Study On Stabilization Of Antibacterial Effect Of Nisin Based On Complex Coacervation

Nisin as a safe and efficient antibacterial peptide has been widely used in dairy products,meat products and alcoholic beverages.However,due to the poor stability of Nisin in the food system,its antibacterial ability is reduced,which further restricts its practical application in food preservation.At present,improving the antibacterial stability of Nisin in the food environment through complex coacervation has become a research hotspot,but the intrinsic relationship between complex coacervation and antibacterial stability of Nisin needs to be further explored.In this study,Nisin and polyanionic polysaccharide-oxidized curdlan derivative(C6-Cc)are used to construct phase diagrams of Nisin/C6-Cc composite system under different conditions.The phase behavior transformation and mico-nano assembly behavior of Nisin/C6-Cc composite system were analyzed by using turbidity tracking,light scattering and microscopy techniques from macro and micro perspectives.The non-covalent forces that maintained the stability of Nisin/C6-Cc complex coacervation and structural characteristics of Nisin/C6-Cc composites were studied.On this basis,physical stability and antibacterial stability of Nisin/C6-Cc complexes were systematically evaluated.This study will help to clarify the internal relationship between complex coacervation and antibacterial stability of Nisin,and broaden its application in food preservation.The main results are as follows:(1)The phase behavior transformation and micro/nano-assembly behavior of the complex coacervation formed by Nisin and C6-Cc were revealed through turibidity tracking and microscope techniques.The results showed that pH_cand pH_?1of the Nisin/C6-Cc complex(pH_c:soluble complex aggregates begin to form;pH_j(16):insoluble complex aggregates begin to form)shifted to higher pH with the increase of mass ratio of Nisin/C6-Cc,and the forming region of complex aggregates was broadened by increasing mass ratio.Meanwhile,the influence of ionic strength on the Nisin/C6-Cc complex coacervation showed multiple effects.When the ionic strength was 20 m M,the electrostatic complexation betwen Nisin and C6-Cc was promoted.However,when the ionic strength was greater than 20 mM,the electrostatic complexation betwen Nisin and C6-Cc was interfered,resulting in a narrower forming region of complex aggregates.When the Nisin/C6-Cc complex systems were at different phase transition points,their microstructures presented obviously different morphologies,among which the complex aggregates showed a tighter microstructure at pH_opt(pH_opt:the maximum point at which insoluble complex aggregates).(2)Fourier-transform infrared spectroscopy analysis showed that electrostatic attraction interaction was a dominant force of the Nisin/C6-Cc composite aggregates,and the hydrogen bond was the secondary force.Surface hydrophobicity and effects of salt ion types on the turbidity and Zeta potential of the complexe aggregates were analyzed.It was found that hydrophobic and electrostatic interactions showed greater impacts on the complex coacervation than hydrogen bonds.Rheological analysis showed that the complex aggregates formed by electrostatic attraction interactions possessed pronounced viscoelasticity.Thermogravimetric analysis revealed that the thermal stability of the Nisin/C6-Cc complex aggregates was greatly improved after complex coacervation.In addition,transmission electron microscopy observation demonstrated that the Nisin/C6-Cc complex aggregates that stabilized by non-covalent forces exhibited relatively uniform spherical structures.(3)The effects of pH,ionic strength,temperature and storage conditions on the stability of Nisin/C6-Cc composite aggregates were studied by dynamic light scattering technology.The results showed that the polydispersibilty index values of Nisin/C6-Cc composite aggregates were in the range of 0.25?0.28 when the pH value was less than 5,showing a higher pH stability.Nisin/C6-Cc complex aggregates can maintain good resistance against salt within the range of 0-100 m M.When the mass ratios were 1:4 and 1:8(w/w),the Nisin/C6-Cc composite aggregates can remain stable in the temperature range of 25-121?.When the mass ratio was 1:14(w/w),the complex aggregates were stable ranging from 60°C to 100°C.Compared with the storage condition at 25°C,the Nisin/C6-Cc composite aggregates stored at 4°C showed better storage stability.(4)The Nisin/C6-Cc complex aggregates exhibited strong antibacterial stability in the pH range of 1-4,the diameters of the inhibition zone were between 11.5 mm and 13.0 mm,and the minimum inhibitory concentration(MIC)value was 0.71mg/m L.Nisin/C6-Cc complex aggregates showed strong antibacterial stability in the ionic strength range of 0-200 m M,and its maximum inhibition zone diameter was13.2 mm,and the MIC value was 0.71 mg/m L.Nisin/C6-Cc complex aggregates at mass ratios of 1:4 and 1:8 can maintain strong antibacterial stability at 25-121?.Moreover,the Nisin/C6-Cc complex aggregates at mass ratios of 1:4 and 1:8 still maintained strong antibacterial stability after one month of storage at 4°C and 25°C,respectively,but when the mass ratio was 1:14,its antibacterial stability was apparently reduced.