اصلاح شیمیایی کیتوزان از طریق واکنش گلیکوزیلاسیون غیرآنزیمی به‌منظور بهبود خواص ضدمیکروبی و آنتی ‏اکسیدانی آن

واکنش گلیکوزیلاسیون غیرآنزیمی (مایلارد) به‌طور گسترده‌ای جهت بهبود ویژگی‌های بیولوژیکی و عملکردی پروتئین‌ها و پلی‏ساکاریدها استفاده شده است. در این پژوهش، پری‏بیوتیک اینولین و قندهای پیش‏ساز آن (گلوکز و فروکتوز) از طریق واکنش مایلارد با کیتوزان گلیکوزیله گردید. تغییرات ph، محصولات حد واسط و نهایی، تغییرات ساختاری، ظرفیت آنتی‌اکسیدانی و ویژگی ضدمیکروبی سامانه‌های کانژوگه مورد بررسی قرار گرفت. فرآیند حرارتی سبب کاهش معنی‌دار ph سامانه‌های کیتوزان ساکارید گردید. بالاترین شدت ترکیبات حد واسط (a 294nm) و کمترین شدت قهوه‌ای شدن (a 420nm) در سامانه کیتوزان فروکتوز مشاهده شد؛ در حالیکه کانژوگه‏ کیتوزان اینولین بیشترین a 420nm و کمترین نسبت تبدیل محصولات میانی به نهایی (a 294nm/a 420nm) را نشان داد. اتصال کووالانی کیتوزان به ساکاریدهای اینولین، فروکتوز و گلوکز با استفاده از طیف‌سنج فروسرخ تبدیل فوریه (ftir) تایید گردید. واکنش مایلارد منجر به تشکیل محصولاتی با فعالیت آنتی‌اکسیدانی قابل‌توجه شد و به استثنای سامانه کیتوزان گلوکز، سایر نمونه‌های کانژوگه حداقل غلظت مهارکنندگی و کشندگی کمتری نسبت به نمونه‌های غیرکانژوگه نشان دادند که بیانگر بهبود فعالیت ضدمیکروبی کیتوزان بعد از واکنش با اینولین و فروکتوز می‌باشد. مطابق نتایج، کانژوگه‌های مایلارد، به‌ویژه اینولین کیتوزان می‌تواند نوع جدیدی از ترکیبات زیست‌فعال بر پایه‌ی پری‏بیوتیک با ویژگی‌های آنتی‌اکسیدانی و ضدمیکروبی جهت استفاده در صنایع غذایی باشد.

Chemical modification of chitosan through nonenzymatic glycosylation reaction to improve its antimicrobial and antioxidative properties

Introduction: Lipid oxidation leads to the generation of offflavors 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 nonenzymatic 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, antibrowning, 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 filmforming 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 chitosansugar 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 4004000 cm1 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 trichloroacetic 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 oneway 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 chitosansaccharide 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 chitosanfructose conjugate, which was likely attributed to the lower reactivity of fructose. Chitosaninulin conjugate presented the highest A 420nm and lowest intermediatetofinal 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 cm1 in chitosan spectrum decreased and shifted to 1578 cm1 (in chitosanfructose conjugate), 1579 cm1 (in chitosanglucose conjugate), and 1580 cm1 (in chitosaninulin conjugate), indicating the stretching CN 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 chitosansaccharide systems improved after the thermal process. Although, chitosanglucose and chitosanfructose conjugates had significantly higher reducing power than unconjugated counterparts, but chitosaninulin conjugate showed nonsignificantly improved antioxidant activity compared to its nonheated 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 chitosanglucose conjugate, which showed no differences in MIC and MBC compared with its nonheated 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 chitosansaccharide Maillardbased conjugates, particularly inulinchitosan one could be used in the food sector as a novel prebioticbased active biocompound with antioxidant and antimicrobial features.;