Hans N. Englyst

Simplified method for the measurement of total non-starch polysaccharides by gas-liquid chromatography of constituent sugars as alditol acetates

A procedure is described for the measurement and characterisation of total non-starch polysaccharides in plant foods by gas-liquid chromatography of individual sugars as alditol acetates. Starch, including that resistant to gelatinisation in boiling water, is dispersed with dimethyl sulphoxide and then hydrolysed with α-amylase and pullulanase. Starch-free material is hydrolysed with sulphuric acid and the released neutral sugars are measured as alditol acetates by using N-methylimidazole in order to catalyse the acetylation. Uronic acids are measured by a spectrophotometric method. The procedure is a modification of a more comprehensive method reported previously. It is relatively rapid and simple compared with gravimetric procedures, is applicable to a wide range of foodstuffs, can be carried out without special expertise in carbohydrate chemistry and is suitable for routine laboratory use.

Determination of the non-starch polysaccharides in plant foods by gas-liquid chromatography of constituent sugars as alditol acetates

A method is reported for the measurement of non-starch polysaccharides (NSP) from plant foods. NSP are the major components of “dietary fibre.” The polysaccharides are divided into cellulose and non-cellulosic material and the constituent sugars are determined by gas-liquid chromatography. Starch is removed, after gelatinisation, by incubation with hog pancreatic α-amylase together with pullulanase. The enzyme preparations are shown to be specific for the hydrolysis of α-1,4- and α-1,6-glucosidic bonds, and not to affect NSP. The starch-free material is then analysed by three separate but complementary procedures: (A) hydrolysis with 1 M sulphuric acid after solubilisation of cellulose with 12 M sulphuric acid; (B) hydrolysis with 1 M sulphuric acid; and (C) extraction with phosphate buffer at pH 7 and 100 °C, solubilisation of cellulose with 12 M sulphuric acid and then hydrolysis with 1 M sulphuric acid. Neutral sugars are measured by gas-liquid chromatography as alditol acetates and uronic acids by a colorimetric method. Starch made resistant to α-amylase digestion by food processing is identified by additional steps in procedure B, and measured as “resistant starch.” Procedure A gives total NSP and procedure B neutral non-cellulosic polysaccharides. A value for cellulose is obtained as the difference between glucose measured in procedures A and B. Procedure C gives NSP insoluble in phosphate buffer at pH 7. Soluble NSP is the difference between total NSP and insoluble NSP. Results for the NSP analysis of selected foods are given.

Prebiotic digestion and fermentation.

Prebiotics, as currently conceived of, are all carbohydrates of relatively short chain length. To be effective they must reach the cecum. Present evidence concerning the 2 most studied prebiotics, fructooligosaccharides and inulin, is consistent with their resisting digestion by gastric acid and pancreatic enzymes in vivo. However, the wide variety of new candidate prebiotics becoming available for human use requires that a manageable set of in vitro tests be agreed on so that their nondigestibility and fermentability can be established without recourse to human studies in every case. In the large intestine, prebiotics, in addition to their selective effects on bifidobacteria and lactobacilli, influence many aspects of bowel function through fermentation. Short-chain fatty acids are a major product of prebiotic breakdown, but as yet, no characteristic pattern of fermentation acids has been identified. Through stimulation of bacterial growth and fermentation, prebiotics affect bowel habit and are mildly laxative. Perhaps more importantly, some are a potent source of hydrogen in the gut. Mild flatulence is frequently observed by subjects being fed prebiotics; in a significant number of subjects it is severe enough to be unacceptable and to discourage consumption. Prebiotics are like other carbohydrates that reach the cecum, such as nonstarch polysaccharides, sugar alcohols, and resistant starch, in being substrates for fermentation. They are, however, distinctive in their selective effect on the microflora and their propensity to produce flatulence.

Determination of dietary fibre as non-starch polysaccharides with gas–liquid chromatographic, high-performance liquid chromatographic or spectrophotometric measurement of constituent sugars

Methods for the measurement of dietary fibre as non-starch polysaccharides (NSP) are described. A common enzymic removal of starch and acid hydrolysis of the NSP to their constituent sugars are followed by one of three alternative techniques, gas-liquid chromatography, high-performance liquid chromatography or spectrophotometry, for measurement of the released sugars. The results obtained by the three methods are in good agreement for a wide range of raw and processed foods. NSP compose approximately 90% of the plant cell-wall material and are therefore a good index of this material. Values for NSP therefore provide a good marker for a diet rich in fruit, vegetables and high-extraction cereal products associated with health and recommended in dietary guidelines. Values for total, soluble and insoluble NSP may be obtained with any of the end-point techniques, and the detailed information obtained from the chromatographic methods is useful in studies of the relationship between the intakes of various types of NSP and health. The causes of some potential interferences in the spectrophotometric assay, especially from processed foods, have been identified and eliminated. The rapid spectrophotometric version is suitable for food labelling purposes and for quality control, and the changes described have made it more robust.

Glycaemic index of cereal products explained by their content of rapidly and slowly available glucose

Elucidating the role of carbohydrate quality in human nutrition requires a greater understanding of how the physico-chemical characteristics of foods relate to their physiological properties. It was hypothesised that rapidly available glucose (RAG) and slowly available glucose (SAG), in vitro measures describing the rate of glucose release from foods, are the main determinants of glycaemic index (GI) and insulinaemic index (II) for cereal products. Twenty-three products (five breakfast cereals, six bakery products and crackers, and twelve biscuits) had their GI and II values determined, and were characterised by their fat, protein, starch and sugar contents, with the carbohydrate fraction further divided into total fructose, RAG, SAG and resistant starch. Relationships between these characteristics and GI and II values were investigated by regression analysis. The cereal products had a range of GI (28-93) and II (61-115) values, which were positively correlated (r(2)) 0.22, P<0.001). The biscuit group, which had the highest SAG content (8.6 (SD 3.7) g per portion) due to the presence of ungelatinised starch, was found to have the lowest GI value (51 (SD 14)). There was no significant association between GI and either starch or sugar, while RAG was positively (r(2)) 0.54, P<0.001) and SAG was negatively (r(2)) 0.63, P<0.001) correlated with GI. Fat was correlated with GI (r(2)) 0.52, P<0.001), and combined SAG and fat accounted for 73.1% of the variance in GI, with SAG as the dominant variable. RAG and protein together contributed equally in accounting for 45.0 % of the variance in II. In conclusion, the GI and II values of the cereal products investigated can be explained by the RAG and SAG contents. A high SAG content identifies low-GI foods that are rich in slowly released carbohydrates for which health benefits have been proposed.

Measurement of rapidly available glucose (RAG) in plant foods: a potential in vitro predictor of the glycaemic response.

The glycaemic index (GI) is an in vivo measurement based on the glycaemic response to carbohydrate-containing foods, and allows foods to be ranked on the basis of the rate of digestion and absorption of the carbohydrates that they contain. GI values are normalized to a reference amount of available carbohydrate and do no reflect the amounts of carbohydrate normally present in foods; for example, a food with a low content of carbohydrates will have a high GI value if that carbohydrate is digested and absorbed rapidly in the human small intestine. This is potentially confusing for a person wishing to control his or her blood glucose levels by the choice of foods. The rate and extent of starch digestion in vitro has been measured using a technique that classifies starch into three major fractions: rapidly digestible starch (RDS), slowly digestible starch (SDS) and resistant starch (RS). In addition, this technique gives a value for rapidly available glucose (RAG), which includes RDS, free glucose and the glucose moiety of sucrose. When the values for thirty-nine foods were expressed on the basis of the available carbohydrate content of these foods, highly significant (P < 0.001) positive correlations were observed between GI and both RDS and RAG. The measurement of RAG in vitro provides values for direct calculation of the amount of glucose likely to be rapidly absorbed in the human small intestine and, thus, to influence blood glucose and insulin levels. These values can be used to compare foods, as eaten, on an equal-weight basis. Food-table RAG values would allow simple calculation of the total amount of RAG provided by single foods, by whole meals and by whole diets. Studies are planned in which RAG and the glycaemic response in man will be measured for identical food products.

The classification and measurement of dietary carbohydrates

Abstract Major advances in our understanding of the digestive physiology of dietary carbohydrates and their potential benefit to health require new and more informative techniques to replace the traditional ‘by difference’ measurement. The human diet contains a range of chemically distinct carbohydrates and research, as well as labelling for dietary carbohydrates including dietary fibre, should be based on the classification and measurement of chemically identified components. Such values do not become obsolete and can be used in different combinations for different purposes. We present a new scheme for carbohydrate classification, including a new class of short-chain carbohydrates (SC). The classification and measurement of nutritionally important types of starch includes its division into rapidly digestible starch (RDS), slowly digestible starch (SDS) and resistant starch (RS). In addition, a new category of rapidly available glucose (RAG) is described, which is the amount of glucose from free sugar and starch that is rapidly available for absorption. Values for dietary fibre based on the measurement of plant cell-wall NSP aid the consumer in choosing the type of high-fibre diet recommended in the dietary guidelines and are, therefore, appropriate for food labelling. The AOAC Prosky procedure, in contrast, is not specific for plant cell-wall material but includes substances that are formed by food processing and by treatment of analytical samples. Such values are not suitable for food labelling because they do not aid the consumer in choosing the recommended diet.

Dietary Fiber and Resistant Starch

Plant polysaccharides may be separated into two broad categories. Starch, a ubiquitous storage polysaccharide, is an α-linked glucan and is the major carbohydrate of dietary staples such as cereal grains and potatoes. The nonstarch polysaccharides (NSP) of plants, such as cellulose, pectin, and hemicellulose, are non-α-glucan polysaccharides. The NSP tend to have a structural function and are the principal components of the plant cell wall. Recently NSP have become the objective in the measurement of dietary fiber.

Determination of dietary fibre as non-starch polysaccharides by gas–liquid chromatography

An improved method is described for the measurement of total, soluble and insoluble dietary fibre as non-starch polysaccharides (NSP). An established procedure is modified to allow more rapid removal of starch and hydrolysis of NSP. In its present form the procedure is simpler and more robust than those previously published. In the modified method starch is removed enzymically within 50 min and NSP is precipitated with ethanol and then hydrolysed by treatment with sulfuric acid for 2 h. The constituent sugars can in turn be measured by gas-liquid chromatography, high-performance liquid chromatography or more rapidly by colorimetry. The improved procedure described here for the removal of starch and hydrolysis of NSP applies to all three techniques, but only the method for measurement of sugars by gas-liquid chromatography is described here in full.

Comparative effects of three resistant starch preparations on transit time and short-chain fatty acid production in rats.

Abstract: A high-fiber diet may protect against colon cancer because of the butyrate generated in the colon by bacterial fermentation of nonstarch polysaccharides. Butryrate can reverse neoplastic changes, at least in vitro, and resistant starch (RS) represents a source of butyrate in vivo. We examined the effects of replacing normal maize starch in the diet of rats with three preparations of RS on the amounts of starch, butyrate, and other short-chain fatty acids in the cecum. We examined the effects on fecal bulking and transit time, which have been suggested to protect against colon cancer. The RS preparations that we tested were potato starch, high-amylose maize starch, and an a-amylase- treated high-amylose maize starch. All had major effects on fecal weight and on the weight of the cecum but only slightly shortened transit times. All increased the amount of starch reaching the cecum and increased short-chain fatty acid production in the cecum; potato starch had the greatest effect and high-amylose maize starch the least. Potato starch, unlike high-amylose maize starch, enhanced the proportion of butyrate. Thus there were marked differences among sources of RS, even though these were all classified as RS2. The significance for colon cancer is discussed.