Preparation Of Egg White Protein-based Nisin Nanoparticles And Their Antibacterial And Fresh-Keeping Application

Freshwater fish have the characteristics of rich nutrition and high water content,and they are prone to spoilage after being slaughtered.In order to maintain the quality of fish meat,adding preservatives has become an important means.Nisin is a common biological preservative,which has been widely used in the field of food preservation and preservation.However,Nisin itself also has some shortcomings,such as poor solubility,easy inactivation under medium alkaline,enzyme and high temperature conditions,and interaction with food ingredients,which limits its application range.Therefore,it is necessary to adopt macromolecular embedding technology to broaden the application range of Nisin.Owing to its rich nutrition,digestibility,self-assembly and amphipathic properties,egg white protein has the potential as a nano-delivery carrier.In this paper,we constructed chitosan-coated egg white protein-Nisin nanoparticles through acid heat induction technology,ultrasonic nanotechnology and chitosan surface modification technology,and studied their physical and chemical properties,structural characteristics,antibacterial properties and fresh-keeping effect on grass carp.The main content and results of this paper are as follows:(1)Acid induced EWP nanoparticles(A-NEn)loaded with Nisin and Acid-heat induced EWP nanoparticles(AH-NEn)loaded with Nisin were constructed.The antibacterial effect on Staphylococcus aureus was studied.Through single-factor experiments,it is clear that the suitable preparation conditions for nanoparticles AH-NEn are:pH3.0,temperature 90 ?,and the mass ratio of egg white protein to Nisin 2:1,and the best encapsulation efficiency of nanoparticles AH-NEn is obtained.The loading rates were 89%and 23.4%,and the particle size,PDI and Zeta potential were 112.5 nm,0.249 and 24 mV,respectively.Through the influence of different reactants on the light transmittance of nanoparticles and the measurement of fluorescence spectroscopy,it is determined that the main intermolecular force of nanoparticle A-NEn is electrostatic effect,while the main intermolecular force of AH-NEn is electrostatic effect and disulfide.Bonding,indicating that thermal induction promotes the formation of new intermolecular forces of AH-NEn.The AH-NEn nanoparticles were observed as spherical particles by transmission electron microscope(TEM).The results of chemical structure analysis by infrared spectroscopy(FTIR)and X-ray diffraction(XRD)showed that under acid-heat induced conditions,the encapsulation of egg white protein particles did not affect the chemical structure of Nisin Differential calorimetric scanner(DSC)analysis results show that the egg white protein particles successfully embed Nisin and improve the original thermodynamic properties of egg white protein.The results of antibacterial activity showed that Nisin embedded in egg white protein did not affect the original antibacterial activity of Nisin.(2)Constructed chitosan-coated egg white protein-Nisin nanoparticles and studied their antibacterial effects on S.aureus and Escherichia coli.The egg white protein-Nisin nanoparticles were coated with different concentrations of chitosan.When the chitosan concentration was 2.5 mg/mL,the encapsulation efficiency reached about 95%.Through the addition of different reagents and fluorescence spectroscopy tests,it is clear that the main forces between the molecules after chitosan coating are electrostatic interaction,disulfide bond and hydrophobic interaction,and with the increase of chitosan concentration,the force gradually increases.The particle size increased from 169.30 nm to 547.33 nm,the potential increased from 23 mV to 30 mV,and the PDI increased from 0.22 to 0.58,indicating that as the concentration of chitosan increased,the uniformity and dispersion of the complex gradually decreased.The TEM results showed that the appearance of chitosan also changed with the increase of the concentration of chitosan.When the concentration of chitosan was 2.5 mg/mL,aggregated particles were produced;the results of FTIR and XRD showed that the chitosan modification did not change.The original structure of Nisin in the composite;DSC analysis results show that the thermodynamic properties of the composite after being coated with chitosan are improved.After being coated with chitosan,it not only has an inhibitory effect on the gram-positive bacteria S.aureus,but also has an inhibitory effect on the gram-negative bacteria E.coli,which broadens its antibacterial spectrum(3)The application performance of egg white protein-Nisin nanoparticles before and after the chitosan coating was studied and the effect of its application on the preservation of grass carp was systematically evaluated.The measurement results showed that before coating with chitosan,the order of emulsification and emulsification stability of nanoparticles from weak to strong was AH-En<A-NEn<AH-NEn;the emulsification and emulsification stability after chitosan was coated Both are higher than AH-NEn nanoparticles,and when the chitosan concentration is 1 mg/mL,the emulsifying activity of chitosan-coated egg white protein-Nisin nanoparticles(CS modified AH-NEn,AH-NEn-CS)reaches best value.The antioxidant activity test results show that the antioxidant activity is improved after chitosan coating,and as the concentration of chitosan increases,the antioxidant activity also increases.The storage stability results show that the storage stability of the sample is more stable at 4? than at 25?,and with the increase of chitosan concentration,the stability of the sample is relatively weakened.The antibacterial stability results show that under high temperature,trypsin and high pH environment,egg white protein has a certain protective effect on the antibacterial activity of Nisin.After being coated with chitosan,it also has antibacterial activity against S.aureus,but has no inhibitory effect on E.coli.By studyring the effects of Nisin and different nanoparticles on the pH,color difference,texture,juice loss rate,fat oxidation and the total number of colonies of grass carp under 4? and 25? storage conditions,the results show that the egg white protein after chitosan coating-Nisin nano-particles can slow down the corruption of fish meat,inhibit the growth of microorganisms,and have a prominent effect on the freshness of grass carp fish.