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Challenges with nonfiber carbohydrate methods^1,2

Department of Animal Sciences, University of Florida, Gainesville 32611-0910

	Abstract

Top Abstract Introduction Defining Carbohydrate Fractions Extractions Mono- + Oligosaccharide/Low... Starch Analysis Soluble Fiber Analysis Implications Literature Cited

 
Nonfiber carbohydrates (NFC) encompass a compositionally and nutritionally diverse group exclusive of those carbohydrates found in NDF. Their content in feeds has often been described as a single value estimated by difference as 100% of dry matter minus the percentages of CP, NDF (adjusted for CP in NDF), ether extract, and ash. A calculated value was used because of difficulties with assays for individual NFC, but it does not differentiate among nutritionally distinct NFC. Errors in NFC estimation can arise from not accounting for CP in NDF and when multipliers other than 6.25 are appropriate to estimate CP. Analyses that begin to distinguish among NFC are those for starch, soluble fiber (non-NDF, nonstarch polysaccharides), and low molecular weight carbohydrates (mono- and oligosaccharides). Many starch analyses quantify -glucans through specific hydrolysis of -(1 4) and -(1 6) linkages in the glucan, and measurement of released glucose. Incomplete gelatinization and hydrolysis will lead to underestimation of starch content. Starch values are inflated by enzyme preparations that hydrolyze carbohydrates other than -glucan, measurement of all released monosaccharides without specificity for glucose, and failure to exclude free glucose present in the unhydrolyzed sample. Soluble fiber analyses err in a fashion similar to NFC if estimation of CP requires multipliers other than 6.25, or if contaminants such as CP and starch have not been properly accounted. Depolymerization and incomplete precipitation can also decrease soluble fiber estimates. The low molecular weight carbohydrates have been defined as carbohydrates soluble in 78 to 80% ethanol, which separates them from polysaccharides. They can be measured in extracts using broad-spectrum colorimetric assays (phenol-sulfuric acid assay or reducing sugar analysis of acid hydrolyzed samples) or chromatographic methods. Limitations of the colorimetric assays include lack of differentiation among mono- and oligosaccharides and differences in efficacy of measuring total carbohydrate. More sensitive and precise chromatographic methods require expensive equipment and specialized expertise. Current methods for NFC can separate nutritionally relevant fractions, but questions remain as to which fractions merit analysis and what analyses to use. These issues must be resolved in order to soundly evaluate and explore the roles of carbohydrates in diets.

Key Words: Analytical Methods • Carbohydrates • Fiber • Starch • Sugars

	Introduction

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Since the 1800s, chemists have attempted to characterize nutritionally relevant fractions in feeds. Perceived as the most digestible of the carbohydrates, nitrogen-free extract (NFE) and its successor, the NFC, have been calculated by difference owing to their compositional diversity, and focus on other feed characteristics such as protein and energy contents. It was recognized that dividing carbohydrates into crude fiber and NFE was inadequate for nutritionally functional description, nor was it a satisfactory basis for the analysis of feeds (Gaillard, 1958). The same inadequacy applies to partitioning carbohydrates into NDF and NFC.

Separation of the NFC into more nutritionally relevant fractions through analyses for its components, such as organic acids, mono- and oligosaccharides (low molecular weight carbohydrates), starch, and soluble fiber, advances the analyses’ applicability for diet formulation, and alters the types of errors that affect the measures. It must be borne in mind that the covalent, ionic, and physical relationships of molecules in native plant material that are relied upon to influence solubility of carbohydrates for separation may not hold uniformly across plant materials or in processed foods to which specific carbohydrates are added individually.

To attain a viable, relevant analytical system for food and feed carbohydrates, there are questions that must be decided: What carbohydrate fractions have the nutritional relevance to merit analysis? Which analyses are appropriate to define and measure a carbohydrate fraction in a given matrix? What magnitude of error from interfering factors and errors inherent within the assays are acceptable? The answers may vary with the intended use of the values: food/feed labeling, research, or ration formulation. The following discussion on NFC methods is not intended to be encyclopedic in scope, but will address assays that may have application for field and research uses.

	Defining Carbohydrate Fractions

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In practice, carbohydrate fractions are defined by the chemical or enzymatic methods used for their analysis. Their partitioning relies upon differences in solubility and enzymatic specificity. Therefore, it is essential that the procedures be sufficiently specific to accomplish the desired separations to describe nutritionally relevant fractions. In common usage, NFC encompass organic acids, monosaccharides, oligosaccharides, fructans, starch, pectic substances, (1 3)(1 4)-ß-glucans, and other carbohydrates exclusive of the hemicelluloses and cellulose found in NDF (Figure 1). A subset of this group, the nonstructural carbohydrates (NSC), are comprised of carbohydrates from the cell contents including monosaccharides, oligosaccharides, fructans and starch. Among the NFC, monosaccharides, maltose, and starch are digestible by mammalian enzymes, and sucrose and lactose may be digested, but with some variation in this capacity among species and individuals. In contrast, mammals and other simple-stomached animals do not themselves possess the enzymes to hydrolyze fructans, mixed linkage ß-glucans, pectic substances and oligosaccharides, thereby allocating these carbohydrates and other nonstarch polysaccharides to the category of dietary fiber. Microbes in the gut may ferment dietary fiber carbohydrates to yield microbial products that may have nutritional value to the animal.

View larger version (27K): [in this window] [in a new window]   Figure 1. Plant carbohydrate fractions. ADF = acid detergent fiber, ß-glucans = (1 3) (1 4)-ß-D-glucans, NDF = neutral detergent fiber, NDSF = neutral detergent-soluble fiber (includes all nonstarch polysaccharides not present in NDF), NFC = non-NDF carbohydrates.

	Extractions

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Grinding increases the uniformity of samples for subsampling and improves the ease of extraction. Recommendations are that samples for extraction be ground through a 40-mesh or 0.5-mm screen, or through a 1-mm screen before extraction. The finer grind has the potential to improve extraction, but may result in filtration difficulties due to plugging of filters with fine material. Selection of grinding specifications should balance the needs of efficient extraction with other factors that can generate artifacts or error within the assay.

Distilled water, buffers, and aqueous ethanol solutions have been used to extract NFC, but they differ in the carbohydrates they extract and this differs by sample type. Water has the potential to extract low molecular weight carbohydrates (organic acids, monosaccharides, oligosaccharides), as well as polysaccharides (fructans, pectic substances, dextrins, etc.). Cold water is used for complete extraction of fructans (Wylam, 1954). However, cold water can solubilize a portion of the polyuronide (presumably from pectic substances) in vegetables or fruits (0.23 to 0.92% of sample dry matter), and an increased amount is soluble in boiling water (0.30 to 5.60% of sample dry matter) (Bailey et al., 1978). Solubilization of polyuronide was used by the analysts as indication that extraction methods were removing an identifiable polysaccharide that was not a part of the NSC. The measurement of extracted pectins as polyuronide underestimates the amount of polysaccharide extracted because it does not account for the neutral sugar side chains associated with the polyuronide backbone, nor for other nonpectic polysaccharides. The solubility of dextrins in water is problematic when analyzing cooked products (Southgate, 1976), if the analyst intends to include these branched alpha-glucans with the starch fraction. Water does not offer the specificity to separate low molecular weight carbohydrates from polysaccharides. The magnitude of the error will vary by substrate. Southgate (1976) recommended that water be used as an extractant if no interfering substances co-extract with the low molecular weight carbohydrates of interest.

Acetate and phosphate buffers are capable of extracting polysaccharide (Monro, 1991) and so their use for separation of polysaccharides from low molecular weight carbohydrates is not recommended. Their respective pH also modifies what materials they extract. Pectins may be hydrolyzed to lower molecular weight products in neutral and weakly acidic solutions at elevated temperatures (Albersheim, 1959). That acetate buffer (0.1 M, starting pH 4.0) extracted less polyuronide than did phosphate buffer (0.1 M, starting pH 6.0) when incubated at 100°C for 1 h was attributed to depolymerization of pectin through pH dependent ß-elimination at the elevated pH (Monro, 1991). In the same study, it was noted that increasing the molarity of the acetate buffer from 0.005 to 0.20 increased the amount of polyuronide extracted, whereas the phosphate buffer achieved its maximal extraction at about 0.1 M. Due to reduced extraction, or depolymerization, the use of these buffers would seem to have the potential for inaccurate estimation of NFC or soluble fiber.

Although alcohol extractions have been used since the 13th century, their deliberate use for plant analysis extends back to the late 1700’s when Hilaire-Marie Roulle recommended the separation of plant constituents using different solvents including alcohol (McCollum, 1957). Currently, aqueous ethanol solutions are used to separate low molecular weight carbohydrates from polysaccharides and proteins, with 78 to 80% ethanol the concentrations most commonly invoked for this purpose (Asp, 1993). The separation is not precise by degree of polymerization. Differing ethanol concentrations extract different amounts of carbohydrates, with lower concentrations extracting more material (Smith, 1973). Some arabinans and the glucofructans are soluble in approximately 70% ethanol (Medcalf and Cheung, 1971), and fructans are variably soluble in 80 to 90% ethanol, presumably on the basis of their degree of polymerization (Wylam, 1954; Smith and Grotelueschen, 1966). Small quantities (0.3% of dry matter) of uronic acid have been reported to be extracted by hot 80% ethanol (Bailey et al., 1978). Like water and buffer solutions, aqueous ethanol does not readily separate the mono- and disaccharides from larger oligosaccharides that are less likely to be digestible by mammalian enzymes. However, oligosaccharides do not tend to predominate in the majority of feeds.

The ability of microbes to grow in the extracting solution may reduce the carbohydrate content of the solution to be analyzed. Intuitively, the amount of carbohydrate consumed by microbes is likely related to the type of carbohydrate, the nature of the extracting solution, the length and temperature of incubation, and the potential for inoculation. Thomas (1977) noted that to minimize losses to microbes in extracts from a 1 h cold-water extraction of herbage, samples were processed immediately after extraction or a preservative was added. Incubation of purified glucose in distilled water at 40°C for 1 h did not result in a decrease in the quantity of glucose, however, likelihood of inoculation was low (Hall, unpublished). Completing carbohydrate analyses from start to finish in a timely fashion decreases risk of microbial degradation of carbohydrate. Caution is advised to avoid degradation of aqueous standard solutions of carbohydrates over time, even if they are stored at cool temperatures. Distilled water saturated with benzoic acid (2.7 g of benzoic acid/L, Handbook of Phys. and Chem., 1944; add in excess, stir overnight at ambient temperature) has commonly been used to prolong the usable life of carbohydrate standard solutions. However, the lifespan is not infinite, and the compatibility of benzoic acid with the analyses applied to the solution should be evaluated. The preservative action of ethanol solutions eliminates concerns about microbial depredations.

	Mono- + Oligosaccharide/Low Molecular Weight Carbohydrate Analysis

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In mixed feeds, the most commonly used methods for low molecular weight carbohydrates are reducing sugar, condensation, enzymatic, and chromatographic analyses. For standard feed analysis, the first three show the most promise for throughput and expense, whereas the automated chromatographic techniques may be most suitable for research and analysis of sample types sufficiently uniform and of a volume to make it feasible, or of an importance to make it desirable. The sensitivity and specificity for mono- and oligosaccharides of relatively newer methods such as high pH anion exchange chromatography with pulsed amperometric detection holds promise for further fractionation of oligosaccharides (Kennedy and Pagliuca, 1994). However, this and other chromatographic methods lack appropriate standards for the larger oligosaccharides and are typically considerably more expensive than the colorimetric assays.

Generally speaking, for all assays requiring a standard curve, a linear, five point curve including a zero concentration point consisting of distilled water plus reagents is recommended. Use of reagent blanks is suggested if they are feasible to provide a subtraction for background absorbance (e.g., in the glucose oxidase-peroxidase assay, but not in the phenol-sulfuric acid assay). Strict standardization of procedures to provide reliable results is recommended.

Reducing Sugar Analysis
The ability of free monosaccharides to reduce alkaline solutions of metallic salts to yield the free metal or its oxide has been used as a method of carbohydrate analysis since 1841 (McCollum, 1957). To effectually measure total low molecular weight carbohydrates, extracted carbohydrates must be hydrolyzed to yield monosaccharides, or only the monosaccharides initially present and the reducing end of carbohydrate chains will be detected. Accordingly, unhydrolyzed sucrose is not measurable with this assay. Fructose is more sensitive to destruction during hydrolysis than glucose, and different hydrolysis procedures are recommended to prevent extensive destruction of the carbohydrates of interest (Southgate, 1976). The specificity of this assay for low molecular weight carbohydrates depends upon the carbohydrate complement in the extract, as polysaccharides extracted and hydrolyzed with the low molecular weight carbohydrates will also be measured.

Protein and naturally occurring reducing substances are among the materials that interfere with this assay. Southgate (1976) recommended that the bulk of the protein be removed before attempting analysis, but did not provide indication of what concentrations of protein caused what degree of problem. Molasses is an example where noncarbohydrate-reducing substances accounting for approximately 10% of dry matter could inflate reducing sugar values (Binkley and Wolfram, 1953). Alcohol cannot be present in extracts used for reducing sugar analysis (Southgate, 1976).

Condensation Reactions
The condensation reactions of carbohydrate, phenolic compound, and strong acid are commonly used to measure carbohydrates. The reactions are made more specific for particular carbohydrates through heating conditions, the strength of acid (Southgate, 1976), and selection of the phenolic compound used. For example, resorcinol is used for ketoses (Kulka, 1956) and carbazole for uronic acids (Bitter and Muir, 1962). Phenol reacts with a broad spectrum of carbohydrates, though each provides a different standard curve, and hexoses have a different wavelength of maximal absorbance than do pentoses or uronic acids (Dubois et al., 1956). Certain wavelengths of light can degrade the chromogens formed in these assays, and samples should be shielded from sunlight.

The phenol-sulfuric acid assay can detect a wide variety of carbohydrates in aqueous or ethanolic solutions. Standardization of methods of acid addition and mixing are essential for the success of the assay. Prior hydrolysis of samples is not necessary. The use of preservatives such as sodium azide "disturbs" the assay, and should not be used to preserve carbohydrate standard solutions (Buysse and Merckx, 1993). The ability of the phenol-sulfuric acid assay to detect a variety of carbohydrates also makes it prone to contamination from ubiquitous carbohydrates such as cellulosic lint. Rinsing test tubes with distilled water and allowing them to dry inverted on a rack before use, as well as running triplicate test tubes of a sample to allow for selective omission of tubes with absorbance values that are obviously much higher than the other replicates reduces the contamination issue. The carbohydrate used for the standard should represent the predominant carbohydrate in the extract, typically lactose for milk products, or sucrose for plant materials. The work of Buysse and Merckx, (1993) on the phenol-sulfuric acid assay indicates that sample absorbance changes linearly with the concentration of ethanol (0 to 80% v/v water) for glucose, fructose and sucrose. Their work also indicates that a phenol concentration can be selected to yield the same absorbance from these low molecular weight carbohydrates on an equal monomer weight basis. Their recommended phenol concentrations (w/w) were 28% for 80% ethanol and 18% for water.

Enzymatic: Glucose Oxidase-Peroxidase
Enzymatic analyses can be used as selective tools for the measure of specific carbohydrates. The most common example of this is the glucose oxidase-peroxidase assay for glucose. Whether combined with a colorimetric analysis (Karkalas, 1985) or with a peroxide-detecting probe (YSI Incorporated, Yellow Springs, OH), the assay is very specific for glucose. Its usefulness for measurement of low molecular weight carbohydrates is quite limited, as no carbohydrates other than free glucose are detected.

	Starch Analysis

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Excepting polarimetry used on cereal grain samples, most starch analyses are enzymatic, relying on the specificity of enzymes to separate starch from other glucose-containing carbohydrates. The steps followed in a starch assay are generally gelatinization, hydrolysis, and measurement of end products. Critical elements for accurate starch analysis are:

1. Complete gelatinization of starch,
   
2. Specificity of enzymes,
   
3. Complete hydrolysis of starch to glucose,
   
4. Specific measurement of glucose yield from hydrolyzed starch, and
   
5. Minimization of interference.
   

Gelatinization involves the dissolution of hydrogen bonds among and within starch molecules to open the molecules up to hydration and enzymatic hydrolysis. Before gelatinization, starch in unprocessed granules is crystalline. The linear portions of starch molecules are aligned and hydrogen bonded to each other, excluding water and resisting enzymatic activity. That crystalline structure must be disrupted for complete enzymatic hydrolysis of the starch to take place in a reasonable amount of time. Gelatinization is typically accomplished with heating (90 to 100°C) with water, or, alternatively, with use of a base (e.g., potassium hydroxide) followed by neutralization (Englyst et al., 1982). Incomplete gelatinization can lead to incomplete hydrolysis of starch to glucose.

Enzymatic hydrolysis of only the -(1 4) (linear chain) and -(1 6) (branches) linkages present in starch is the element that makes a method specific for starch and -glucans, and excludes other glucose-containing carbohydrates from the analysis. Heat-stable -amylase (an endoamylase), which can be added during the gelatinization step, and amyloglucosidase (an exoamylase), which hydrolyzes -glucan to glucose, are commonly used. Care must be taken to assure that a given enzyme is incubated at the correct pH and temperature to optimize its ability to hydrolyze the substrate. The optimal pH are close to neutrality for the heat stable -amylase, and 4.5 to 5.0 for amyloglucosidase, but these values should be verified with the specifications provided by the manufacturer of the enzyme. An acetic acid buffer solution (0.10 M) adjusted to pH 4.5 is commonly used for amyloglucosidase incubations. Since starch is estimated as the glucose released by enzymatic hydrolysis, the hydrolysis must be complete, or starch content will be underestimated. Presence of other enzymes such as invertase (hydrolyzes sucrose), or cellulase (hydrolyzes cellulose) that release glucose through hydrolysis of nonstarch substrates inflate the analyzed starch value.

Measurement of the products of starch hydrolysis should be accomplished with an analysis specific for glucose, such as a glucose oxidase-peroxidase assay (Karkalas, 1985). Starch content is calculated as glucose content times 0.9, which allows for the molecular weight of one molecule of water (18 g/mole) to be subtracted for the weight of each molecule of glucose (180 g/mole). This accounts for the removal of water during the covalent bonding of glucose molecules to form starch. Glucose should be used as the standard for the end product assay. Use of starch as the standard is not recommended because it relies upon complete recovery (starch measured/actual starch), and presumes similar recoveries for starch from all sources. Use of glucose as a standard removes the question of percent recovery. Carrying purified starch as a control sample in analyses allows assessment of enzyme, assay conditions, and technical efficacy.

An alternative method for end product determination measures reducing sugars. This approach carries a greater risk of accounting monosaccharides not derived from starch as starch. Such sugars may be present in the sample as monosaccharides, or released by hydrolysis of nonstarch carbohydrates. Particularly when the enzyme preparations used contain invertase, fructanase, or other nonamylolytic enzymes, measurement of hydrolysis products from sucrose, fructans, and other carbohydrates can significantly inflate starch estimates (Hall et al., 2000). Accordingly, this method is not recommended for use in starch determination.

Interfering substances include any substance that increases or decreases the measured starch estimate from its correct value. The method of end product measurement used, either for glucose or reducing sugars, determines what substances interfere. Some commercial amyloglucosidase preparations, especially those for dietary fiber analysis, contain glucose and should not be used for starch analysis. Even noncarbohydrate substances that absorb at the appropriate wavelength in colorimetric analyses can unduly alter starch values. Interference from low molecular weight carbohydrates can be reduced or eliminated by preextracting them with 80% ethanol:water (v/v) before starch analysis and analyzing the residue. Alternatively, free glucose can be measured in a sample blank untreated with enzymes and the value subtracted from the amount of glucose released by hydrolysis. This approach requires that the enzymes used on samples be of sufficient purity so that they do not hydrolyze nonstarch carbohydrates to any appreciable extent and thereby add to the free glucose pool. This can be tested by analyzing samples of sucrose, cellobiose or cellulose with the starch method using the selected enzymes; they should not yield appreciable glucose. The extent to which one should be concerned about interfering substances will depend upon the type of sample analyzed. Mature grain samples and silage samples will likely have little sugar remaining to interfere with starch analysis. Interfering carbohydrates may be an issue in byproduct feeds such as bakery waste, almond hulls, and citrus pulp. The entire starch assay should be performed from hydrolysis through detection of end products in one day to minimize microbial degradation of products.

	Soluble Fiber Analysis

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Soluble fiber, the nutritional entity, is a compositionally diverse fraction in foods and feeds. Soluble fiber encompasses the nonstarch, non-NDF polysaccharides including pectic substances, (1 3)(1 4)-ß-glucans, fructans, and gums. The term "soluble fiber" begs the question, "Soluble in what solvent and under what conditions?" The current AOAC soluble dietary fiber method (Prosky et al., 1992) solubilizes the carbohydrates in phosphate buffer, and hydrolyzes -glucans with amyloglucosidase. After filtration to separate it from insoluble dietary fiber, solubilized dietary fiber is precipitated from the phosphate buffer extract with 78% ethanol and is determined gravimetrically with correction for protein and ash. As previously described, phosphate buffer may depolymerize some of the pectic polysaccharides of interest (Monro, 1991). Additionally, reliance on ethanol precipitation for recovery may result in incomplete precipitation of soluble fiber and coprecipitation of other organic compounds (Mañas and Saura-Calixto, 1993). The recovery of soluble dietary fiber after precipitation with 78% ethanol or dialysis (12,000 to 14,000 molecular weight cutoff) were 5.34% and 12.33% of dry matter, respectively for orange peel. The magnitude of difference between the recovery methods differed by foodstuff (Mañas and Saura-Calixto, 1993).

An alternative measure of soluble fiber is that of neutral detergent-soluble fiber, or those nonstarch non-NDF polysaccharides soluble in neutral detergent plus heat-stable, -amylase. The polysaccharides found in starch, soluble fiber, and insoluble fiber comprise the carbohydrate constituents of the residue remaining after extraction with 80% ethanol (Theander and Westerlund, 1986). Rather than attempting precipitation of soluble fiber after its extraction, soluble fiber may be determined gravimetrically as the difference between the weight of the 80% ethanol-insoluble residue and those of starch and insoluble fiber (NDF), with corrections for protein and ash (Hall et al., 1999). This system of analysis allows concurrent determination of starch, soluble fiber, and insoluble fiber. Although the constituent assays have good repeatability and are simpler than the soluble dietary fiber method, estimation of soluble fiber by difference leaves the potential for errors from the component assays to accumulate in the estimate. The errors inherent in correcting for protein as N x 6.25, with the incorrect presumption that 6.25 is the correct multiplier for all samples (Jones, 1931), affects both soluble fiber methods.

	Implications

Top Abstract Introduction Defining Carbohydrate Fractions Extractions Mono- + Oligosaccharide/Low... Starch Analysis Soluble Fiber Analysis Implications Literature Cited

 
Debate continues over which carbohydrate fractions are nutritionally relevant and merit analysis, and which methods to use. For carbohydrates exclusive of NDF, arguments can be made for the nutritional relevance and needed analysis of mono- and oligosaccharides, starch, and soluble fiber. Additional partitioning may be needed for use with animals that do not have a pregastric fermentation compartment. Regarding the methodology, the use of 80% ethanol is preferable to water for more certain partitioning of polysaccharides and low molecular weight carbohydrates. It is crucial that the assays used are chemically and enzymatically appropriate to accomplish the tasks to which we apply them, and that we recognize and account for artifacts. Each method has its limitations, and extensive comparisons of methods are lacking for some carbohydrate fractions. Continued work to refine a coherent system of carbohydrate analysis for defining nutritionally relevant carbohydrate fractions is warranted.

	Footnotes

 
¹ Presented as a symposium paper at the 2002 Animal Science and Dairy Science Joint Annual Meeting, Quebec City, Canada.

² The author wishes to acknowledge D. Ben-Ghedalia, G. Fahey, and G. Varga for their thoughtful comments and input to this paper.

³ Correspondence: P.O. Box 110910 (phone: 352-392-1958; fax: 352-392-5595; E-mail: hall{at}animal.ufl.edu).

Received for publication September 14, 2002. Accepted for publication March 3, 2003.

	Literature Cited

Top Abstract Introduction Defining Carbohydrate Fractions Extractions Mono- + Oligosaccharide/Low... Starch Analysis Soluble Fiber Analysis Implications Literature Cited

 


Albersheim, P. 1959. Instability of pectin in neutral solutions. Biochem. Biophys. Res. Comm. 1:253–256.

Asp, N-G. 1993. Nutritional importance and classification of food carbohydrates. Pages 121–126 in Plant Polymeric Carbohydrates. F. Meuser, D. J. Manners, and W. Seibel, ed. Royal Soc. Chem., Cambridge, U.K.

Bailey, R. W., A. Chesson, and J. Monro. 1978. Plant cell wall fractionation and structural analysis. Am. J. Clin. Nutr. 31:S77–S81.[Abstract]

Binkley, W. W., and M. L. Wolfram. 1953. Composition of cane juice and cane final molasses. Scientific Research Report Series No. 15 in Advances in Carbohydrate Chemistry Vol. VIII. Sugar Research Foundation, Inc. Academic Press, Inc., New York.

Bitter, T., and H. M. Muir. 1962. A modified uronic acid carbazole reaction. Anal. Biochem. 4:330–334.[Medline]

Buysse, J., and R. Merckx. 1993. An improved colorimetric method to quantify sugar content of plant tissue. J. Experimental Botany 44:1627–1629.[Abstract/Free Full Text]

Dubois, M., K. A. Gilles, J. K. Hamilton, P. A. Rebers, and F. Smith. 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28:350–356.

Englyst, H., H. S. Wiggins, and J. H. Cummings. 1982. Determination of the nonstarch polysaccharides in plant foods by gas-liquid chromatography of constituent sugars as alditol acetates. Analyst 107:307–318.[Medline]

Gaillard, B. D. E. 1958. A detailed summative analysis of the crude fibre and nitrogen-free extractives fractions of roughages. I. proposed scheme of analysis. J. Sci. Food Agric. 3:170–177.

Hall, M. B., W. H. Hoover, J. P. Jennings, and T. K. Miller Webster. 1999. A method for partitioning neutral detergent soluble carbohydrates. J. Sci. Food Agric. 79:2079–2086.

Hall, M. B., J. P. Jennings, B. A. Lewis, and J. B. Robertson. 2000. Evaluation of starch analysis methods for feed samples. J. Sci. Food Agric. 81:17–21.

Handbook of Chemistry and Physics. 1944. 28th ed. C. D. Hodgman, Ed. Chemical Rubber Publishing, Co., Cleveland, OH.

Jones, D. B. 1931. Factors for converting percentages of nitrogen in foods and feeds into percentages of proteins. Circular No. 183. USDA, Washington, DC.

Karkalas, J. J. 1985. An improved enzymatic method for the determination of native and modified starch. J. Sci. Food Agric. 36:1019–1027.

Kennedy, J. F., and G. Pagliuca. 1994. Chapter 2. Oligosaccharides. Pages 43–72 in carbohydrate analysis—a practical approach. 2nd ed. M. F. Chapling and J. F. Kennedy, Ed. Oxford University Press, Inc., New York.

Kulka, R. G. 1956. Colorimetric estimation of ketopentoses and ketohexoses. Biochem. J. 63:542–548.[Medline]

Mañas, E., and F. Saura-Calixto. 1993. Ethanolic precipitation: a source of error in dietary fibre determination. Food Chem. 47:351–355.

McCollum, E. V. 1957. A history of nutrition. The Riverside Press, Cambridge, MA, Houghton Mifflin Company, Boston.

Medcalf, D. G., and P. W. Cheung. 1971. Composition and structure of glucofructans from durum wheat flour. Cereal Chem. 48:1–8.

Monro, J. A. 1991. Dietary fiber pectic substances: source of discrepancy between methods of fiber analysis. J. Food Comp. Anal. 4:88–99.

Prosky, L., N-G. Asp, T. F. Schweizer, J. W. Devries, and I. Furda. 1992. Determination of insoluble and soluble dietary fiber in foods and food products: collaborative study. J. AOAC 75:360–367.

Smith, D. 1973. Chapter 3. The nonstructural carbohydrates. Chemistry and Biochemistry of herbage, Vol. 1. G. W. Butler and R. W. Bailey, ed. Academic Press, London.

Smith, D., and R. D. Grotelueschen. 1966. Carbohydrates in grasses. I. sugar and fructosan composition of the stem bases of several northern-adapted grasses at seed maturity. Crop Sci. 6:263–266.

Southgate, D. A. T. 1976. Determination of food carbohydrates. Applied Science Publishers, Ltd, London.

Theander, O., and E. A. Westerlund. 1986. Studies on dietary fiber. 3. Improved procedures for analysis of dietary fiber. J. Agric. Food Chem. 34:330–336.

Thomas, T. A. 1977. An automated procedure for the determination of soluble carbohydrates in herbage. J. Sci. Food Agric. 28:639–642.

Van Soest, P. J., J. B. Robertson, and B. A. Lewis. 1991. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74:3583–3597.[Abstract]

Wylam, C. B. 1954. Analytical studies on the carbohydrates of grasses and clovers. IV.—Further developments in the methods of estimation of mono- and di- and oligo-saccharides and fructosan. J. Sci. Food Agric. 5:167–172.

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