Oligosaccharide Chitosan: Viscosity, Molecular Weight, Antibacterial Activity, and Impact of γ Radiation

Chitosan is a bioactive polymer produced from shrimp and crab shells, etc. According to VASEP (Vietnam Association of Seafood Exporters and Producers), the production of raw shrimp cultured in Vietnam was about 800,000 tons in 2018. Therefore, the shrimp processing industry has generated about 320,000 tons of wastes, including heads and shells. If wastes are not utilized and managed in proper ways, it can lead to serious environmental problems. In our study, shrimp shells were used to produce chitosan and further obtained oligochitosan for application in food preservation. The cobalt-60 radiation technology has been used to segment chitosan into oligochitosan. The radiation dose applied to chitosan solution was in the range of 25 ÷ 50 kGy and in the range of 66 ÷ 166 kGy for chitosan flakes. The results showed that the chitosan solution had higher segmental efficiency compared to that of chitosan flakes. The antibacterial activities of oligosaccharide chitosan segmented from chitosan flakes were higher than those of oligosaccharide chitosan segmented from chitosan solution. The highest antibacterial activities were observed in the oligochitosan segmented from chitosan flakes at the radiation dose of 66 kGy for all tested bacteria: E. coli O157:H7, Salmonella typhimurium, Listeria monocytogenes, Staphylococcus aureus, Bacillus subtilis. In addition, oligochitosan segmented from chitosan flakes at the radiation dose of 66 kGy had higher antibacterial activities on bacteria gram (-) than bacteria gram (+). The strongest antibacterial activities on L. monocytogenes and B. subtilis at the concentration of 0.3125%.

Oligochitosan is believed to have antibacterial activities. Therefore, some researches have been carried out on using oligosaccharide chitosan in vegetable preservation and antifungal, etc [7][8][9][10]. The advantage of using cobalt-60 radiation technology to segment chitosan is that after segmentation oligochitosan products can be used immediately in the preservation process without purification or removal of impurities.
The aim of this study was to the impact of cobalt-60 radiation on the viscosity, the molecular weight, and antibacterial activity of oligosaccharide chitosan.

Material
+ Chitosan: Chitosan was whiteness, fine flakes, 0.51% of protein, 0.5% of ash, the water content of 13.2%, deacetyl degree of 93.5%, soluble degree of 99.5%, the viscosity of 1050 cPs, and turbidity of (NTU) 12 The chemicals in the analysis were purchased from Sigma -Aldrich, except for distilled water and 96% ethanol of Vietnam.

Methodologies
The segment chitosan flakes and chitosan solution in 1% acetic acid were by using gamma 60Co radiation equipment (GC-5000) (Made in India) at the Da Lat Nuclear Research Institute. Radioactivity used was at 4.000 Ci, dose rate of ~ 3,6 kGy/hour, operating in semi-automatic mechanism. Radiation chamber volume of 4.4 liters. The radiation temperature was controlled by water at temperature of 25°C [10].
The irradiation of chitosan flakes and chitosan solutions was by various doses for collecting oligosaccharide chitosan, for example, 10, 30, 70, 166 kGy for chitosan flakes and 10, 15, 20, 25 and 50 kGy for chitosan solutions. After the irradiation, all samples were filtered, segmented by different solvents (methanol and acetone), and evaluated on its viscosity and molecular weight.
The irradiation of chitosan solution and chitosan flakes was at 50 kGy and by various doses (66 kGy, 76 kGy, 100 kGy, 158 kGy, and 166 kGy), respectively, for collecting the solution of antibacterial oligosaccharide chitosan that evaluated by using the method of minimum inhibitory concentration (MIC).

Data Analysis
The data sets presented were mean of three different replicates. Means were calculated and graphed by using MS. Excel 2007 software.

The Viscosity
When the radiation dose increased, the viscosity was decreased for chitosan solution preparing from chitosan of flakes and solution. The solution viscositiy of flakes chitosan was higher than one of solution chitosan. The concentration of chitosan also decided the viscosity of the radiated chitosan concentration. The concentration of chitosan flakes affected the viscosity of soluton more than chitosan solution, as in Figures 1 and 2.

The Molecule Weight
The radiation dose affected the weight molecular of chitosan (p<0.05). The molecular weight of chitosan decreased with increasing the radiation dose for both chitosan flakes and chitosan solution (Figures 3 and 4, respectively). The molecular weight of chitosan flakes decreased from about 549 kDa to 67 kDa (decreasing 8.2 times) at the radiation dose of 166 kGγ ( Figure 3). It means that chitosan had segmented into smaller molecular weight chitosan (called oligosaccharide chitosan) and similar to those notices of Ulanski et al. (1992) [14] and Le Hai et al. (2003) [10]. After radiation, the molecular weight was 94 kDa at the radiation dose of 10 kGy, which decreased 5.8 times compared to the control sample. At radiation dose of 15 kGy, 20 kGy, 25 kGy, and 50 kGy, the molecular weight of chitosan corresponded about 83 kDa, 52 kDa, 34 kDa, and 17 kDa, respectively, for solution chitosan (Figure 4). It means the γ60 radiation segmented chitosan in forms of either chitosan flakes or chitosan solution. The γ60 radiation impacted on solution chitosan more than chitosan flakes.
Oligosaccharide chitosan in solution was continuously precipitated in different solvents, for example, methanol and acetone. The results showed acetone precipitated the lowest oligosaccharide chitosan of the molecular weight and the content, compared to methanol solvent. The molecular weight of oligosaccharide chitosan was arranged in the increasing order for a molecular weight as follows: acetone, the methanol to chitosan solution ratio (9/1 (v/v)), and the methanol to chitosan solution ratio (2/1 (v/v)). The average molecular weight was in the range of 15 -70 kDa for the 1 st segment 1 and 11 -50 kDa for the 2nd segment. The average molecular weight of 3rd segment was under 11 kDa (Figures 5 and 6). The content of oligosaccharide chitosan in the precipitation segment by using the methanol to chitosan solution ratio (2/1 (v/v)) was 40 and 1.8 times, compared to the precipitation segment by using acetone and the methanol to chitosan solution ratio (9/1 (v/v)), respectively. The oligosaccharide chitosan in 1 st , 2 nd , and 3 rd segment was black color, gelatinous form, and ivory-white color powder, respectively. The oligosaccharide chitosan in the 3 rd segment solubilized well in water with a molecular weight under 11 kDa. The solution viscosity was a positive correlation to the chitosan concentration and a negative correlation to radiation dose for both chitosan flakes and chitosan solution.
Note: PĐi: Segment i, and i was from 1 to 3.
The oligosaccharide chitosan from chitosan flakes possessed higher antibacterial activity than that from chitosan solution at the same concentration. The antibacterial activity of oligosaccharide chitosan at the radiation dose of 66 kGγ was higher than that at the radiation dose. The oligosaccharide chitosan affected gram-negative bacteria more than gram-positive bacteria, and the highest impact on L. monocytogenes and B. subtilis, compared to other bacteria. The minimum inhibition concentration of oligosaccharide chitosan on L. monocytogenes and B. subtilis was 0.3125%. For most other bacteria strains, the minimum inhibition concentration of oligosaccharide chitosan was in the range of 0.625 to 1.25%. Numerous previous studies noticed on antibacterial activity of oligosaccharide chitosan, as in [15,16]. Antibacterial activity of oligosaccharide chitosan depended on the molecular weight of oligosaccharide chitosan, the chitosan destroying method, and the material extracting of chitosan [17,18]. The difference caused the diverse on antibacterial activity of oligosaccharide chitosan. The antibacterial mechanism of oligosaccharide chitosan was to base on the membrane disruption [15], the inhibition of RNA and protein synthesis, the breakage of the intracellular component [16], the ionic interaction [19], and free amino groups of oligosaccharide chitosan [20]. According to [21], cationic formation based on the amino protonation leading the bacterial killing. All mechanisms had a common to be interaction with the cell membrane of bacterial and mechanism inhibition in microorganisms. Antimicrobial activity depended not only on the physical chemistry properties of chitosan and their derivatives, but also on the concentration of metal ions (K + , Na + , Mg 2+ , and Ca 2+ ) [22].

Conclusion
The γ60 radiation destroyed chitosan to oligosaccharide chitosan that got the molecular weight under 100 kDa. The viscosity and the molecular weight of oligosaccharide chitosan were negative proportional to the radiation dose, positively proportional to the chitosan concentration. Acetone and the mixture of methanol and aqueous were useful in the segment of oligosaccharide chitosan. The average molecular weight of the 3 rd segment was under 11 kDa. Oligosaccharide chitosan produced by radiation of chitosan flakes (dried chitosan) had higher antibacterial activity than that of oligosaccharide chitosan produced by radiation of chitosan solution. The radiated oligosaccharide chitosan at the radiation dose of 66 kGy had the highest antibacterial activity. Oligosaccharide chitosan at the radiation dose of 66 kGy had antibacterial activity on all tested bacteria strains: E. coli O157: H7, Salmonella typhimurium, L. monocytogenes, S. aureus, and B. subtilis. Antibacterial activity on bacteria gram (-) was higher than bacteria gram (+), in particular, the antibacterial activity was observed on L. monocytogenes and B. subtilis, which are most common spoilage bacteria on the food system. Oligosaccharide chitosan at the concentration of 0.3125% inhibited L. monocytogenes and B. subtilis.