Preparation And Characterization Of Nisin-Loaded Rhamnolipid/Chitosan Complex Aggregates And Its Antibacterial Effect

Nisin is a natural,safe and efficient food preservative,which can inhibit the growth and reproduction of most Gram-positive bacteria.It has been widely applied in foods.However,due to its poor antibacterial stability and narrow antibacterial spectrum,the antibacterial effect of Nisin is seriously affected in practical applications.Microencapsulation of Nisin by complex coacervation is one of the important strategies to solve the above problems.Therefore,in this paper,Nisin and anionic surfactant rhamnolipid（RL）as raw materials were studied.The phase diagram of Nisin/RL complex coacervation system under different conditions was constructed through complex coacervation reaction,and the phase behavior transition law and micro-nano assembly behavior of the Nisin/RL complex coacervate system were systematically revealed;the effect of non-covalent forces on maintaining the stability of Nisin/RL complex coacervates during complex coacervation was investigated.On this basis,the Nisin/RL/chitosan（CS）complex coacervate was constructed to investigate the physicochemical and structural characteristics,functional properties,in vitro release properties and antibacterial activity of Nisin/RL/CS complex coacervate,and its practical application in beef preservation was evaluated preliminarily.The main results are as follows:（1）The transformation law in phase behavior of complex coacervation between RL and Nisin and the micro-nano assembly behavior were revealed by turbidity tracking and microscopic techniques.Results showed that the higher Nisin content is,the more obvious the complex coagulation reaction is.When the mass ratio of RL and Nisin is 1:14,the turbidity curve turns and obvious phase separation occurs.The increasing of Nisin content will lead to the higher p H_c and p H_φ(p H_c:soluble complex aggregates begin to form.p H_φ:insoluble complex aggregates begin to form)of Nisin/RL complex coagulation.The microscopic morphology of Nisin/RL complex aggregates showed obvious differences at different p H key points.In particular,the microstructure at p H_opt（the maximum point of insoluble complex coagulation formation）was relatively tight.The complex coacervation process of RL and Nisin was mainly attributed to electrostatic interactions,while hydrophobic interactions and hydrogen bonds played a litter role.（2）The structural characteristics,release characteristics and storage stability of Nisin/RL complex aggregates were studied by physical and chemical methods with the help of microscopic techniques.Results showed that with the increase of the mass ratio of RL to Nisin and the p H value,the encapsulation efficiency and drug loading of Nisin/RL complex coacervates increased first and then decreased.When the mass ratio of RL to Nisin was 1:14 and p H 4.0,the encapsulation efficiency and drug loading of Nisin/RL complex aggregates were 67.25%and 44.83%,respectively.The complex coacervation reaction improved the thermal stability and storage stability of Nisin/RL complex coacervates,but decreased the crystallinity.The Nisin/RL complex coacervates had good sustained release properties,and the release efficiency increased with the increase of mass ratio.（3）On the basis of Nisin/RL complex aggregates,Nisin/RL/CS complex aggregates were constructed.The physicochemical and structural characteristics,in vitro release behavior,storage stability and antibacterial activity of Nisin/RL/CS complex aggregates under different p H conditions were studied.Results showed that when the volume ratio of Nisin/RL complex aggregates to CS was 1:1 and the p H at the p H_opt point（p H 4.3）,the encapsulation efficiency and drug loading of Nisin/RL/CS complex aggregates were 59.17%and 29.58%,respectively.Compared with Nisin/RL complex aggregates,Nisin/RL/CS complex coacervates had better in vitro sustained-release performance and storage stability,but the crystallinity was decreased.Nisin/RL complex aggregates only had antimicrobial activity against Staphylococcus aureus（S.aureus）,while Nisin/RL/CS complex aggregates exhibited antibacterial activity against S.aureus and Escherichia coli（E.coli）,and the antibacterial effect of Nisin/RL/CS complex coacervates was better than that of Nisin/RL complex coacervates.（4）The preservation effect of Nisin/RL and Nisin/RL/CS complex aggregates on beef was comprehensively evaluated by measuring the p H value,total number of colonies,thiobarbituric acid value,color,texture and other indicators of beef.Results demonstrated that both Nisin/RL and Nisin/RL/CS complex coacervates could effectively maintain the color,texture and sensory quality of beef,and Nisin/RL/CS complex aggregates had superior preservation effect than that of Nisin/RL complex aggregates.