with 0.1 M sodium chloride in 10% acetonitrile at a flow rate of 0.6 ml/min. Two detectors were used consecutively, namely ultraviolet at 270 nm and a refractive index detector. Standards of sodium polystyrene sulfonate were used to determine the molecular weight ranges of the separated HMW of beet and cane process samples (Vercellotti, et al., 1996). Other tests included phenol sulfuric acid test for total carbohydrate concentration, Folin-Ciocalteu test for polyphenol/protein reactive material (Lowry, et al., 1951), pectin by m-hydroxydiphenyl (Kintner and Van Buren, 1982), tests for color, total polysaccharide, starch and dextran. RESULTS AND DISCUSSION Table 1 shows a comparison of the total polysaccharide content in cane and beet processing samples.

3 Since cane manufacture to refined sugar is a two-step process, the cane data shown here represent the first stage, from raw juice to raw sugar. The value shown for white sugar represents the average value of several representative white sugars. Table 2 shows representative color for beet and cane processing. The data shown in both tables are the average values across the season in one beet factory and one cane factory. Table 1. Polysaccharides in beet and cane sugar processing (Representative values) Sample/process Beet Cane Raw Juice/mixed juice Thin/clarified juice Thick juice/evaporator syrup Raw sugar (cane) White sugar Molasses ,411 Table 2. Color in beet and cane sugar processing (Representative values) Sample/process Beet Cane Raw Juice/mixed juice ,848 Thin/clarified juice ,388 Thick juice/evaporator syrup ,131 Raw sugar (cane) White sugar Molasses 37,038 81,298 The data in Tables 1 and 2 show dramatic differences in the levels of polysaccharide and color in cane and beet processing. Beet is much lower in both. Raw cane juice has double the polysaccharide content; it is reduced by half in clarification, whereas beet polysaccharide is reduced by 75%. White cane sugar has about twice as much polysaccharide as beet white sugar. The story is the same for color, even more so. Cane raw juice has 10 times more color than beet raw juice. There is essentially no change in the color level during clarification and evaporation in in both cane and beet processing. The color differential between evaporator syrup and raw cane sugar is 14:1; the color differential in beet is 71:1. Cane molasses color is more than twice that of beet molasses.

4 The data in Table 2, showing essentially no change in the color of raw beet juice and thin juice does not tell the whole story, however. Although the colors are about the same, the color of the raw juice is of a different character than the thin juice. The raw beet juice was lavender in color. We have noted beet juice that is dark purple to black, due to the oxidation of polyphenolic compounds; however, this set was not so dark. During carbonation, the color increased to 2145 IC in the first carbonation juice, a 150% increase in color. After purification, the color dropped to 1373, but it was of a completely different character than the raw juice, being a light brown color, representing the formation of alkaline degradation colorant caused by invert degradation reactions during carbonation. There is no change during cane juice processing because the clarification process is very mild -- the ph is raised only to about and every attempt is made to avoid excessive invert degradation. Invert levels are considerably higher in cane than in beet. In cane raw juice, glucose and fructose are each about % on solids (5% invert). In beet raw juice, we noted about 1.20% glucose and 0.62% fructose. After clarification in cane, there was little change in the invert level. After clarification (purification) in beet, the fructose was completely destroyed and the glucose was unchanged or slightly higher. GPC of Cane and Beet HMW. Figure 1 shows the overlaid chromatograms of the HMW material in beet processing, including raw juice, thin juice, thick juice, standard liquor, white sugar and molasses. The picture clearly shows how color formed: Raw juice contained a very high molecular weight peak, probably pectinaceous material, which was almost completely destroyed after purification. There was subsequent formation of a set of peaks at a lower MW range. These continued throughout the process, increasing during evaporation, and accumulating in molasses. The same set of colorants, with possibly a bias toward a slightly higher molecular weight were occluded in the white sugar crystal. This pattern was very consistent throughout the season. There were some changes in the details of the pattern, especially for raw juice, but not in the over-all picture, as shown in Figure 2, which highlights the raw juice and the white sugar HMW colorants across the season. Figure 1. Formation of HMW colorant in beet processing. Figure 2. Comparison of the HMW colorant in raw beet juice and the colorant that subsequently ends up in the white sugar crystal. By contrast, raw sugar colorant, as shown in Figure 3, is almost identical to that in the incoming raw cane juice, although the polysaccharide composition does change (see next section.) Figure 3. GPC of nondialyzable fraction of Louisiana raw sugar, detection at 270 nm.

5 Comparison of the Polysaccharide Composition. The monosaccharide composition of various beet and cane process samples shows that both are hemicellulosic arabinogalactan material. The composition of the indigenous polysaccharide from both beet and cane are shown in Table 3. A highly purified sample of cane ISP from crusher juice was shown to be mostly arabinogalactan (Roberts, et al., 1976). Later work showed the presence of aconitic acid tightly bound to the ISP in some cane varieties grown in Louisiana. Table 3. Composition of cane indigenous polysaccharide and beet indigenous polysaccharide. Cane Rha Ara Xyl Man Gal Glc Aconitic * * ** Beet Rha Ara Xyl Man Gal Glc Aconitic Thin juice Raw juice * Nondialyzable fraction of juice, not purified indigenous sugarcane polysaccharide * * Purified indigenous sugarcane polysaccharide 1 From Vogel and Schiweck, From Clarke, et al., 1989 (depectinated raw juice) Table 4 shows the composition of beet process samples. There is little change throughout the process, with the white sugar showing an increase in the glucose content and a decrease in the arabinose content, but still essentially an arabinogalactan polysaccharide in composition. Table 4. Monomer composition of polysaccharide in nondialyzable fraction in beet processing samples Sample Rhamnose Arabinose Xylose Mannose Galactose Glucose Raw juice Thin juice Thick juice Std. liquor White Sugar Molasses

6 Table 5 shows the monosaccharide composition of the nondialyzable fraction of several raw cane sugars. There is general consistency to the results in raw sugars from all over the world. The polysaccharide portion is largely glucose, with somewhat significant amounts of ISP sugars and considerable aconitic acid in some, but not all, the sugars. An earlier study has shown that the concentration of aconitic acid is correlated to the maturity of the cane at harvest (Godshall, et al., 2001). The presence of starch and dextran also contribute to the higher glucose content in the raw sugar fractions. Table 5. Monomer composition of polysaccharide portion of the nondialyzable fraction of raw cane sugar (Godshall, et al., 2001) Sample Rha Ara Xyl Man Gal Glc Aconitic Ivory Coast Brazil Florida Louisiana Dom. Rep Australia Table 6 compares the monomer composition of the nondialyzable fractions of cane and beet white sugars. The cane sugars show compositions similar to the raw sugar, showing that little change occurs in spite of the refining process. The ISP portion is reduced, while the glucose portion is increased to close to 90%. By contrast, the beet sugars show a pattern similar to raw juice, with a higher glucose content. Table 6. Comparison of the monomer composition of nondialyzable fraction of cane and beet white sugars. Cane Rhamnose Arabinose Xylose Mannose Galactose Glucose Louisiana Florida South Africa Central Am Beet Rhamnose Arabinose Xylose Mannose Galactose Glucose Nov. 8, Dec. 23, Feb. 27,

7 Table 7 shows the distribution of polysaccharide monomers in a Louisiana raw cane sugar when it is treated with the strongly basic ion exchange resin, IRA 900 in the chloride form. The eluate is the resin product, which contains the sucrose fraction. This shows that much of the acidic arabinogalactan cane polysaccharide was removed by the resin, leaving behind the glucose-enriched polymers, probably dextran and soluble starch, along with a trace of the arabinogalactan hemicellulose. The polysaccharides remaining in the eluate have an affinity for the sucrose crystal, and will end up in the refined cane sugar, as shown by the very similar composition of the polysaccharide material inside the refined sugars, shown in Table 6. Table 7. Distribution of polysaccharide monomers between eluate and regenerate from IRA-900 strong anionic exchange resin. Sample Rhamnose Arabinose Xylose Mannose Galactose Glucose Eluate Regenerate Differences in Molecular Weight of Beet and Cane HMW. GPC shows that cane sugar colorants have a wider range of molecular weight and an over-all higher molecular weight than beet sugar. Table 8 lists the molecular weight ranges of various cane and beet processing samples. In general, the data show that the MW of the colorants in cane is higher than that of cane colorants CONCLUSIONS This study has presented an overview of some differences between the HMW substances in cane and beet processing. Both colorants (detected at 270 nm) and polysaccharides (detected with RI) were examined. Cane processing has significantly higher quantities of incoming color and polysaccharide than beet processing. This may explain, in part, why color elimination is so much higher in beet than in cane, almost 100:1 in beet and 10:1 in cane. In this study, polysaccharide elimination in beet (thick juice to white sugar) was 12:1 and in cane (evaporator syrup to raw sugar) was only 6:1. Thus, the elimination of polysaccharide in beet processing is twice that of cane. In this study the elimination of color was 71:1 in the beet and 14:1 in the cane. Both cane and beet polysaccharides are arabinogalactans, but the nature of the polysaccharide incoming with raw beet juice is changed, as is the colorant, by carbonation and purification, so that the remaining colorant is a lower molecular weight and more easily eliminated. It is probably mostly made up of alkaline degradation products of invert. A portion of this colorant does go into the white beet sugar crystal. The greatest difference between cane and beet colorants is that the color that comes in with the cane juice remains largely unchanged throughout the milder process of making raw sugar. They are

8 mainly plant pigments associated with polysaccharide. These pigments would appear to have a much greater affinity for the white sugar crystal than the colorants produced during beet processing. Table 8. Molecular weight ranges of various cane and beet processing samples. Cane Crusher Juice Raw Beet Juice MW ranges % of Total MW Ranges % of Total 782, , , , , , , , ,000-31, ,500 2 <12, <12,000 1 Raw Cane Sugar (Gabon) Beet Thin Juice MW Ranges % of Total MW Ranges % of Total 840, , , , , , ,800-37, ,000-49, , , <12, <12, White Cane Sugar White Beet Sugar MW Ranges % of Total MW Ranges % of Total >1,000, , , , ,000-30, ,000-57, , ,000-18, <12, <12,

9 REFERENCES Baunsgaard, D., Andersson, C.A., Arndal, A., and Munck, L., Multi-way chemometrics for mathematical separation of fluorescent colorants and colour precursors from spectrofluorimetry of beet sugar and beet sugar thick juice as validated by HPLC analysis, Food Chem., 2000, 70, Baunsgaard, D., Nørgaard, L., and Godshall, M.A., Specific screening for color precursors and colorants in beet and cane sugar liquors in relation to model colorants using spectrofluorometry evaluated by HPLC and multiway data analysis, J. Agric. Food Chem., 2001, 49, Broughton, N.W., Sargent, D., Houghton, B.J., and Sissons, A., Studies of the colour of U.K. beet white sugar, Proc. Sugar Processing Research Conference, 1986, pp Broughton, N.W., Sargent, D., Houghton, B.J., and Sissons, A., The inclusion of colour and ash components in U.K. beet white sugar, CITS, 1987, pp Clarke, M.A., Godshall, M.A., Blanco, R.S., and Perret, G.T., Beet sugar colorant: recent studies, Zuckerindustrie, 1989, 114, Clarke, M.A., Roberts, E.J., Godshall, M.A., and Miranda, X.M.,The polysaccharides of sugarbeet, 1991, Proc. 19 th General Assembly CITS, 1991, Courtois, J. and Rousseau, G., Macromolecules in sugar factory, presentation to CITS Scientific Committee, May 2001, Paris. Fares, K., R Zina, Q., El Amrani, M., Crepeau, M-J., and Thibault, J-F., Extraction and degradation of polysaccharides from sugar beet roots under factory conditions, presentation to CITS Scientific Committee, May 2001, Paris. Godshall, M.A., Clarke, M.A., Dooley, C.D. and Roberts, E.J., Large colorant and polysaccharide molecules in raw cane sugars, Proc. Sugar Industry Technologists, 1987, 46, Godshall, M.A., Clarke, M.A., Dooley, C.C., and Roberts, E.J., High molecular weight color in refineries, Proc. Sugar Processing Research Conference, 1988, pp Godshall, M.A., Clarke, M.A., Dooley, C.D., and Blanco, R.S., Progress in beet sugar colorant research, J. Sugar Beet Research, 1991, 28, Godshall, M.A., Isolation of a high molecular weight colorant from white beet sugar, Proc Sugar Processing Research Conference, 1992, pp Godshall, M.A., Buchler, I.P., Boyle, H.G., and Clarke, M.A., Changes in white sugars under accelerated storage conditions, Proc., 1998 Sugar Processing Research Conference, 1998a, pp Godshall, M.A., Roberts, E.J., and Miranda, X.M., Composition of the soluble, nondialyzable components in raw cane sugar, J. of Food Processing and Preservation, 2001, 15,

10 Godshall, M.A., Buchler, I.P., and Vercellotti, J.R., Identification of esterified phenolic and organic acids in the high molecular weight colorant polysaccharide fraction in cane sugar processing, 1998 Sugar Processing Research Conference, 1998b, pp Godshall, M.A., and Baunsgaard, D., The nature of colorant, Proc. Conf. on Sugar Processing Research, 2000, pp Godshall, M.A., Roberts, E.J., and Miranda, X.M., Composition of the soluble, nondialyzable components in raw cane sugar, J. Food Processing and Preservation, 2001, 25, Gross, D., and Coombs, J., Enzymic colour formation in beet and cane juices, Internat. Sugar J., 1976, 78, and Kintner, III, P.K., and Van Buren, J.P., Carbohydrate interference and its correction in pectin analysis using the m-hydroxydiphenyl method, J. Food Sci., 1982, 47, Lindeman, P.F., and O Shea, M.G., High molecular weight (HMW) colorants and their impact on the refinability of raw sugar. A study of Australian and overseas raw sugars, Proc. Australian Soc. Sugar Cane Technol., 2001, 23, Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. chem, 1951, 193, Mantovani, G., et al., Colouring matter inclusions in sucrose crystals, Proc. Internat. Soc. Sugar Cane Technol., 1986, 19, Maurandi, V., Colour precursors in sugar beet juices. Flavonoids, L Industria Saccarifera Italiana, 1988, 81, Rhinefeld, E., Bliesener, K.M., Borass, B. and Poltrock, U., Studies on the browning of technical sugar juices with special reference to phenolic compounds, Zuckerind., 1984, 109, Richmond, J.A., Observations on color developed in stored sugar samples, Raw Sugar Quality and White Sugar Quality, Proc. of SPRI Workshops, ed., M.A. Clarke, 1990, pp Roberts, E.J., Godshall, M.A., Carpenter, F.g., and Clarke, M.A., Composition of soluble indigenous polysaccharides from sugar cane, Internat. Sugar J., 1976, 78, Shore, M., Broughton, N.W., Dutton, J.V., and Sissons, Factors affecting white sugar colour, Sugar Technol. Rev., 1984, Tu, C.C., Kondo, A.., and Sloane, G.E., The role of high and low molecular weight colorants in sugar color, Sugar J., 1977, 40(2), Vercellotti, J.R., Clarke, M.A., and Edye, L.A., Components of molasses: I. Sugarcane molasses: Factory and seasonal variables, Proc Sugar Processing Research Conference, 1996, pp. 321-

11 349. Vogel, M. and Schiweck, H., Polysaccharides in juices and syrups from sugarbeet: Isolation and characterization, Zucekrindustrie, 1988, 113, Wintrom-Olsen, B., Enzymic colour formation in sugar beet, Internat. Sugar J., 1981, 83, Winstrom-Olson, B., Madsen, R.F., and Kofod Nielsen, W., Sugar beet phenols. Investigation of phenolic compounds from sugar beet in relation to the formation of colour, Part I, Internat. Sugar J., 1979, 81, ; Part II, Ibid, 1979, 81,