Evaluation of diffusion and dilution methods to determine the antimicrobial activity of water-soluble chitosan derivatives.

AIMS: Chitosan has gained wide applications in the food industry and biomedical field owing to its biodegradability, biocompatibility, nontoxicity and its antimicrobial activity against a wide spectrum of micro-organisms. However, the methods used to investigate antimicrobial effects of chitosan vary considerably among studies, making comparisons difficult. METHODS AND RESULTS: One diffusion (disc diffusion) and two dilution (agar dilution and broth microdilution) methods commonly used in clinical laboratories to assess microbial susceptibility/resistance to antimicrobial agents were comparatively used to determine the antimicrobial activity of two water-soluble chitosan derivatives (molecular weights of 43 and 67 kDa) against 31 representative foodborne pathogens. When tested at 1.6% for the 43-kDa chitosan and 3.2% for the 67-kDa chitosan, by disc diffusion, approximately 10- to 11-mm-diameter inhibition zones were observed for all of the bacterial groups, except for Salmonella tested for the 67-kDa chitosan where no inhibition zone was observed. By agar dilution and broth microdilution, the minimal inhibitory concentration (MIC) values varied largely dependent upon the molecular weight of chitosan, bacterial genus/species and the testing method. The agreement between MIC values obtained by the two methods was poor, with broth microdilution generally having lower MIC values than agar dilution. Regardless of the testing method, Salmonella strains were the least susceptible among Gram-negative strains for both chitosans, followed by Escherichia coli and Vibrio. CONCLUSIONS: Besides chitosan's molecular weight and bacterial genus/species, the antimicrobial activity of chitosan was also influenced largely by the susceptibility testing method used. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study that comparatively evaluated these diffusion and dilution methods, particularly two quantitative methods (agar dilution and broth microdilution), to assess the antimicrobial activity of two water-soluble chitosans against a large number of foodborne pathogens. The study highlights the need for standardized methods to be used in evaluating chitosan's antimicrobial properties in future studies.

Selected quality characteristics of fresh-cut sweet potatoes coated with chitosan during 17-day refrigerated storage.

Selected quality characteristics of fresh-cut sweet potatoes (FCSP) coated with chitosan were evaluated during 17-d refrigerated storage. The FCSP cubes were coated with a solution (1%, w/v) of chitosan having 470 or 1110 kDa. Color (L*, a*, b*) values of uncoated and chitosan-coated FCSP during storage were generally affected by storage time as well as coating treatments (P < 0.05). No significant changes in color lightness (L*) of 470 kDa-coated FCSP were observed during the 17-d storage. During days 3 to 17, 470 kDa-coated FCSP had significantly higher redness (a*) and yellowness (b*) values than did uncoated and 1110 kDa-coated FCSP. Texture firmness of uncoated and chitosan-coated FCSP exhibited minimal changes during the 17-d storage. Although actual weight loss values (%) of uncoated and chitosan-coated FCSP were not significantly different at day 17, the weight loss difference (%) between day 3 and day 17 for uncoated FCSP (3.02%) was slightly higher compared to those (2.24% to 2.26%) of chitosan-coated FCSP. The initial total aerobic count was 4.7 log(10) CFU/g which then gradually increased to 8.54 and 9.67 log(10) CFU/g after 17 d of storage for 470 kDa-coated and uncoated FCSP, respectively. After day 6, the total aerobic counts of uncoated FCSP were higher than those of 470 kDa-coated FCSP. The yeast and mold count of chitosan-coated FCSP was about 2.5 log(10) CFU/g at day 17. Overall, consumers could not differentiate between 470 kDa-coated FCSP at day 17 and uncoated FCSP at day 0.

Plasticizer types and coating methods affect quality and shelf life of eggs coated with chitosan.

Effects of different plasticizer types (glycerol, propylene glycol, and sorbitol) and coating methods (brushing, dipping, and spraying) on the internal quality and shelf life of chitosan-coated eggs were evaluated during 5 wk of storage at 25 degrees C. The Haugh unit and yolk index values suggested that chitosan coating, irrespective of the plasticizer types, extended the shelf life of eggs by almost 3 wk at 25 degrees C compared with noncoated eggs. After 5 wk of storage, plasticizer types did not significantly affect the quality (weight loss, Haugh unit, and yolk index) of chitosan-coated eggs. However, there was an observable trend indicating that use of sorbitol rather than propylene glycol and glycerol as a plasticizer was better in reducing weight loss (whole egg) of chitosan-coated eggs during a 5-wk storage. After a 5-wk storage, there were no significant differences in weight loss and weight of albumen and yolk among chitosan-coated eggs, regardless of the coating methods. However, both brushing and dipping methods yielded chitosan-coated eggs with better yolk (higher yolk index values) and albumen (lower pH) qualities than did the spraying method. During 3 to 5 wk of storage, the Haugh unit values of chitosan-coated eggs by the brushing method were higher than or comparable to those by dipping or spraying. Therefore, coating of eggs with chitosan using sorbitol as a plasticizer and by the brushing method may offer a protective barrier in preserving the internal quality and thus extending shelf life of eggs.

Chitosan treatments affect growth and selected quality of sunflower sprouts.

The effects of chitosan molecular weights, solvent types, and concentrations of chitosan solution, and seed soaking times on growth and selected quality of sunflower sprouts were investigated. Among 5 chitosans tested (746, 444, 223, 67, and 28 kDa), 28 kDa chitosan exhibited the highest DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity both at 0.1% and 1.0% concentrations. Optimal conditions selected for cultivation of sunflower sprouts involved soaking seeds in 0.5% chitosan with 28 kDa (dissolved in 0.5% lactic acid) for 18 h. After cultivation for 6 d at 20 degrees C, sunflower seeds soaked in chitosan solution for 18 h under the optimal conditions yielded sprouts with 12.9% higher total weight and 16.0% higher germination rate, compared with those of seeds soaked in water for 18 h (control). Furthermore, the total amino acid content of the former sprouts (12098 mg/100 g) was slightly higher than that of the latter (12057 mg/100 g). Sprouting of sunflower seeds improved DPPH radical scavenging activity, probably due to the increased total phenolic, melatonin, and total isoflavone contents. Similarly, chitosan-treated sprouts exhibited slightly improved DPPH radical scavenging activity, probably due to slightly increased total phenolic and melatonin contents, and moderately increased total isoflavone content compared with those of the control. Chitosan treatment increased the total isoflavone content of sprouts by 11.8%, due mainly to the increased daidzein content, compared with that of the control.

Applications of chitosan for improvement of quality and shelf life of foods: a review.

Chitosan is a modified, natural biopolymer derived by deacetylation of chitin, a major component of the shells of crustacean. Recently, chitosan has received increased attention for its commercial applications in the biomedical, food, and chemical industries. Use of chitosan in food industry is readily seen due to its several distinctive biological activities and functional properties. The antimicrobial activity and film-forming property of chitosan make it a potential source of food preservative or coating material of natural origin. This review focuses on the applications of chitosan for improvement of quality and shelf life of various foods from agriculture, poultry, and seafood origin.

Antioxidant activities of different colored sweet bell peppers (Capsicum annuum L.).

Antioxidant compounds and their antioxidant activity in 4 different colored (green, yellow, orange, and red) sweet bell peppers (Capsicum annuum L.) were investigated. The total phenolics content of green, yellow, orange, and red peppers determined by the Folin-Ciocalteau method were 2.4, 3.3, 3.4, and 4.2 micromol catechin equivalent/g fresh weight, respectively. The red pepper had significantly higher total phenolics content than the green pepper. Among the 4 different colored peppers, red pepper contained a higher level of beta-carotene (5.4 microg/g), capsanthin (8.0 microg/g), quercetin (34.0 microg/g), and luteolin (11.0 microg/g). The yellow pepper had the lowest beta-carotene content (0.2 microg/g), while the green one had undetectable capsanthin and the lowest content of luteolin (2.0 microg/g). The free radical scavenging abilities of peppers determined by the 2,2'-diphenyl-1-picrylhydrazyl (DPPH) method were lowest for the green pepper (2.1 micromol Trolox equivalent/g) but not significantly different from the other 3 peppers. All 4 colored peppers exhibited significant abilities in preventing the oxidation of cholesterol or docosahexaenoic acid (DHA) (C22:6) during heating. However, these 4 peppers did not show significant differences in their abilities in preventing cholesterol oxidation. The green pepper showed slightly higher capability in preventing the oxidation of DHA compared to the other 3 peppers.

Effective deacetylation of chitin under conditions of 15 psi/121 degrees C.

Deacetylation of chitin under autoclaving conditions (15 psi/121 degrees C) was evaluated for the preparation of chitosan under different NaOH concentrations and reaction times. Deacetylation was effectively achieved by treatment of chitin under elevated temperature and pressure with 45% NaOH for 30 min and a solids/solvent ratio of 1:15. Treated chitosan showed similar nitrogen content (7.42%), degree of deacetylation (90.4%), and molecular mass (1560 kDa) but significantly higher viscosity values (2025 cP) compared with those (7.40%, 87.6%, 1304 kDa, and 143 cP, respectively) of a commercial chitosan. Reduction of the solids/solvent ratio from 1:15 to 1:10 did not affect degree of deacetylation, viscosity, and molecular mass of chitosan.

Application of chitosan for treatment of wastewaters.

Research has clearly demonstrated that the biopolymer chitosan (deacetylated chitin) can be used as an effective coagulating agent for organic compounds, as a chelating polymer for binding toxic heavy metals, as well as an adsorption medium for dyes and small concentrations of phenols and PCBs present in various industrial wastewaters. In these specific applications, chitosan appears more effective than other polymers such as synthetic resins, activated charcoal, and even chitin itself. In addition, the amino group in chitosan is an effective functional group that can be altered chemically for production of other chitinous derivatives with specific useful characteristics as effective absorptive agents. Chitosans exhibiting different physicochemical characteristics, i.e., molecular weight, crystallinity, deacetylation, particle size, and hydrophilicity, differ in their effectiveness as waste treatment agents. The specific relationship between methods and the particular crustacean species used in preparation of chitosan for wastewater treatment needs further examination. Use of bioremediation approaches, combined with immobilization of specific microorganisms on immobilized chitinous columns, is an extremely promising area of current research and actual plant operation.