Mechanisms and kinetics of nisin release from calcium alginate films
Open Access
- Author:
- Lalpuria, Minal Prakash
- Graduate Program:
- Food Science
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- May 22, 2012
- Committee Members:
- Ramaswamy C Anantheswaran, Dissertation Advisor/Co-Advisor
Ramaswamy C Anantheswaran, Committee Chair/Co-Chair
John Coupland, Committee Member
Ali Demirci, Committee Member - Keywords:
- antimicrobial packaging
nisin
alginate
diffusion - Abstract:
- Several studies have shown that calcium alginate matrices containing nisin effectively inhibits microbial growth in various foods such as beef, poultry, milk, and mushrooms, but they only present qualitative information on overall effectiveness of these systems on microbial inhibition. In order to design an effective antimicrobial packaging system for commercial applications, it is important to study the impact of various factors that govern the structure and properties of calcium alginate films and the diffusion of nisin from these films. It is known that nisin is a cationic polypeptide, whereas alginate is an anionic polymer. Ionic interactions between nisin and alginate might hamper the release of nisin from the alginate matrix, leading to reduced antimicrobial activity. Hence, it is essential to characterize interactions between nisin and alginate and to investigate the antimicrobial activity of nisin released from calcium alginate films. The overall goal of this research was to study the mechanisms and kinetics of nisin release from calcium alginate films. The objectives were to: (a) modify agar diffusion assay for better quantification of nisin; (b) determine factors affecting the release of nisin from calcium alginate films; (c) investigate the interaction between nisin and alginate; (d) determine the kinetics of release; (e) evaluate the physical, mechanical and optical properties of these films; and (f) investigate antimicrobial activity of the released nisin. Agar diffusion bioassay, using Micrococcus luteus, is a commonly used method for quantification of nisin concentration. However, the correlation between inhibition zone and nisin concentration is not well established at higher nisin concentrations. The objective of this study was to investigate the effect of different well size (small and large) and pre-diffusion times (0, 24, 48 and 72 h) at 4°C on the sensitivity, precision and accuracy of nisin quantification. Regression analysis indicate that small wells were better than large wells because of smaller standard deviation, higher predictive accuracy, and better discrimination between mean inhibition zones at neighboring nisin concentrations. Statistical analysis by Tukey’s test showed that pre-diffusion resulted in significantly different inhibition zones at different nisin concentations. Pre-diffusion also improved sensitivity of the assay at different nisin concentrations. Different regression models were considered to explore the relationship between inhibition zone and nisin concentration for all pre-diffusion times. A spline model was the best fit model and 48 h was the best pre-diffusion time. Factors affecting the release of nisin from calcium alginate films were studied by the disc agar diffusion bioassay. Calcium alginate films were formed by two methods – cross-linking before drying (CBD) and cross-linking after drying (CAD) – using various alginate compositions, cross-linking times and Ca2+concentrations. The method of film formation, cross-linking time, and Ca2+concentration had a significant effect (p<0.05) on nisin release. Alginate composition did not affect nisin release significantly (p>0.05). SEM images showed that film formation method influenced the microstructure of films considerably. Films made by the CBD method and cross-linked with high Ca2+ concentration had the highest nisin release. The binding between nisin and alginate was investigated for various concentrations and compositions of sodium alginate and in the presence of Ca2+. Six concentrations of sodium alginate (1.67-33.33 mg/ml) were added to nisin solution (0.5 mg/ml) and the free nisin remaining was measured by HPLC. Results indicated that nisin binds with alginate, but this binding was reversible in the presence of Ca2+. The composition of alginate did not have a significant effect (p>0.05) on the nisin-sodium alginate binding. However, alginate with higher guluronic acid content binds less nisin in the presence of Ca2+. Diffusion of nisin from calcium alginate films cross-linked with various concentrations of Ca2+ (0.18-1.25 M) was investigated. The effect of cross-linking on the degree of swelling, mechanical properties, and optical properties of calcium alginate films was evaluated. The amount of nisin released from calcium alginate films increased significantly (p<0.05) with Ca2+ concentration. The mechanism involved in the diffusion process was investigated using the Power law model. Nisin diffusion from films cross-linked with 0.18M Ca2+ exhibited anomalous behavior, all other films exhibited near-Fickian behavior. Nisin release from these films fitted well to Fick’s second law of diffusion (R2>0.90). The effective diffusivity of nisin into water at 21°C was of the order 10−8 cm2/s and did not change significantly (p>0.05) with Ca2+ concentration. This released nisin retained its antimicrobial activity. Films cross-linked with low Ca2+ concentrations underwent significant swelling. The transparency, tensile strength and elastic modulus of the films decreased, whereas elongation at break increased, with increasing Ca2+ concentration.