Elsevier

Carbohydrate Research

Volume 346, Issue 11, 16 August 2011, Pages 1307-1310
Carbohydrate Research

A new method for the quantification of chitin and chitosan in edible mushrooms

https://doi.org/10.1016/j.carres.2011.03.040Get rights and content

Abstract

Along with β-glucans, chitin is the dominant component of the fungal cell wall. Chitosan, the deacetylated form of chitin, has found quite a number of biomedical and biotechnological applications recently. Mushroom chitin could be an important source for chitosan production. A direct determination of chitin and chitosan in mushrooms is of expedient interest. In this paper, a new method for the quantification of chitin and chitosan is described. This method is based on the specific reaction between polyiodide anions and chitosan and on measuring the optical density of the insoluble polyiodide–chitosan complex. After deacetylation, chitin can also be quantified. The specificity of the reaction is used to quantify the polymers in the presence of complex matrices. With this new spot assay, the chitin content of mycelia and fruiting bodies from several basidiomycetes and an ascomycete were analysed. The presented method could also be used for the determination in other samples as well. The chitin content of the analysed species varies between 0.4 and 9.8 g chitin per 100 g of dry mass. Chitosan could not be detected in our mushroom samples, indicating that the glucosamine units are mostly acetylated.

Introduction

Alongside β-glucans, chitin is an important component of the fungal cell wall. It is also a main component of the exoskeleton of Arthropoda. The polymer is characterized by β-(1→4)-branched N-acetylglucosamine units.1 Partial deacetylation of this biopolymer yields chitosan. Preparation of chitosan from chitin sources has been discussed recently.2 In recent years, health benefits and technical uses of chitosan and N-acetylglucosamine oligomers have been discussed.3 The wide variety of possible applications in the food industry includes, for example, functional foods, packaging materials and filtration devices.4 Thus, the cell wall of mushrooms could be an important source for chitosan production. Basidiomycetes like Lentinula edodes or Grifola frondosa are well known for their application in various medical domains. β-Glucans, in particular, have been proposed as the active immunostimulating agents.5 Furthermore, after isolation of the glucans, the remaining dietary fibre, consisting mainly of chitin, could be used for chitosan preparation as well. Unfortunately, chitin is insoluble in most solvents, so that direct detection is difficult,6 but it is possible to quantify it indirectly as chitosan or N-acetylglucosamine. In a modification of a method of Tsuji et al.7 for chitosan determination,8 chitin was determined by converting chitin to chitosan with concentrated potassium hydroxide and hydrolysing chitosan to glucosamine. With this method, the chitin content of a number of mushrooms has been determined.9, 10

Another indirect method is based on a complete hydrolysis of chitin to N-acetylglucosamine and its quantification.11 Vetter and Siller,12 and Vetter13 report on the use of this method for successful quantification of mushroom chitin.

Another method was used by Johnson and Chen.14, 15 They optimized the colorimetric assay of Svennerholm for glucosamine quantification.16 After acid hydrolysis of chitin, they quantified the released glucosamine with 4-(N,N-dimethylamino)benzaldehyde colorimetrically.

The detection of chitosan is in principle simple because of its solubility in acid solutions. Therefore, it is possible to use colorimetric tests. Recently, several methods have been described that are based on a reaction of the free amino group of the glucosamine unit with a specific reagent. Unfortunately, a cross reaction with the amino groups of amino acids or proteins is possible. Thus, the protein content in mushroom cell walls leads to complications in the chitosan detection. Larionova et al. developed a method using o-phthalaldehyde to quantify chitosan.17 Here, cross-reactions with other free amino groups occur as well. They therefore, calculated their results from standard curves of chitosan, bovine serum albumin and insulin. Another determination method is based on the reaction of the amino group of the polyglucosamine with ninhydrin. However, other free amino groups react, too.18

Our group wanted to develop a new, simple and direct determination of chitosan that relies on the formation of a coloured complex of chitosan with Lugol’s solution. Lugol’s solution is used in microscopy as a specific dye for chitin in cell walls.

The complex of Lugol’s solution and chitosan is insoluble. Therefore, a spot assay was developed. After pipetting the samples on plates, the optical density of the chitin–polyiodide complex was measured by a photographic technique which is used for quantitative analysis of TLC plates as well.

Section snippets

Chemicals

Chitin and chitosan (purity 99%) were obtained from Sigma–Aldrich (Seelze, Germany) for the calibration standard and model reactions. Thin-layer plates (Macherey Nagel Polygram Sil G 0.2 mm (Art. Nr. 805013), Macherey Nagel Alugram RP18W/UV254 0.15 mm (Art. Nr. 818146), Macherey Nagel Polygram CEL300 0.1 mm (Art. Nr. 801012)), Schleicher & Schuell Gel-Blotting paper GB 002 and Macherey-Nagel MN 615 filtration paper for the spot assay were bought from Carl Roth Gmbh (Karlsruhe, Germany).

Mushroom samples

The

General parameters and method validation

We tested several concentrations of Lugol’s solution (data not shown), but a 1% solution was found to be preferable. We also tested other reagents, such as acid fuchsin, for detection, but cross-reactions and lower sensitivity of the other reagents made Lugol’s solution the best. In addition, spraying the detection reagent seems to be more practical and leads to better results than immersing the plates. Several foils and papers were tested to optimize this spot assay. Concentrations in the

Discussion and conclusions

In the present work, a reliable and specific determination for chitin and chitosan has been achieved. The average chitin content in the mushroom species analysed is about 2.5 g for the mycelia and 3.5 g for the fruiting bodies. Some exceptions occur: The fruiting bodies of A. bisporus, F. velutipes and P. eryngii have notably higher chitin contents. Of the mycelia studied, those of A. bisporus and P. eryngii contain the highest amounts of chitin. As the culture conditions for all mycelia are

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