Preparation And Research On Antimicrobial Activity Of Quaternized Chitooligosaccharide

In recent years, the increasing incidents of public health are of great concern. How to effectively control the growth and spread of harmful bacteria is becoming an important topic, as it is closely related to human health. Therefore, the antibacterial materials with high safety, biodegradability, nontoxic nature, and no irritation to human body have become a popular research topic. Chitosan(including chitooligosaccharide) is one kind of renewable natural polymer, which has many unique properties, such as good biocompatibility, biodegradability, biological activity and so on. In this thesis, chitooligosaccharide was used as a raw material to synthesize a series of quaternized chitooligosaccharide, and the antibacterial properties and possible antibacterial mechanism of these chitosan derivatives have been detailed explored.Low molecular weight(Mw < 2 KDa) Chitooligosaccharide is used to synthesize hydroxypropyl trimethyl ammonium chloride chitooligosaccharides(HTCOS). Through adjusting reaction time and raw material ratio, a serial of HTCOS with different substitution degrees are obtained. Using titration and 1H-NMR spectra, the substitution degrees are calculated, and the result illustrates that the substitution degrees are positively correlated with reaction time and quantity of glycidyl trimethyl ammonium chloride(GTMAC). Turbidimetry, growth curve assay and colony counting method are used to characterize the antibacterial properties, which shows that HTCOS have inhibitory effects on both S. aureus(Gram-negative bacteria) and E. coli(Gram-positive bacteria), and inhibition on S. aureus is better than that of E. coli. With the degrees of substitution increased, the antibacterial ability for HTCOS is weaken. As a concenquence, chitooligosaccharide has the best antibacterial effect. Combined with theoretical knowledge and experimental results, we assume that the electrostatic interaction of chitooligosaccharide and HTCOS with bacterial membranes occur and they attach to the bacterial surface, and then "wrap" on or "insert" to bacteria to inhibit bactera growth.Chitosanoligosaccharide(Mw = 6 KDa), which is produced by chitosan degradation, is also used to synthesize HTCOS. 1H-NMR spectra is used to calculate the substitution degrees of products synthesized under different reaction conditions, while turbidimetry method is used to determine the minimum inhibitory concentration, dynamical antimicrobial activities. It is shown that this kind of Chitosanoligosaccharide has weak inhibitory effects, and its quaternized derivatives exhibit no inhibition but promotion on bacteria growth to some extent. It is suspected that the oxidizability of hydrogen peroxide leads to the destruction of amino group and carboxyl group on Chitosanoligosaccharide, which results in the loss of antibacterial effect.In order to further explore the influence of molecular weight on antibacterial effect, chitosan with molecular weight between 50 KDa and 100 KDa was also choosen to prepare quaternized derivatives. Compared with other derivatives with different molecular weight, low molecular weight chitosan is relatively easy to form quaternized chitosan. The antibacterial ability of quaternized chitosan is determined by the method of plate culture counting. As a result, it demonstrated that quaternized chitosan with low molecular weight has little or even no inhibition on E.coli; while its inhibitory effect on S.aureus is apparent, and there is a negative correlation between the degrees of substitution and inhibitory effects.In summary, the antimicrobial effects of the chitosan(chitosanoligosaccharide) and their quaternary ammonium salt are determined by many factors, such as molecular weight, water solubility, concentration, p H value, and bacteria species and so on. In the case of chitosan, with increased substitution degrees, the water solubility is improved, and the static interaction with bacterial membranes is enhanced. As to chitosanoligosaccharide which has lower molecular weight and better water solubility,when the substitution degrees increase, the static interaction with bacterial membranes shows an opposite trend because the electrostatic effect is insufficient to offset the loss of entropy. The different dominant mechanism of antimicrobial mechanism and the dominant factors under different conditions cause antimicrobial difference; hence, it is difficult to obtain a unified and clear law. It is important to take all factors into consideration and find a balance point rather than adjusting one single factor in the research of these antibacterial materials.