Construction Of Bioactive Films With Lignin/chitosan Based On Solvent And Self-assembly Strategy And Its Application In Fish Preservation

Food packaging has the advantages of protecting food,facilitating transportation and promoting sales.However,the traditional plastic packaging fabricated with petroleum-based polymer causes potential hazard to ecological environment and food safety.Recently,the exploitation of new biodegradable packaging materials based on the abundant natural renewable resources is one of the effective ways to solve this issue.Chitosan is an ideal raw material due to its excellent film forming property and broad spectrum bacteriostasis.Nevertheless,the pure chitosan film exists some drawbacks such as brittleness,poor water resistance,and insufficient biological activity.The addition of plasticizers and active substances are general required to improve the physical and functional properties of the films,which have been studied in various food packaging films.In this paper,the potential application of lignin as functional additive in food packaging was systematically studied using chitosan as matrix.Deep eutectic solvent(DES)was firstly used to clarify the effect of compatibility between lignin and chitosan matrix.Then,lignin nanoparticles(LNPs)were produced by DES combined with antisolvent method,and the effect of LNPs content on the physical properties and biological activities of composite films were clarified.Subsequently,lysozyme-lignin nanoparticles(LyzLNPs)were prepared by electrostatic co-assembly,which were used to construct the bilayer chitosan active films to explore their active release characteristics.Finally,the optimized nanobilayer active films were applied to evaluate the preservation effect of refrigerated fish.The main contents and results were as follows:(1)The preparation and characterization of DES plasticized lignin/chitosan composite membrane were explicated.Firstly,the influence of organic acid(succinic,adipic,tartaric,malic and citric acids)structures on the properties of the cross-linked chitosan films were analyzed.Fourier transform infrared spectroscopy(FTIR)and X-ray photoelectron spectroscopy(XPS)spectra confirmed different degrees of amination reaction occurred in the cross-linking films compared with the acetic acid-chitosan film,without esterification reaction.In addition,the swelling degree,contact angle and water vapor permeability of the crosslinked films were lower than those of the Cs-GA.Malic or citric acid/chitosan membranes were softer and had higher water resistance,while adipic acid/chitosan membranes had higher free radical scavenging activity(52.19 ± 3.90%).Afterwards,citric acid/adipic acid-chitosan(CA)or malic acid/adipic acid-chitosan(MA)composite membrane solution were considered as the film forming matrix,and the effects of DES(10-30 wt%)and lignin(1 wt%)on the properties of the composite films were studied.The results of atomic force microscopy(AFM),scanning electron microscope(SEM)and X-ray diffractometer(XRD)showed that the addition of DESx or DESx L resulted in a large number of small particles with different sizes on the cross-section images of films,and the surface roughness values of films were increased.The films containing30 wt% DES presented a smoother and denser texture,contributing to the dispersion of lignin in the matrix.The mechanical data identified the plastication of DES,which could significantly enhance the elongation at break of composite films.Increasing the addition of DESx or DESx L,the water contact angle and water vapor permeability of CA films improved gradually,while those of MA films increased first and then decreased.Furthermore,the addition of lignin could raise the hydrophobicity,oxidation resistance,and antibacterial activity of the films.(2)The preparation and characterization of lignin nanoparticles/chitosan bioactive membranes were fabricated.The formation mechanism of lignin nanoparticles(LNPs)prepared by DES combined with anti-solvent method was elucidated and the enhancement properties of LNPs as nano-filler for chitosan films were investigated.The results of dynamic/static light scattering,transmission electron microscopy(TEM)and SEM traced the formation of LNPs underwent an intrinsic structural transformation from solution,local clusters,irregular particles or molten spheres to stable colloidal spheres with the increase of water content.According to the factors affecting the preparation of LNPs,the optimized preparation conditions of LPNs with an average particle size of 57.16±1.43 nm were obtained under the conditions of initial lignin concentration of 5 mg/m L,dropping rate of 1 m L/min,and water content of 80 vol %.There was no significant difference in size and PDI after 28 days of storage at p H = 4.5.Compared with lignin,LNPs had higher thermal stability and free radicals scavenging ability,as well as lower minimum inhibitory concentration against staphylococcus aureus.Otherwise,the incorporation of LNPs resulted in the lower swelling rate,water vapor permeability,and good integrity at any p H(3.0,7.0 and 11.0)of films.SEM,XRD and mechanical results of the films showed that the LNPs were uniformly distributed in the film matrix,reducing the diffraction peak intensity and broaden its amorphous region of films.As a result,the tensile strength of composite films was significantly increased by about 40%.With the increase of LNPs content,the DPPH,ABTS free radical scavenging ability,and ultraviolet barrier properties of the composite films increased gradually,whereas only the bacteriostatic circle diameter of the Cs-LNPs1 broadened.(3)The electrostatic-modulated lysozyme-lignin nanoparticles/bilayer chitosan active films were constructed.Based on the water sensitivity,mechanical properties and barrier properties of the bilayer films,the film forming solution ratio of the bilayer membrane was determined to be 3:2.A lower WS and swelling ratio(SD)of bilayer films were obtained after neutralization treatment.The barrier properties of water molecules and oxygen were significantly enhanced at 4°C and 25°C,and the gained films still maintained good tensile properties in wet state.Encapsulation efficiency,loading rate,particle size,and zeta potential results reveled that lysozyme-lignin nanoparticles(Lyz-LNPs)with an average particle size of164.8 ± 5.93 nm were obtained at 4:1 mass ratio of Lyz: LNPs and 5 min ultrasonic time.Also,the encapsulation and loading efficiency were 99.23 ± 0.42 % and 79.38 ± 0.34 %,respectively.With increasing Lyz-LNPs in films,SEM and XRD results presented the surface of bilayer active films changed from “wavy” shaped projection,small particles evenly distributed,to a smooth and flat morphology,and the intensity of the crystal diffraction peak of the film decreased gradually.However,no significant changes were observed in the thermal stability.Compared with the control group,the addition of nanoparticles showed more than doubled DPPH free radical scavenging rate of the film,and further enhanced the inhibition of the film on Escherichia coli and Staphylococcus aureus under the synergic action of Lyz and LNPs.(4)The preservation effect of nano-bilayer active film on fish was evaluated.The fish fillets wrapped by the optimized nano-bilayer active film were used to test the quality changes during storage at 4℃ for 8 d.The results showed that the film had no adverse effect on the appearance of fish and could maintain good sensory quality at the end of storage.Compared with the control group,the minimum p H value of fish in MA-N32-3% group delayed for 2 days,and the p H was significantly decreased in late storage,which effectively slowed down the accumulation of alkaline organic matter.After 8 days of storage in MA-N32 and MA-N32-3%groups,salt-soluble protein content in fish decreased by 47.8% and 28.9%,and TCA-dissolved peptide content decreased by 15.5% and 23.7%,respectively,indicating that film treatment could effectively inhibit protein degradation or enzymatic hydrolysis to produce small molecule peptides.Through the determination of thiobarbiturates(TBARS),bioamines and the total number of colonies,the results showed that the film treatment groups could delay the rate of fat oxidation,reduce the generation of reactive oxygen species,and thus slow down the process of fat oxidation during fish refrigeration.Additionally,film containing active nanoparticles had an active slow-release effect during the period of cold storage,which could further reduce the rate of fat oxidation,delay the secretion rate of amino acid decarboxylase,decrease the formation of characteristic bioamines in the late period of cold storage,and produce a synergistic antibacterial effect on the growth of microorganisms during the period of fish storage.As a result,fish fillets treated with nano-bilayer active film exhibited good quality after 8 days of storage,which was a simple and effective means of preservation.