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X.T. Tolipov
DETERMINATION OF INULIN IN PLANTS
The exact and reliable detection of sugar monomers and fructans provides important information for the evaluation of carbohydrate metabolism in plants and animals. Using the HPLC method; a large number of samples and single sugars; with both high sensitivity and selectivity; may be analysed. It was shown that the described method—using a Nucleosil column loaded with Pb ions; a refractive index detector (RID); and HPLC-grade water as the elu-ent—gives precise and reproducible results regarding the detection of individual sugars in extracts ofplants and feed materials. The method can be applied for the detection of sucrose; maltose; lactose; xylose; glucose; galactose; arab-inose; fructose; ribose; and mannitol. Furthermore; depending on the plant material; the sugars verbascose; stachyose; and raffinose can be separated. The peaks were well resolved and the reproducibility of the analysis; with 94-108% of recovery (RC) and relative standard deviation (RSD) of up to 5%; was very good. The method was successfully applied to a variety of green forages and samples of sugar beet pulp silages. It is also possible to determine fructan with inulin as a standard; together with the other sugars; or alone by a different protocol and column.
Keywords: single sugar; monosaccharides; glucose; fructose; sucrose; fructan; high performance liquid chromatography.
Carbohydrates comprise a variety of sugars, mainly lower-molecular carbohydrates which share certain traits regarding chemical structure and reactivity. Carbohydrates form the largest portion of the organic matter in plants and feed materials, so are of great importance as storage compounds, structure elements, and energy sources. They can be assigned to different groups based on certain traits, such as chemical structure (e.g., mono-, di-, oligo-, and polysaccharides, sugar alcohols, sugar phosphates), physico-chemical properties (e.g., redox potential, hydrolytic stability, solubility in different solvents), and function (e.g., storage or matrix/cell wall carbohydrates). Regarding the detection of the carbohydrate fractions in plant and feed materials, it is advisable to divide
© Tolipov X.T., 2021.
them into non-structural (NSC) and structural (SC) carbohydrates. The SC comprise hemicelluloses, celluloses, and pectins, as well as plant gums. In contrast to cellulose, hemicelluloses are the polysaccharide part of the plant which can be more easily accessed by chemicals, e.g., by extraction with bases. Mono-, di-, and oligosaccharides, starch, and fructans represent the major compounds of the NSC fraction.
The detection of sugar monomers and fructans provides important information for the evaluation of carbohydrate metabolism in plants and animals. Single sugars can be analysed by enzymatic or chromatographic methods. Although enzymatic assays are characterised by high specificity and sensitivity, only one sugar species can be detected. Hall [1] reported that reducing sugars and condensation assays are widely used for the analysis of water soluble carbohydrates in feeds. They require the hydrolysis of oligo- and polysaccharides to monomers which will be measured, which means that specific carbohydrates are not detected. In contrast, chromatographic methods (gas-chromatography, GC, and high-performance liquid chromatography, HPLC), offer the advantage of the simultaneous detection of a variety of sugars with rapid, specific, and sensitive measurement. HPLC is mainly used for sugar analysis in foods [2,3]. Furthermore, HPLC is the most common chromatographic method for analysing these compounds in plants and feed materials. An overview, with references, of HPLC methods which are used for sugar analysis in forages is given in Table 1
For the detection of sugar monomers by HPLC, they must be present in a soluble state. In most cases water or ethanol is used as the extraction solvent.
However, which sugar fractions can be separated by chromatography is affected by the solvent used, and its concentration. While aqueous extracts may contain mono-, di-, and oligosaccharides, and fructans [10,11,12], only a small proportion of lower-molecular fructans dissolve in 80% ethanol [5,13]. The lower the concentration of ethanol in the extracting agent, the more higher-polymeric fructans will be extracted [14].
For the separation of single sugars by HPLC, various columns and detectors can be used. According to Scott [2], columns with polymer-based cations have low detection limits and can be employed in combination with a refraction index (RI) detector. The individual sugars form complexes with cations, e.g., Ca2+ and Pb2+, whose binding strength is affected by the chemical structure of the sugar and the cation species. Deionised water is used as an eluent, and a flow gradient is not needed. In most cases, the column is heated to a temperature of 85 °C. However, Scott [2] reported problems with using this method to separate sucrose from maltose and mentioned, despite reasonable column stability, damage to the column by organic acids, methanol, and salts.
Table 1
Methods to determine individual sugars in plants
ISSN 2223-4047
Вестник магистратуры. 2021. № 4-1 (115)
Glucose, fructose, and sucrose belong to the group of the most frequently-occurring lower-molecular carbohydrates in plants. Other monosaccharides, which can be detected after hydrolysis of cell-wall components of plants for food and feed use, are summarised in Table 2.
Tab le 2. Cell wa I monosaccharides in plants for feed.
Plant Materiii Glu' Fiuc' Kyi 3 Rib-* Ага ^ Ga|6 Man' Rham® Reference
Grasses Legumes X X X X X X [15]
Tra:cal legumes X X X X X X [16]
Glasses XXX X X X X [1F]I1
Diwisel» X X X X X X [1ЩО
silage, cereal grains X X X X X X [20.21]
Sorghurrfsudan grass XXX [22;
1 Slucnse;1 fructose;3 xylose;; '¡bose; - arab nose;6 galactose;; mannose;s rhamnose:: maize, cereals. rice. different vegetables, f si/. bread. ard others: ''■ wheal straw, wheat bran, surf ower leavES. sugarcane ;=;=sse. soy bean hulls, sugar beet, peanut rrea, and cftnjsiraster; " add tonally 4-c-melhyl glucuronic acid;add 1 anally fucose. X: occurrence of sucstarces.
Fructans represent the most prominent storage carbohydrates in temperate and cool zone grasses [23]. Fruc-tans are mainly determined in feeds and forages for horses as they can cause, if overfed, diseases, such as laminitis, in this species [24]. In the literature, five types of fructans in plants are described [25], all with p-linkage: inulin p 2-1, levan p 2-6, branched p 2-1 and 2-6, inulin neoseries p 2-1, and levan neoseries p 2-6. The HPLC methods mostly use inulin as a standard. According to Apolinario et al. [26], the extraction, isolation, and characterisation of inulin-type fructans have gained attention in recent years due to their wide distribution in nature, and their significant role in industry.
Conclusions: The HPLC method using a Nucleosil column loaded with Pb2+ ions, a RI detector, and HPLC-grade water as an eluent gives precise and reproducible results regarding the detection of individual sugars in extracts of plants and feed materials. The method can be used for the detection of sucrose, maltose, lactose, xylose, glucose, galactose, arabinose, fructose, ribose, and mannitol. Furthermore, depending on the plant material, the sugars verbascose, stachyose, and raffinose can be separated. The method was extended to the analysis of fructan with inulin as standard. The peaks were well resolved and the reproducibility of the analysis with 94-108% recovery (RC) and, a relative standard deviation (RSD) of up to 5%, was very good.
References
1. Zhiqian, L.; Mouradov, A.; Smith, K.F.; Spangenberg, G. An improved method for quantitative analysis of total fructans in plant tissues. Anal. Biochem. 2011
2. Li, S.; Bai, X.; Dong, M.; Li, N.; Li, T. Rapid simultaneous analysis for three sugars fraction in enzymatic degradation of stachyose. Adv. J. Food Sci. Technol. 2014
3. Duarte-Delgado, D.; Narvaez-Cuenca, C.-E.; Restrepo-Sanchez, L.-P.; Kushalappa, A.; Mosquera-Vasquez, T. Development and validation of a liquid chromatographic method to quantify sucrose, glucose and fructose in tubers of Solanum tuberosum Group Phureja. J. Chromatogr. B 2015, 975,
4. Smith, D. Influence of drying and storage conditions on nonstructural carbohydrate analysis of herbage tissue—A review. Grass Forage Sci. 1973, 28,
ТОЛИПОВ ХУРШИД ТУЛКИНЖОН УГЛИ - магистр химии, Ферганский государственный университет, Узбекистан.
