Chemical modification of chitosan through non-enzymatic glycosylation reaction to improve its antimicrobial and anti-oxidative properties

مجله پژوهشهای علوم و صنایع غذایی ایران

Article 9, Volume 16, Issue 1 - Serial Number 61, March and April 2020, Pages 117-129   

Document Type: Research Article

DOI: 10.22067/ifstrj.v16i1.75740

Authors

Fakhri Shahidi^* ; Farideh Tabatabaei Yazdi; Majid Nooshkam; Zahra Zareie; Fereshte Fallah

Department of Food Science and Technology, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran.

Abstract

Introduction: Lipid oxidation leads to the generation of off-flavors and potential toxic compounds. Synthetic antioxidants are frequently applied for inhibiting this reaction, however; there is a concern regarding to the potent toxic effects of synthetic antioxidants on human health. The non-enzymatic glycosylation reaction (Maillard reaction) has been broadly used to ameliorate the biological and functional features of proteins and polysaccharides. The Maillard reaction produces products with versatile functions such as antioxidant, antimicrobial, antihypertensive, anti-browning, and prebiotic properties. In this regard, the Maillard reaction products (MRPs) can be used in the food industry to inhibit the oxidation reaction due to their superb antioxidant effect. In this study, chitosan was glycosylated with inulin, fructose, and glucose. Chitosan is a chitin derivative with cationic nature having antimicrobial, antioxidant, metal chelation, and film-forming features. Inulin is recognized as a prebiotic sugar with vast applications in food and pharmaceutical sciences. The purpose of this study was to chemically modify chitosan through the Maillard reaction in order to boost its antioxidant and antimicrobial properties.
 
Materials and methods: Chitosan (0.5% w/v) was dissolved in 1.0% v/v acetic acid solution followed by stirring for 1.0 h at room temperature. Afterwards, sugars inulin, glucose, and fructose were separately added to the chitosan solution at final concentration of 1.0% w/v. The obtained solutions were then stirred until complete sugar dissolution. The pH of solution was adjusted to 6.07 by adding 2.0 M sodium hydroxide and then the chitosan-sugar Maillard conjugates were fabricated through autoclaving the solutions at 121 °C. Changes in pH after the reaction were measured using a pH meter. The extent of the Maillard reaction was estimated via measuring the absorbance of the conjugated solutions at 294 nm (the intermediate products) and 420 nm (final products). Fourier transform infrared (FTIR) spectroscopy at transmission mode and 400-4000 cm-1 was employed to evaluate the structural changes of chitosan upon conjugation. Antioxidant activity of the conjugates was evaluated based on the reducing power assay. One mL of the samples was charged with 1.0 mL of distilled water and 1.0 mL of potassium ferricyanide (1.0% w/v). The solution was mixed and incubated at 50 °C for 20 min. After adding 2.5 mL of tri-chloroacetic solution (10% w/v), the obtained solution was centrifuged at 5000 g for 5.0 min. Afterwards, 2.0 mL of the supernatant was mixed with 2.0 mL of distilled water and 1.0 mL of ferric chloride (0.1% w/v). The solution was stand for 10 min at ambient temperature and then its absorbance was recorded at 700 nm. Antimicrobial effect of the conjugates against pathogenic microorganisms (E. coli, S. aureus, B. subtilis, P. aeruginosa, A. niger, and C. albicans) was measured according to the minimum inhibitory (MIC) and microbiocidal (MBC) concentrations. SPSS software (version 21) and one-way ANOVA were applied for data analysis. Duncan’s multiple range test was employed to determine the differences between means.
 
Results & discussion: The Maillard reaction led to a significant decrement in pH value of chitosan-saccharide systems, mainly due to the covalent coupling of amino groups of chitosan to carbonyl groups of reducing sugars in conjugation with the production of acetic and formic acids. The highest intermediate compounds (A 294nm) and lowest browning intensity (A 420nm) observed in chitosan-fructose conjugate, which was likely attributed to the lower reactivity of fructose. Chitosan-inulin conjugate presented the highest A 420nm and lowest intermediate-to-final ratio (A 294nm/A 420nm), probably due to the lower inulin molecules and subsequently carbonyl groups compared to fructose and glucose. These groups may react with amino groups of chitosan at initial reaction times, leading more conversion rate of the intermediate compounds to the final ones. FTIR spectra of the chitosan and conjugates revealed that absorbance peak at 1661 cm-1 in chitosan spectrum decreased and shifted to 1578 cm-1 (in chitosan-fructose conjugate), 1579 cm-1 (in chitosan-glucose conjugate), and 1580 cm-1 (in chitosan-inulin conjugate), indicating the stretching C-N group and -C=N group and the formation of Schiff base (-C=N) between reducing end of the saccharides and amino groups of chitosan. Reducing power of the chitosan-saccharide systems improved after the thermal process. Although, chitosan-glucose and chitosan-fructose conjugates had significantly higher reducing power than unconjugated counterparts, but chitosan-inulin conjugate showed non-significantly improved antioxidant activity compared to its non-heated mixture. Antioxidant activity of the Maillard conjugates was ascribed from the electron donating ability of their hydroxyl and pyrrole groups. The conjugates had lower MIC and MBC in comparison to their unconjugated pairs, except for chitosan-glucose conjugate, which showed no differences in MIC and MBC compared with its non-heated mixture. Antimicrobial property of the Maillard products, especially melanoidins has been attributed to their metal chelating features; melanoidins exert a bacteriostatic effect at low concentration and bactericidal effect at high levels through sequestering ionic iron from medium and magnesium from outer membrane, leading to the cell membranes destabilization. Additionally, antioxidant capacity, high surface activity, and inhibiting effect towards catabolic enzymes have been reported as another antimicrobial mechanisms of the Maillard products. In general, it can be concluded that chitosan-saccharide Maillard-based conjugates, particularly inulin-chitosan one could be used in the food sector as a novel prebiotic-based active bio-compound with antioxidant and antimicrobial features.
 

Keywords

Inulin; Antioxidant; antimicrobial; Chitosan; Melanoidin; Maillard reaction

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