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Determination of soluble fiber components: (1- > 3; 1- > 4)-β-d-glucans and pectins
Animal Feed Science and Technology, 23 (1989) 15-25
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Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
D e t e r m i n a t i o n of Soluble Fiber Components: (1- > 3; 1- > 4)- fl-D-Glucans and Pectins*
J.L. JERACI and B.A. LEWIS
Division of Nutritional Sciences, Cornell University, Ithaca, N Y 14853 "U.S.A.)
INTRODUCTION
The physiological effects associated with some of the more easily hydrated and soluble components of plant cell walls have led to increased research on these dietary fibers both in humans and animals. In humans, some components of the water-soluble fiber complex appear to have an effect on adult-onset diabetes, ischemic heart disease and cancer. The water-soluble fiber fractions in animal diets affect serum cholesterol and lipid levels, as well as contributing to reduced growth in some species. These effects are usually associated with high molecular weight, linear polysaccharides which give viscous solutions. Burnett (1966) showed that pectin and barley in the diet of chicks gave rise to wet sticky feces and reduced growth performance. Incorporation of viscosity-reducing enzymes into the pectin and barley diets improved the chick performance. Repeated demonstration of these effects with barley (Rickes et al., 1962; Gohl et al., 1978; Hesselman et al., 1982; Newman and Newman, 1987) have implicated the (1-> 3; 1-> 4)-fl-D-glucans as the major factor. Furthermore, pectin, barley and oats in the diet lower serum cholesterol levels in the rat, chicken and swine (Fisher and Griminger, 1967; Chen et al., 1981; Quereshi et al., 1982; Ahrens et al., 1986; Fadel et al., 1987). Studies of oat and barley fractions, and dietary supplementation with enzymes which hydrolyze soluble fl-glucan show that the fl-glucan fraction is, in part, responsible for this cholesterol-lowering effect. Varietal differences and environmental growing conditions all influence the physiological effects of barley and oat diets. This *This research was supported in part by NCI Contract No. N01-CN-45182.
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could be attributed to the quantity of fl-glucan present, but may also reflect differences in the chemical structure and physical properties of the fl-glucans. This paper focusses on the pectins and cereal (1-> 3; 1-> 4)-fl-D-glucans and reviews the methods of analysis in plant tissue. C E R E A L (1- > 3; 1- > 4)-fl-D-GLUCANS
These glucans are high molecular weight (MW) linear polymers of )~-Dglucopyranose linked at carbons 3 or 4 by glycosidic bonds (Fig. 1 ). The ratio of 3-1inked to 4-1inked residues is variously reported in the range of 1 to 2-3. Strong intermolecular hydrogen bonding is precluded by the conformational effect of the two linkages and, unlike cellulose, these glucans are soluble in water or dilute alkali. Since these polymers are linear and of high MW, they give highly viscous solutions. This viscosity appears to be partially responsible for the physiological effects. Although most cereals (Gramineae) contain these fl-glucans, barley and oats are the major sources with reported values for the fl-glucans of 4-12%. By contrast, wheat contains 1% or less fl-glucan. Variety (Henry, 1986) and, to a lesser extent, growing conditions affect the amount of fl-glucan in the seed. fl-Glucans are located in the endosperm cell walls of the Gramineae along with an arabinoxylan and a small amount of protein and cellulose (Table 1 ). In barley, the endosperm cell wall is composed of 70-75% fl-glucan and 2025% arabinoxylan (Fincher, 1975; Forrest and Wainwright, 1977). The remaining 5 % consists of cellulose, ( 1- > 3) -fl-D-glucans, glucomannans and arabinogalactans. Interestingly, pectic polymers (~-galacturonans) appear to be virtually absent from Gramineae cell walls. Within the endosperm, the fl-glucan in barley is relatively uniformly distributed across the cells and cell wall, whereas in certain varieties of oats, the thickened cell walls of the sub-aleurone Glucose
l
Cellulose Complex (Trichoderma and Penicillium)
p
13
13
p
t3
p
p
13
"--P3 G 1"-~,4G 1"-~3Gl-'~4G1"-~"4G1-'3G1 "-'~"4G 13-~'3G1
t
t
t
°
( 1 - > 3 ; , 1 - > 4 ) - IB- b - Glucon
l
~-Glucanase {B. subtilis)
#
P
G1 3 G +
+
P
G 1 4G1 P
G1 [3G1]
P
n-1
3G
3G
Fig. 1. Structure of ( 1- > 3; 1- > 4 ) -fl-D-glucan and the products of enzymolysis.
17 TABLE 1 Composition of cereal endosperm cellwalls Source
fl-glucan (%)
Arabino-xylan ( % )
Reference
Barley
70-75% 74.6, 75.2
20-25% 19.7, 18.4
Fincher (1975) Forrest and Wainwright (1977)
85%
Mares and Stone (1973)
24%
Smith and Stone (1973)
Wheat
7-8%
Rye
region show higher concentrations of fl-glucan (Wood and Fulcher, 1978, 1983; Wood et al., 1983; Wood, 1984). With oats then, it is possible to prepare an oat bran fraction with an enhanced content of fl-glucan as well as of insoluble fiber. Products of this type should be o f value for h u m a n consumption. T h e r e is some evidence that the fl-glucan is covalently bound to a peptide or protein in the cell wall. Proteases degrade fl-glucan preparations (Forrest and Wainwright, 1977), and a carboxypeptidase appears to facilitate solubilization of the fl-glucan from the cell wall at the onset of germination of barley (Barnforth et at., 1979). In the past, barley glucans have been studied extensively because of their involvement in the brewing industry and their physiological role in some lives t o c k a n d poultry. With increased interest in the physiological effects in humans, there should be an increasing research effort on oat glucan. At this time, our knowledge of oat fl-glucan is more limited. Extraction conditions
The apparent content of fl-glucan in any sample is strongly dependent on the extraction conditions and the endogenous fl-glucanases and proteases, as well as the cereal variety and growing conditions. This is of particular concern with neutral aqueous extractions since the fl-glucanases are rapidly activated, increasing the solubilityofthe fl-glucans and altering their physical properties. Based on this observation, the nutritive quality of barley for swine and chickens can be improved by aqueous pre-treatment (Gohl et al., 1978). Most current methods for analysis of the fl-glucan content depend on solubilization of the polymers. Within the endosperm cell wall, the fl-glucans exist as a family of fl-glucans or fl-glucan-protein complexes. The latter appear to be covalently associated. Forrest and Wainwright (1977) demonstrated two discrete high MW fractions in barley cultivars, Maris Otter and Julia. They extracted approximately 55-59% of the fl-glucan with water at 65°C and an additional 37-42% with N sodium hydroxide. T r e a t m e n t of these fractions
18 with thermolysin or by hydrazinolysis lowered their MW from ~ 33-39 X 106 to ~ 1 X 106 Da suggesting covalent bonding between fl-glucan and a protein. Covalent bonding by peptide, N-glycosidic, or through protein-ferulic acid bonds would be consistent with this evidence. From this study and others (Fincher, 1975; Forrest and Wainwright, 1977), it is apparent that hot water is a relatively poor solvent for quantitative extraction of these glucans. Alkali, however, is relatively effective extracting 95 % or more of the fl-glucan or fl-glucan-protein complex from barley (Forrest and Wainwright, 1977; Palmer and Mackenzie, 1986). Other hydrogen-bond breaking reagents such as urea (Costello and Stone, 1968; Smith and Stone, 1973) improve the aqueous extraction of fl-glucans, but fail to insure quantitative recovery and, in general, are probably inferior to alkali (J.M. Carr, S. Glatter, J.L. Jeraci and B.A. Lewis, unpublished data). Voragen et al. (1987) achieved a good extraction of fl-glucan from barley with 4-methylmorpholine-N-oxide with no apparent degradation. Hydrazine completely disrupts plant cell walls and has been used extensively to give good recoveries of fl-glucan (Martin and Bamforth, 1981 ). Since these conditions permit hydrazinolysis of peptide or N-glycosidically linked fl-glucan-protein complexes, this method can give essentially quantitative recovery of the glucan as shown by Forrest and Wainwright (1977). However, Palmer and Mackenzie (1986) demonstrated, for several barley cultivars, that alkali extraction is as effective as hydrazine and that losses of up to 12% occurred with the hydrazine treatment. Their reaction conditions were more drastic than those used by Forrest and Wainwright (1977) which may explain the variation in results. The toxicity of hydrazine also makes it less desirable. Various acidic extraction conditions have also been used together with specific assays for fl-glucan. Ahluwalia and Ellis (1984) employed a brief hot perchloric-acid treatment to extract both starch and fl-glucan for specific analysis by amyloglucosidase and cellulase (PeniciUium funiculosum), respectively. Hydrolysis of sucrose or other fructose-containing oligosaccharides under these conditions could lead to high blanks. If these extraction conditions are suitable for other cereals as well, this method would represent the quickest and simplest procedure for routine assay of total fl-glucans. Extraction under more mild conditions (1.5 N hydrochloric acid, 40 °C ) removes only a fraction of the fl-glucan (Smith et al., 1980a) and is not applicable for total fl-glucan.
Assay methods Enzymic assays The availability of specific fl-glucanases capable of hydrolyzing the mixed linkages of fl-glucans to glucose or low MW oligosaccharides, have made enzymic assays the preferred route for quantitation of fl-glucans, Most assays use a microbial source of enzyme (Table 2). Glucose released directly by the en-
19 TABLE 2 Survey of enzymic assays for determination of ( 1- > 3; 1- > 4)-]~-D-glucans in cereal grains Enzyme source (Product of reaction)
Quantitation method 1
Reference
Bacillus subtilis (Oligosaccharides) (Oligosaccharides) (Oligosaccharides)
Acid hydrolysis + G0P 2 Reducing sugar assay Reducing sugar assay fl-glucosidase + GOP
Anderson et al. (1978) Henry (1984) Henry and Blakeney (1986) McCleary and Glennie-Holmes (1985)
Trichoderma reesei Glucose Glucose Glucose Glucose
Hexokinase/Glc-6-PO4 dehydrogenase or GOP Hexokinase/Glc-6-PO4 dehydrogenase or GOP Hexokinase/Glc-6-PO4 dehydrogenase or GOP G0P
Martin and Bamforth (1981) Bamforth (1981) Gill et al. (1982) Prentice et al. (1980) Prentice (1982) Bourne et al. (1982)
Glucose
NA
Penicillium funiculosum Glucose Hexokinase/Glc-6-PO4 dehydrogenase Glucose Hexokinase/Glc-6-PO4 dehydrogenase Glucose GOP
Rhizomucor pusillus (Oligosaecharides)
Acid hydrolysis + GOP
Bamforth (1983) Ahluwalia and Ellis (1984) Glatter and Lewis, unpublished data
Aman and Heshelman, (1985)
1Method for assay of glucose: oligosaccharides were hydrolyzed to glucose with acid or fl-D-glucosidase. The reducing sugar assay used p-hydroxybenzoic acid hydrazine as the reagent. 2GOP = glucose oxidase/peroxidase.
zyme preparation or following acid or fl-glucosidase hydrolysis of the oligosaccharides (Fig. 1) can be estimated by the glucose oxidase reagents or by hexokinase/glucose-6-phosphate dehydrogenase. Colorimetric reducing sugar assays have also been used (Henry and Blakeney, 1986). The method of Anderson et al. (1978) has been used extensively. The BaciUus subtilis endo- ( 1- > 3; 1- > 4 ) -fl-D-glucan hydrolase (E.C.3.2.1.73), which is free of amylase activity, specifically hydrolyses a (1- > 4) -linkage adjacent to ( 1- > 3 ) -linked fl-glucopyranose units giving a series of ( 1- > 3; 1 - > 4 ) -linked oligosaccharides (Fig. 1 ) which are then acid hydrolyzed to glucose. The validity of this procedure rests on the absence of amylases in the fl-glucanase preparation and requires a preliminary extraction of the sample with 80% ethanol to remove endogenous glucose oligosaccharides and interfering endogenous hydrolases. The oligosaccharides released from the fl-glucans by both a commercial B. subtilis preparation (Anderson et al., 1978) and the purified enzyme (Huber and Nevins, 1977) have been characterized and found to be consistent with the action pattern of the enzyme.
20 McCleary and Glennie-Holmes (1985) have simplified this method and improved the specificity by using a purified fl-glucanase followed by a fl-glucosidase for hydrolysis of the oligosaccharides instead of acid hydrolysis. The glucose released has also been estimated colorimetrically by reaction with phydroxybenzoic acid hydrazide (Blakeney and Mutton, 1980; Henry and Blakeney, 1986). A preliminary reduction of endogenous reducing sugars with sodium borohydride has been recommended for samples, such as malt, which contain large quantities of sugars (Henry and Blakeney, 1986). In addition to eliminating the 80% alcohol-extraction step, the borohydride may also inactivate the endogenous carbohydrases. Cellulases from fungi offer an alternative to the bacterial glucanases (Fig. 1 ). Martin and Bamforth (1981) introduced a procedure based on the cellulase from Trichoderma reesei which completely hydrolyzes fl-glucan to glucose. Commercial preparations of this enzyme contain variable amounts of amyloglucosidase which is inactivated by heat treatment (Bamforth, 1981; Bourne et al., 1982; Gill et al., 1982; Prentice, 1982). Careful control is required to avoid inactivation of the cellulase also. The cellulase in P. funiculosum preparations is considerably more heat stable than that from T. reesei permitting a larger margin of safety during heat inactivation of the amyloglucosidase (Bamforth, 1983). This enzyme system appears to be the method of choice at this time. The fl-glucan-hydrolyzing cellulases can be readily purified further by ionexchange chromatography (S. Glatter and B.A. Lewis, unpublished data; Wood and McRae, 1982); however, for routine assays the heat-treated preparation appears to be adequate. These cellulases have the advantage that the fl-glucan is essentially completely hydrolyzed to glucose, thus avoiding an acid-hydrolysis step. Assuming adequate inactivation of amyloglucosidase by the heat treatment there is little interference from other glucose-containing oligosaccharides or polysaccharides. This assay method proposed by Bamforth (1983) using the P. funiculosum cellulase and the modification of McCleary and Glennie-Holmes (1985) using B. subtilis fl-glucanase appear to offer the most specific and direct assays for fl-glucans. In all the assays reviewed here, a variety of extraction conditions have been used including alkali (Palmer and Mackenzie, 1986), perchloric acid (Ahluwalia and Ellis, 1984) and hydrazine (Forrest and Wainwright, 1977) or direct contact of the enzyme with the ground sample (Anderson et al., 1978). At this time, it is not apparent which procedure (s) would be most reliable as a routine assay for total fl-glucans in the different cereals. Selective precipitation Compounds which are direct dyes for cellulose may also show a strong affinity for the mixed linkage fl-D-glucans. Two dyes, Congo Red and Calcofluor, have been used in studies of the fl-glucans. Of these, the optical brightener
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Calcofluor White M2R New (4,4'-bis [ (4-anilino-6- [bis(2-hydroxyethyl ) amino ] -s-triazin-2-yl ) amino ] -2,2' -stilbenedisulfonic acid disodium salt) has been used as a fluorochrome in microscopic studies of cereal cell walls (Hughes and McCully, 1975; Fulcher et al., 1977). Since it has strong binding capability and specificity for the fl-linked glucans, it will also bind to the polysaccharide in solution to give a gel-like precipitate of the complex, free from non-complexing polysaccharides (including starch), protein and salts (Wood and Fulcher, 1978). Therefore, Calcofluor has been used in isolation and purification of the soluble fl-glucans and as a specific assay. The precipitated complex can be redispersed and the glucan hydrolyzed for quantitation of the liberated glucose (Wood and Weisz, 1984). However, the procedure is dependent on closely controlled pH and ionic strength to insure quantitative complexation. Alternatively, the complex can be analyzed by fluorescence spectroscopy (Jensen and Aastrup, 1981). The enhanced fluorescence (4-7-fold) associated with complexation (Wood, 1980; Wood et al., 1983) has been particularly useful in microscopy to locate cell wall fl-glucans in plant tissue.
Viscosity The viscosity of extracts has frequently been used as an indicator of soluble fl-glucan content because of the high viscosity of fl-glucan solutions (Greenberg, 1974; Greenberg and Whitmore, 1974). Ullrich et al. (1986), however, have shown that while viscosity of acidic extracts correlate more closely than alkaline extracts with fl-glucan content, viscosity measurements do not give good estimates of fl-glucan from barleys. This may reflect structural differences in the fl-glucans since it has been shown that cOntaminating protein contributes little to the overall viscosity (Smith et al., 1980a).
Near infrared spectroscopy (NIR) NIR, which permits direct analysis of the ground sample, has now been applied to the estimation of total fl-glucan (Henry, 1985). PECTINS
The pectins are a family of polysaccharides having a (1-> 4)-~-D-galacturonic acid backbone with occasional ~-L-rhamnose units interspersed within the linear chain. At various stages of maturity of the plant the pectin is partially in the methyl ester form and may contain some acetyl groups. In the plant tissue, the galacturonan is physically or covalently associated with neutral polysaccharides. This complex of polysaccharides is frequently referred to as the pectic substances. The pectins predominate in the primary cell wall and middle lamella of the plant cell wall and are thought to promote cellular adhesion. Since they have a tendency to degrade under neutral or alkaline aqueous
22
conditions and because of their unique association with the other pectic substances, it is difficult to extract the pectin galacturonan in its native form. Extractants frequently include chelating agents to dissociate the pectin from its insoluble calcium salts or have an alkaline pH which promotes degradation by a fl-elimination reaction. For total pectin, extraction is not necessary. The sample can be solubilized directly in 72% sulfuric acid for the colorimetric assays or in hydrochloric acid, a hydroidic acid in the decarboxylation procedure.
Analysis Methods for quantitation of pectin are based on decarboxylation of the galacturonosyl units by strong acid or are based on a reaction with aromatic chromogens to give a colorimetric assay. Selective precipitation of the soluble pectins with rare earths has been investigated (P.J. van Soest, unpublished data), but as yet it has not been developed into a satisfactory assay.
Carbazole reaction This is a classic colorimetric reaction for uronic acids in which the galacturonic acid-containing sample is digested in hot concentrated sulfuric acid and the resulting product, 5-formyl-furancarboxylicacid, is reacted with carbazole to give a color which is read at 530 nm. The modification of Bitter and Muir (1962), which enhances the response of uronic acids, is frequently used. The advantages of the method are its simplicity and stability of color. Neutral sugars, when present in the sample, give rise to additional absorbence which can cause a significant error. All uronic acids react, but with variable response. Galacturonic acid gives about 90% of the response given by glucuronic acid which means that hemicellulose uronic acid, if present, will contribute to the apparent pectin content. Automated applications of the carbazole procedure are available.
m-Hydroxydiphenyl (3-phenylphenol) reaction In order to enhance the specificity for uronic acids, Blumencrantz and Asboe-Hansen (1973) substituted 3-phenylphenol for the carbazole in the method just described. This increased the sensitivity for uronic acids by 2-3 times and decreased the interference by neutral sugars. This method is widely used as is the modification proposed by Thibault (1979). Several parameters were adjusted by Thibault and he showed that omission of tetraborate decreased the interference by neutral sugars. Bucher (1984) re-examined the parameters of the reaction conditions in order to enhance the specificity for galacturonic acid and pectin, and observed that omission of tetraborate also increased the response from galacturonic acid.
23
Decarboxylation Hot 12% hydrochloric acid (constant boiling) decarboxylates uronic acids to give stoichiometric amounts of carbon dioxide under carefully controlled conditions. An early quantitative procedure was devised by Whistler et al. (see Whistler and Feather, 1962 ) which uses a carrier gas and an absorption train to sweep out impurities and trap the carbon dioxide which is then titrated. This general procedure has been improved and automated (Theander and Westerlund, 1986a). Recent modifications use changes in conductivity for quantitation of the galacturonic acid and hydriotic acid instead of the hydrochloric acid. The decarboxylation method measures the total of all uronic acids, but does not give differing responses for the different uronic acids as found for the colorimetric methods (Theander and Westerlund, 1986a). Using the procedure of Theander and Aman (1979), Theander and Westerlund (1986b) have reported values that range from 20.9% (SD 0.6) for sugar beet fiber to 1.6% (SD 0.1) for wheat bran.
REFERENCES Ahluwalia, B. and Ellis, E.E., 1984. A rapid and simple method for the determination of starch and fl-glucan in barley and malt. J. Inst. Brew., 90: 254-259. Ahrens, F., Hagemeister, H.. Pfeuffer, M. and Barth, C.A., 1986. Effects of oral and intracecal pectin administration on blood lipids in mini pigs. J. Nutr., 116: 70-76. Aman, P. and Heschelman, K., 1985. An enzymic method for analysis of total mixed-linkage flglucan in cereal grains. J. Cereal Sci., 3: 231-237. Anderson, M.A., Cook, J.A. and Stone, B.A., 1978. Enzymatic determination of 1,3:l,4-fl-glucans in barley grain and other cereals. J. Inst. Brew., 84: 233-239. Bamforth, C.W., 1981. The estimation of fl-glucan in barley. J. Inst. Brew., 87: 276. Bamforth, C.W., 1983. Penicillium funiculosum as a source of fl-glucanase for the estimation of barley fl-glucan. J. Inst. Brew., 89: 391-392. Bamforth, C.W., Martin, H.L. and Wainwright, T., 1979. A role for carboxypeptidase in the solubilization of barley fl-glucan. J. Inst. Brew., 85: 334-338. Beresford, G. and Stone, B.A., 1983. (1- > 3 ), (1- > 4)-fl-D-Glucan content of Triticum grains. J. Cereal Sci., 1: 111-114. Bitter, T. and Muir, H.M., 1962. A modified uronic acid carbazole reaction. Anal. Biochem., 4: 330-334. Blakeney, A.B. and Mutton, L.L., 1980. A simple colorimetric method for the determination of sugars in fruits and vegetables. J. Sci. Food Agric., 31: 889-897. Blumencrantz, N. and Asboe-Hansen, G., 1973. A new method for quantitative determination of uronic acids. Anal. Biochem., 54: 484-489. Bourne, D.T., Powlesland, T., Smith, D. and Morgan, A., 1982. The estimation of fl-glucan in barley. J. Inst. Brew., 88: 135-136. Bucher, A.C., 1984. A comparison of solvent systems for extraction of pectic substances from fruits and vegetables. M.S. Thesis, Cornell University, NY. Burnett, G.S., 1966. Studies of viscosity as the probable factor involved in the improvement of certain barleys for chickens by enzyme supplementation. Br. Poult. Sci., 7: 55-75. Chen, W-J.L., Anderson, J.W. and Gould, M.R., 1981. Effects of oat bran, oat gum and pectin on lipid metabolism of cholesterol-fed rats. Nutr. Rep. Int., 24: 1093-1098.
24 Costello, P.R. and Stone, B.A., 1968. Isolation and analysis of cereal endosperm cell walls. Proc. Aust. Biochem. Soc., 1: 43. Fadel, J.G., Newman, R.K., Newman, C.W. and Barnes, A.E., 1987. Hypocholesterolemic effects of beta-glucans in different barley diets fed to broiler chicks. Nutr. Rep. Int., 35: 1049-1058. Fincher, G.B., 1975. Morphology and chemical composition of barley endosperm cell walls. J. Inst. Brew., 81: 116-122. Fisher, H. and Griminger, P., 1967. Cholesterol-lowering effects of certain grains and oat fractions in the chick. Proc. Soc. Exp. Biol. Med., 126: 108-111. Forrest, I.S. and Wainwright, T., 1977. The mode of binding of fl-glucans and pentosans in barley endosperm cell walls. J. Inst. Brew., 83: 279-286. Fu!cher, R.G., Setterfield, G., McCully, M.E. and Wood, P.J., 1977. Observations on the aleurone layer. II. Fluorescence microscopy of the aleurone-sub-aleurone junction with emphasis on possible fl-1, 3-glucan deposits in barley. Aust. J. Plant Physiol., 4: 917-928. Gill, A.A., Morgan, A.G. and Smith, D.B., 1982. Total fl-glucan content of some barley cultivars. J. Inst. Brew., 88: 317-319. Gohl, B., Alden, S., Elwinger, K. and Thomke, S., 1978. Influence of fl-glucanase in feeding value of barley for poultry and moisture content of excreta. Br. Poult. Sci., 19: 41-47. Greenberg, D.C., 1974. fl-Glucan and extract viscosity. J. Inst. Brew., 80: 435. Greenberg, D.C. and Whitmore, E.T., 1974. A rapid method for estimating the viscosity of barley extracts. J. Inst. Brew., 80: 31-33. Henry, R.J., 1984. A simplified enzymic method for the determination of (1->3) (1->4)-flglucans in barley. J. Inst. Brew., 90: 178-180. Henry, R.J., 1985. Near-infrared reflectance analysis of carbohydrates and its application to the determination of ( 1- > 3 ) ( 1- > 4 ) -fl-D-glucan in barley. Carbohydr. Res., 141: 13-19. Henry, R.J., 1986. Genetic and environmental variations in the pentosan and fl-glucan contents of barley, and their relation to malting quality. J. Cereal Sci., 4: 269-277. Henry, R.J. and Blakeney, A.B., 1986. Determination of total p-glucan in malt. J. Inst. Brew., 92: 354-356. Hesselman, K., Elwinger, K. and Thomke, S., 1982. Influence of increasing levels of fl-glucanase on the productive value of barley diets for broiler chickens. Anim. Feed Sci. Technol., 7: 351358. Huber, D.J. and Nevins, D.J., 1977. Preparation and properties of a fl-D-glucanase for the specific hydrolysis of fl-D-glucans. Plant Physiol., 60: 300-304. Hughes, J. and McCully, M.E., 1975. The use of an optical brightener in the study of plant structure. Stain Technol., 50: 319-329. Jensen, S.A. and Aastrup, S., 1981. A fluorometric method for measuring 1,3:1,4-fl-glucan in beer, wort, malt and barley by use of Calcofluor. Carlsberg Res. Commun., 46: 87-95. Mares, D.J. and Stone, B.A., 1973. Studies on wheat endosperm. I. Chemical composition and ultrastructure of the cell walls. Aust. J. Biol. Sci., 26: 793-812. Martin, H.L. and Bamforth, C.W., 1981. An enzymic method for the measurement of total and water-soluble fl-glucan in barley. J. Inst. Brew., 87: 88-91. McCleary, B.V. and Glennie-Holmes, M., 1985. Enzymic quantification of (1-> 3 ) (1-> 4 )-fl-Dglucan in barley and malt. J. Inst. Brew., 91: 285-295. Newman, R.K. and Newman, C.W., 1987. Beta-glucanase effect on the performance of broiler chicks fed covered and hulless barley isotypes having normal and waxy starch. Nutr. Rep. Int., 36: 693-699. Palmer, G.H. and Mackenzie, C.I., 1986. Levels of alkali-soluble fl-D-glucans in cereal grains. J. Inst. Brew., 92: 461-462. Prentice, N., 1982. Purification of beta-glucan for fl-D-glucan assays. Cereal Chem., 59: 231-232. Prentice, N., Babler, S. and Faber, S., 1980. Enzymic analysis of beta-D-glucans in cereal grains. Cereal Chem., 57: 198-202.
25 Quereshi, A.A., Burger, W.C., Elson, C.E. and Benevenga, N.J., 1982. Effects of cereals and culture filtrate of Trichoderma viride on lipid metabolism of swine. Lipids, 17: 924-934. Rickes, E.L., Ham, E.A., Moscatelli, E.A. and Ott, W.H., 1962. The isolation and biological properties of a fl-glucanase from B. subtilis. Arch. Biochem. Biophys., 96: 371-375. Smith, D.B., Morgan, A.G. and Aastrup, S., 1980a. Variation in the biochemical composition of acid extracts from barleys of contrasting malting quality. J. Inst. Brew., 86: 277-283. Smith, D.B., Morgan, A.G. and Gill, A.A., 1980b. The major biochemical constituents of an acid extract of barley flour as used in viscosity determinations by the falling ball method. J. Inst. Brew., 86: 113-119. Smith, M.M. and Stone, B.A., 1973. Chemical composition of the cell walls ofLolium multiflorum endosperm. Phytochemistry, 12: 1361-1367. Theander, O. and Aman, P., 1979. Studies on dietary fiber, 1. Analysis and chemical characterization of water-soluble and water-insoluble dietary fiber. Swed. J. Agric. Res., 9: 97-106. Theander, O. and Westerlund, E.A., 1986a. Determination of individual components of dietary fiber. In: G.E. Spiller (Editor), Handbook of Dietary Fiber. CRC Press, Cleveland, OH, pp. 57-69. Theander, 0. and Westerlund, E.A., 1986b. Studies on dietary fiber. 3. Improved procedures for analysis of dietary fiber. J. Agric. Food Chem., 34: 330-336. Thibault, J.F., 1979. Automatisation du dosage des substances pectiques par la methode au metahydroxydiphenyl. Lebensm. Wiss. Technol., 12: 247-251. Ullrich, S.E., Clancy, J.A., Eslick, R.F. and Lance, R.C.M., 1986. fl-Glucan content and viscosity of extracts from waxy barley. J. Cereal Sci., 4: 279-285. Voragen, A.G.J., Schols, H.A., Marijs, J. and Rombouts, F.M., 1987. Non-starch polysaccharides from barley: structural features and breakdown during malting. J. Inst. Brew., 93: 202-208. Whistler, R.L. and Feather, M.S., 1962. Carboxyl determinations. Uronic acid carboxyl and total carboxyl. In:. R.L. Whistler and M.L. Wolfrom (Editors), Methods in Carbohydrate Chemistry, I. Academic Press, New York, pp. 464-468. Wood, P.J., 1980. Specificity in the interaction of direct dyes with polysaccharides. Carbohydr. Res., 85: 271-287. Wood, P.J., 1984. Physicochemical properties and technological and nutritional significance of cereal fl-glucans. In: V.F. Rasper (Editor), Cereal Polysaccharides in Technology and Nutrition. American Association of Cereal Chemists, St. Paul, MN. Wood, P.J. and Fulcher, R.G., 1978. Interaction of some dyes with cereal fl-glucans. Cereal Chem., 55: 952-965. Wood, P.J. and Fulcher, R.G., 1983. Dye interactions. A basis for specific detection and histochemistry of polysaccharides. J. Histochem. Cytochem., 31: 823-826. Wood, T.M. and McCrae, S.I., 1982. Purification and some properties of a (1->4)-fl-D-glucan glucohydrolase associated with the cellulase from the fungus PeniciUium funiculosum. Carbohydr. Res., 110: 291-303. Wood, P.J. and Weisz, J., 1984. Use of Calcofluor in analysis of oat beta-D-glucan. Cereal Chem., 61: 73-75. Wood, P.J., Fulcher, R.G. and Stone, B.A., 1983. Studies on the specificity of interaction of cereal cell wall components with Congo Red and Calcofluor. Specific detection and histochemistry of ( 1- > 3 ) (1- > 4)-fl-D-glucan. J. Cereal Sci., 1: 95-100.
15
Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
D e t e r m i n a t i o n of Soluble Fiber Components: (1- > 3; 1- > 4)- fl-D-Glucans and Pectins*
J.L. JERACI and B.A. LEWIS
Division of Nutritional Sciences, Cornell University, Ithaca, N Y 14853 "U.S.A.)
INTRODUCTION
The physiological effects associated with some of the more easily hydrated and soluble components of plant cell walls have led to increased research on these dietary fibers both in humans and animals. In humans, some components of the water-soluble fiber complex appear to have an effect on adult-onset diabetes, ischemic heart disease and cancer. The water-soluble fiber fractions in animal diets affect serum cholesterol and lipid levels, as well as contributing to reduced growth in some species. These effects are usually associated with high molecular weight, linear polysaccharides which give viscous solutions. Burnett (1966) showed that pectin and barley in the diet of chicks gave rise to wet sticky feces and reduced growth performance. Incorporation of viscosity-reducing enzymes into the pectin and barley diets improved the chick performance. Repeated demonstration of these effects with barley (Rickes et al., 1962; Gohl et al., 1978; Hesselman et al., 1982; Newman and Newman, 1987) have implicated the (1-> 3; 1-> 4)-fl-D-glucans as the major factor. Furthermore, pectin, barley and oats in the diet lower serum cholesterol levels in the rat, chicken and swine (Fisher and Griminger, 1967; Chen et al., 1981; Quereshi et al., 1982; Ahrens et al., 1986; Fadel et al., 1987). Studies of oat and barley fractions, and dietary supplementation with enzymes which hydrolyze soluble fl-glucan show that the fl-glucan fraction is, in part, responsible for this cholesterol-lowering effect. Varietal differences and environmental growing conditions all influence the physiological effects of barley and oat diets. This *This research was supported in part by NCI Contract No. N01-CN-45182.
16
could be attributed to the quantity of fl-glucan present, but may also reflect differences in the chemical structure and physical properties of the fl-glucans. This paper focusses on the pectins and cereal (1-> 3; 1-> 4)-fl-D-glucans and reviews the methods of analysis in plant tissue. C E R E A L (1- > 3; 1- > 4)-fl-D-GLUCANS
These glucans are high molecular weight (MW) linear polymers of )~-Dglucopyranose linked at carbons 3 or 4 by glycosidic bonds (Fig. 1 ). The ratio of 3-1inked to 4-1inked residues is variously reported in the range of 1 to 2-3. Strong intermolecular hydrogen bonding is precluded by the conformational effect of the two linkages and, unlike cellulose, these glucans are soluble in water or dilute alkali. Since these polymers are linear and of high MW, they give highly viscous solutions. This viscosity appears to be partially responsible for the physiological effects. Although most cereals (Gramineae) contain these fl-glucans, barley and oats are the major sources with reported values for the fl-glucans of 4-12%. By contrast, wheat contains 1% or less fl-glucan. Variety (Henry, 1986) and, to a lesser extent, growing conditions affect the amount of fl-glucan in the seed. fl-Glucans are located in the endosperm cell walls of the Gramineae along with an arabinoxylan and a small amount of protein and cellulose (Table 1 ). In barley, the endosperm cell wall is composed of 70-75% fl-glucan and 2025% arabinoxylan (Fincher, 1975; Forrest and Wainwright, 1977). The remaining 5 % consists of cellulose, ( 1- > 3) -fl-D-glucans, glucomannans and arabinogalactans. Interestingly, pectic polymers (~-galacturonans) appear to be virtually absent from Gramineae cell walls. Within the endosperm, the fl-glucan in barley is relatively uniformly distributed across the cells and cell wall, whereas in certain varieties of oats, the thickened cell walls of the sub-aleurone Glucose
l
Cellulose Complex (Trichoderma and Penicillium)
p
13
13
p
t3
p
p
13
"--P3 G 1"-~,4G 1"-~3Gl-'~4G1"-~"4G1-'3G1 "-'~"4G 13-~'3G1
t
t
t
°
( 1 - > 3 ; , 1 - > 4 ) - IB- b - Glucon
l
~-Glucanase {B. subtilis)
#
P
G1 3 G +
+
P
G 1 4G1 P
G1 [3G1]
P
n-1
3G
3G
Fig. 1. Structure of ( 1- > 3; 1- > 4 ) -fl-D-glucan and the products of enzymolysis.
17 TABLE 1 Composition of cereal endosperm cellwalls Source
fl-glucan (%)
Arabino-xylan ( % )
Reference
Barley
70-75% 74.6, 75.2
20-25% 19.7, 18.4
Fincher (1975) Forrest and Wainwright (1977)
85%
Mares and Stone (1973)
24%
Smith and Stone (1973)
Wheat
7-8%
Rye
region show higher concentrations of fl-glucan (Wood and Fulcher, 1978, 1983; Wood et al., 1983; Wood, 1984). With oats then, it is possible to prepare an oat bran fraction with an enhanced content of fl-glucan as well as of insoluble fiber. Products of this type should be o f value for h u m a n consumption. T h e r e is some evidence that the fl-glucan is covalently bound to a peptide or protein in the cell wall. Proteases degrade fl-glucan preparations (Forrest and Wainwright, 1977), and a carboxypeptidase appears to facilitate solubilization of the fl-glucan from the cell wall at the onset of germination of barley (Barnforth et at., 1979). In the past, barley glucans have been studied extensively because of their involvement in the brewing industry and their physiological role in some lives t o c k a n d poultry. With increased interest in the physiological effects in humans, there should be an increasing research effort on oat glucan. At this time, our knowledge of oat fl-glucan is more limited. Extraction conditions
The apparent content of fl-glucan in any sample is strongly dependent on the extraction conditions and the endogenous fl-glucanases and proteases, as well as the cereal variety and growing conditions. This is of particular concern with neutral aqueous extractions since the fl-glucanases are rapidly activated, increasing the solubilityofthe fl-glucans and altering their physical properties. Based on this observation, the nutritive quality of barley for swine and chickens can be improved by aqueous pre-treatment (Gohl et al., 1978). Most current methods for analysis of the fl-glucan content depend on solubilization of the polymers. Within the endosperm cell wall, the fl-glucans exist as a family of fl-glucans or fl-glucan-protein complexes. The latter appear to be covalently associated. Forrest and Wainwright (1977) demonstrated two discrete high MW fractions in barley cultivars, Maris Otter and Julia. They extracted approximately 55-59% of the fl-glucan with water at 65°C and an additional 37-42% with N sodium hydroxide. T r e a t m e n t of these fractions
18 with thermolysin or by hydrazinolysis lowered their MW from ~ 33-39 X 106 to ~ 1 X 106 Da suggesting covalent bonding between fl-glucan and a protein. Covalent bonding by peptide, N-glycosidic, or through protein-ferulic acid bonds would be consistent with this evidence. From this study and others (Fincher, 1975; Forrest and Wainwright, 1977), it is apparent that hot water is a relatively poor solvent for quantitative extraction of these glucans. Alkali, however, is relatively effective extracting 95 % or more of the fl-glucan or fl-glucan-protein complex from barley (Forrest and Wainwright, 1977; Palmer and Mackenzie, 1986). Other hydrogen-bond breaking reagents such as urea (Costello and Stone, 1968; Smith and Stone, 1973) improve the aqueous extraction of fl-glucans, but fail to insure quantitative recovery and, in general, are probably inferior to alkali (J.M. Carr, S. Glatter, J.L. Jeraci and B.A. Lewis, unpublished data). Voragen et al. (1987) achieved a good extraction of fl-glucan from barley with 4-methylmorpholine-N-oxide with no apparent degradation. Hydrazine completely disrupts plant cell walls and has been used extensively to give good recoveries of fl-glucan (Martin and Bamforth, 1981 ). Since these conditions permit hydrazinolysis of peptide or N-glycosidically linked fl-glucan-protein complexes, this method can give essentially quantitative recovery of the glucan as shown by Forrest and Wainwright (1977). However, Palmer and Mackenzie (1986) demonstrated, for several barley cultivars, that alkali extraction is as effective as hydrazine and that losses of up to 12% occurred with the hydrazine treatment. Their reaction conditions were more drastic than those used by Forrest and Wainwright (1977) which may explain the variation in results. The toxicity of hydrazine also makes it less desirable. Various acidic extraction conditions have also been used together with specific assays for fl-glucan. Ahluwalia and Ellis (1984) employed a brief hot perchloric-acid treatment to extract both starch and fl-glucan for specific analysis by amyloglucosidase and cellulase (PeniciUium funiculosum), respectively. Hydrolysis of sucrose or other fructose-containing oligosaccharides under these conditions could lead to high blanks. If these extraction conditions are suitable for other cereals as well, this method would represent the quickest and simplest procedure for routine assay of total fl-glucans. Extraction under more mild conditions (1.5 N hydrochloric acid, 40 °C ) removes only a fraction of the fl-glucan (Smith et al., 1980a) and is not applicable for total fl-glucan.
Assay methods Enzymic assays The availability of specific fl-glucanases capable of hydrolyzing the mixed linkages of fl-glucans to glucose or low MW oligosaccharides, have made enzymic assays the preferred route for quantitation of fl-glucans, Most assays use a microbial source of enzyme (Table 2). Glucose released directly by the en-
19 TABLE 2 Survey of enzymic assays for determination of ( 1- > 3; 1- > 4)-]~-D-glucans in cereal grains Enzyme source (Product of reaction)
Quantitation method 1
Reference
Bacillus subtilis (Oligosaccharides) (Oligosaccharides) (Oligosaccharides)
Acid hydrolysis + G0P 2 Reducing sugar assay Reducing sugar assay fl-glucosidase + GOP
Anderson et al. (1978) Henry (1984) Henry and Blakeney (1986) McCleary and Glennie-Holmes (1985)
Trichoderma reesei Glucose Glucose Glucose Glucose
Hexokinase/Glc-6-PO4 dehydrogenase or GOP Hexokinase/Glc-6-PO4 dehydrogenase or GOP Hexokinase/Glc-6-PO4 dehydrogenase or GOP G0P
Martin and Bamforth (1981) Bamforth (1981) Gill et al. (1982) Prentice et al. (1980) Prentice (1982) Bourne et al. (1982)
Glucose
NA
Penicillium funiculosum Glucose Hexokinase/Glc-6-PO4 dehydrogenase Glucose Hexokinase/Glc-6-PO4 dehydrogenase Glucose GOP
Rhizomucor pusillus (Oligosaecharides)
Acid hydrolysis + GOP
Bamforth (1983) Ahluwalia and Ellis (1984) Glatter and Lewis, unpublished data
Aman and Heshelman, (1985)
1Method for assay of glucose: oligosaccharides were hydrolyzed to glucose with acid or fl-D-glucosidase. The reducing sugar assay used p-hydroxybenzoic acid hydrazine as the reagent. 2GOP = glucose oxidase/peroxidase.
zyme preparation or following acid or fl-glucosidase hydrolysis of the oligosaccharides (Fig. 1) can be estimated by the glucose oxidase reagents or by hexokinase/glucose-6-phosphate dehydrogenase. Colorimetric reducing sugar assays have also been used (Henry and Blakeney, 1986). The method of Anderson et al. (1978) has been used extensively. The BaciUus subtilis endo- ( 1- > 3; 1- > 4 ) -fl-D-glucan hydrolase (E.C.3.2.1.73), which is free of amylase activity, specifically hydrolyses a (1- > 4) -linkage adjacent to ( 1- > 3 ) -linked fl-glucopyranose units giving a series of ( 1- > 3; 1 - > 4 ) -linked oligosaccharides (Fig. 1 ) which are then acid hydrolyzed to glucose. The validity of this procedure rests on the absence of amylases in the fl-glucanase preparation and requires a preliminary extraction of the sample with 80% ethanol to remove endogenous glucose oligosaccharides and interfering endogenous hydrolases. The oligosaccharides released from the fl-glucans by both a commercial B. subtilis preparation (Anderson et al., 1978) and the purified enzyme (Huber and Nevins, 1977) have been characterized and found to be consistent with the action pattern of the enzyme.
20 McCleary and Glennie-Holmes (1985) have simplified this method and improved the specificity by using a purified fl-glucanase followed by a fl-glucosidase for hydrolysis of the oligosaccharides instead of acid hydrolysis. The glucose released has also been estimated colorimetrically by reaction with phydroxybenzoic acid hydrazide (Blakeney and Mutton, 1980; Henry and Blakeney, 1986). A preliminary reduction of endogenous reducing sugars with sodium borohydride has been recommended for samples, such as malt, which contain large quantities of sugars (Henry and Blakeney, 1986). In addition to eliminating the 80% alcohol-extraction step, the borohydride may also inactivate the endogenous carbohydrases. Cellulases from fungi offer an alternative to the bacterial glucanases (Fig. 1 ). Martin and Bamforth (1981) introduced a procedure based on the cellulase from Trichoderma reesei which completely hydrolyzes fl-glucan to glucose. Commercial preparations of this enzyme contain variable amounts of amyloglucosidase which is inactivated by heat treatment (Bamforth, 1981; Bourne et al., 1982; Gill et al., 1982; Prentice, 1982). Careful control is required to avoid inactivation of the cellulase also. The cellulase in P. funiculosum preparations is considerably more heat stable than that from T. reesei permitting a larger margin of safety during heat inactivation of the amyloglucosidase (Bamforth, 1983). This enzyme system appears to be the method of choice at this time. The fl-glucan-hydrolyzing cellulases can be readily purified further by ionexchange chromatography (S. Glatter and B.A. Lewis, unpublished data; Wood and McRae, 1982); however, for routine assays the heat-treated preparation appears to be adequate. These cellulases have the advantage that the fl-glucan is essentially completely hydrolyzed to glucose, thus avoiding an acid-hydrolysis step. Assuming adequate inactivation of amyloglucosidase by the heat treatment there is little interference from other glucose-containing oligosaccharides or polysaccharides. This assay method proposed by Bamforth (1983) using the P. funiculosum cellulase and the modification of McCleary and Glennie-Holmes (1985) using B. subtilis fl-glucanase appear to offer the most specific and direct assays for fl-glucans. In all the assays reviewed here, a variety of extraction conditions have been used including alkali (Palmer and Mackenzie, 1986), perchloric acid (Ahluwalia and Ellis, 1984) and hydrazine (Forrest and Wainwright, 1977) or direct contact of the enzyme with the ground sample (Anderson et al., 1978). At this time, it is not apparent which procedure (s) would be most reliable as a routine assay for total fl-glucans in the different cereals. Selective precipitation Compounds which are direct dyes for cellulose may also show a strong affinity for the mixed linkage fl-D-glucans. Two dyes, Congo Red and Calcofluor, have been used in studies of the fl-glucans. Of these, the optical brightener
21
Calcofluor White M2R New (4,4'-bis [ (4-anilino-6- [bis(2-hydroxyethyl ) amino ] -s-triazin-2-yl ) amino ] -2,2' -stilbenedisulfonic acid disodium salt) has been used as a fluorochrome in microscopic studies of cereal cell walls (Hughes and McCully, 1975; Fulcher et al., 1977). Since it has strong binding capability and specificity for the fl-linked glucans, it will also bind to the polysaccharide in solution to give a gel-like precipitate of the complex, free from non-complexing polysaccharides (including starch), protein and salts (Wood and Fulcher, 1978). Therefore, Calcofluor has been used in isolation and purification of the soluble fl-glucans and as a specific assay. The precipitated complex can be redispersed and the glucan hydrolyzed for quantitation of the liberated glucose (Wood and Weisz, 1984). However, the procedure is dependent on closely controlled pH and ionic strength to insure quantitative complexation. Alternatively, the complex can be analyzed by fluorescence spectroscopy (Jensen and Aastrup, 1981). The enhanced fluorescence (4-7-fold) associated with complexation (Wood, 1980; Wood et al., 1983) has been particularly useful in microscopy to locate cell wall fl-glucans in plant tissue.
Viscosity The viscosity of extracts has frequently been used as an indicator of soluble fl-glucan content because of the high viscosity of fl-glucan solutions (Greenberg, 1974; Greenberg and Whitmore, 1974). Ullrich et al. (1986), however, have shown that while viscosity of acidic extracts correlate more closely than alkaline extracts with fl-glucan content, viscosity measurements do not give good estimates of fl-glucan from barleys. This may reflect structural differences in the fl-glucans since it has been shown that cOntaminating protein contributes little to the overall viscosity (Smith et al., 1980a).
Near infrared spectroscopy (NIR) NIR, which permits direct analysis of the ground sample, has now been applied to the estimation of total fl-glucan (Henry, 1985). PECTINS
The pectins are a family of polysaccharides having a (1-> 4)-~-D-galacturonic acid backbone with occasional ~-L-rhamnose units interspersed within the linear chain. At various stages of maturity of the plant the pectin is partially in the methyl ester form and may contain some acetyl groups. In the plant tissue, the galacturonan is physically or covalently associated with neutral polysaccharides. This complex of polysaccharides is frequently referred to as the pectic substances. The pectins predominate in the primary cell wall and middle lamella of the plant cell wall and are thought to promote cellular adhesion. Since they have a tendency to degrade under neutral or alkaline aqueous
22
conditions and because of their unique association with the other pectic substances, it is difficult to extract the pectin galacturonan in its native form. Extractants frequently include chelating agents to dissociate the pectin from its insoluble calcium salts or have an alkaline pH which promotes degradation by a fl-elimination reaction. For total pectin, extraction is not necessary. The sample can be solubilized directly in 72% sulfuric acid for the colorimetric assays or in hydrochloric acid, a hydroidic acid in the decarboxylation procedure.
Analysis Methods for quantitation of pectin are based on decarboxylation of the galacturonosyl units by strong acid or are based on a reaction with aromatic chromogens to give a colorimetric assay. Selective precipitation of the soluble pectins with rare earths has been investigated (P.J. van Soest, unpublished data), but as yet it has not been developed into a satisfactory assay.
Carbazole reaction This is a classic colorimetric reaction for uronic acids in which the galacturonic acid-containing sample is digested in hot concentrated sulfuric acid and the resulting product, 5-formyl-furancarboxylicacid, is reacted with carbazole to give a color which is read at 530 nm. The modification of Bitter and Muir (1962), which enhances the response of uronic acids, is frequently used. The advantages of the method are its simplicity and stability of color. Neutral sugars, when present in the sample, give rise to additional absorbence which can cause a significant error. All uronic acids react, but with variable response. Galacturonic acid gives about 90% of the response given by glucuronic acid which means that hemicellulose uronic acid, if present, will contribute to the apparent pectin content. Automated applications of the carbazole procedure are available.
m-Hydroxydiphenyl (3-phenylphenol) reaction In order to enhance the specificity for uronic acids, Blumencrantz and Asboe-Hansen (1973) substituted 3-phenylphenol for the carbazole in the method just described. This increased the sensitivity for uronic acids by 2-3 times and decreased the interference by neutral sugars. This method is widely used as is the modification proposed by Thibault (1979). Several parameters were adjusted by Thibault and he showed that omission of tetraborate decreased the interference by neutral sugars. Bucher (1984) re-examined the parameters of the reaction conditions in order to enhance the specificity for galacturonic acid and pectin, and observed that omission of tetraborate also increased the response from galacturonic acid.
23
Decarboxylation Hot 12% hydrochloric acid (constant boiling) decarboxylates uronic acids to give stoichiometric amounts of carbon dioxide under carefully controlled conditions. An early quantitative procedure was devised by Whistler et al. (see Whistler and Feather, 1962 ) which uses a carrier gas and an absorption train to sweep out impurities and trap the carbon dioxide which is then titrated. This general procedure has been improved and automated (Theander and Westerlund, 1986a). Recent modifications use changes in conductivity for quantitation of the galacturonic acid and hydriotic acid instead of the hydrochloric acid. The decarboxylation method measures the total of all uronic acids, but does not give differing responses for the different uronic acids as found for the colorimetric methods (Theander and Westerlund, 1986a). Using the procedure of Theander and Aman (1979), Theander and Westerlund (1986b) have reported values that range from 20.9% (SD 0.6) for sugar beet fiber to 1.6% (SD 0.1) for wheat bran.
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