Effect of Chitosan on Membrane Permeability of Suspension-Cultured Glycine max and Phaseolus vulgaris Cells

Abstract

Treatment of suspension-cultured Glycine max cv Harosoy 63 cells with soluble chitosan (20-500 micrograms per milliliter) increased membrane permeability as shown by leakage of electrolytes, protein, and UV absorbing material. Severe damage to the cell membrane by chitosan (100 and 500 ug/ml) was also indicated by reduced staining with fluorescein diacetate and the leakage of fluorescein from preloaded cells. Other basic polymers (poly-L-lysine, histone, DEAE-dextran, protamine sulfate, and glycol chitosan) also increased permeability, whereas the basic monomers L-lysine and D-glucosamine, and acidic or neutral polymers were not active. Chitosan-induced leakage was inhibited by divalent cations, the order of effectiveness being Ba2 > Ca2+ > Sr2 > Mg2+. Na polygalacturonate and Na poly-L-aspartate also reduced polycation-induced leakage, probably by formation of polycation-polyanion complexes. A chitosan-polygalacturonate complex precipitated on mixing solutions of the two polymers containing approximately equal numbers of galacturonate and glucosamine residues, but not with either polymer in excess. A similar concentrationdependent precipitation of chitosan by Na poly-L-aspartate was found. Leakage from Phaseolus vulgaris cv Grandessa cells was also induced by chitosan, and was inhibited by Ca2+ and Na polygalacturonate.Chitosan (,8-1,4- MATERIALS AND METHODSCell Cultures. The cell suspension culture of Glycine max cv Harosoy 63 was a gift from J. Ebel, Freiburg University, Germany and that of Phaseolus vulgaris cv Grandessa (seeds from Bruno Nebelung, Munster, Germany) was initiated by inoculation of callus derived from sterile hypocotyl explants of 6-d-old seedlings. Suspension cultures were grown at 26°C in the dark on a 1.5-cm radius rotary shaker at 120 rpm in Erlenmeyer flasks containing B5 medium (9) which was modified by using 50 jLM FeSO4-EDTA as the source of iron (8), and subcultured at 6-and 12-d intervals, respectively.Chemicals. Chitosan from crab shells (Sigma) was dissolved in 90 ml 0.1 N acetic acid/g chitosan by stirring overnight, centrifuged at 27,000g for 20 min to remove insoluble material, then precipitated by neutralization to pH 8.0 with 5 N NaOH. The precipitate was washed extensively with distilled H20 by centrifugation and freeze-dried. The glucosamine content of this purified preparation was estimated to be 100%o by the method of Ride and Drysdale (23). Aqueous solutions of purified chitosan and glycol chitosan (Sigma) were prepared for use by dissolving 100 mg in 18 ml 0.1 N acetic acid and dialyzing four times against 2 liters of distilled H20. Poly-L-lysine hydrobromide (mol wt, >70,000), DEAE-dextran (approximate mol wt, 500,000), histone (calf thymus, type II), Na polygalacturonate (grade II), Na poly-L-aspartate (mol wt, 20,000-50,000), glucosamine hydrochloride, L-lysine monohydrochloride, L-aspartic acid, FDA, and BSA (fraction V) were from Sigma. Protamine sulfate and galacturonic acid were from Merck (Darmstadt, Germany). Solutions of aspartic acid and galacturonic...

Effect of Tenebrio molitor larvae meal on growth performance, in vivo nutrients digestibility, somatic and marketable indexes of gilthead sea bream (Sparus aurata)

“…One of them is the ability of chitin and its derivatives to activate defence mechanisms of the host organisms by inducing the accumulation of chitinases and other pathogenesis-related proteins (El Ghaouth et al, 1992). Another one is leakage in the cell wall of bacteria due to the interaction between positively charged chitin molecules and the negatively charged surface of the bacteria (Young et al, 1982). In Sparus aurata, Esteban et al (2001) observed an increase in the activity of innate immune system when chitin was incorporated in the diet.…”

A review of the antimicrobial activity of chitosan

“…Early research describing the antimicrobial potential of chitin, chitosan, and their derivatives dated from the 1980-1990s [9][10][11][12][13][14] . Generally, in these studies the chitosan is considered to be a bacteriocidal (kills the live bacteria or some fraction therein) or bacteriostatic (hinders the growth of bacteria but does not imply whether or not bacteria are killed), often with no distinction between activities.…”

Ki̇tosan Ve Anti̇mi̇krobi̇yal Özelli̇kleri̇

“…Antimikrobiyal özelliğinin yanı sıra kitosan gıda sanayinde elma suyu [85] ve sirke [86] üretiminde berraklaştırma maddesi, sosis üretiminde antioksidan [6], liçi [87], elma ve armut suyu [88] ile patateste [89] enzimatik esmerleşmeyi önleyici madde ve meyve sularında asitlik giderici ajan [90] olarak da kullanılmaktadır. Ayrıca kitosan su bağlama ajanı olarak da rol oynayarak birçok enzimi inhibe edebilmektedir [91]. Bilindiği üzere gıdalar farklı bileşiklerin (karbonhidrat, protein, yağ, mineral, vitamin vb.)…”