Study On The Structure And Antibacterial Mechanism Of Agar-sodium Alginate Bilayer Slow-release Antibacterial Film

Polysaccharide-based slow release antibacterial films have become a research hotspot in the field of packaging materials because of its good biocompatibility,biodegradability,slow and sustained release of active ingredients,as well as targeting antibacterial properties to specific food and microorganisms.Taking agar extracted from fresh Gracilaria lemaneiformis and commercial sodium alginate as substrates,a novel agar-sodium alginate bilayer slow-release antibacterial film containing six kinds of plant essential oils was designed and prepared in this study.Based on the differences in the bacteriostatic effect,physical performance and microstructural properties of bilayer films,the influence of the interaction between essential oil-polymer matrix on the structure and properties of the film was explored through the similarities and differences of the chemical composition of the essential oils（EOs）,with the film containing ginger essential oil（GEO）exhibited better comprehensive performance and investigated its effects on the properties,structure and application for beef refrigeration systematically.Meanwhile,the effect of AG-SA antibacterial film and AG-SA antibacterial coating with the same concentration GEO on beef refrigeration was compared.Finally,the film diffusion kinetic models were used to fit the release behavior of GEO released from film matrix under two conditions including immersion release and unilateral release,revealing the main mechanism of GEO released from agar-sodium alginate antibacterial film to different food simulants.The main research contents and conclusions are as follows:1.Preparation of AG-SA bilayer slow-release antibacterial film and comparison of antibacterial propertiesTaking the mechanical properties of commercially available polyethylene（PE）bags as a reference,the optimum formulation of fabricating AG-SA bilayer antibacterial film containing 5%v/v essential oils was obtained by single factor and L₉（3³）orthogonal test,namely,mass ration of AG/SA 2:8,1.8%w/v of AG/SA dosage,25%w/v of glycerol dosage.The growth inhibition effect of AG-SA bilayer antibacterial film on Escherichia coli（E.coli）,Staphylococcus aureus（S.aureus）,Bacillus subtilis（B.subtilis）was significantly better than that of AG-SA monolayer antibacterial film;the addition of six kinds of EOs into films significantly increased the thickness and elongation at break（EB）value of the bilayer film,but the tensile strength（TS）value decreased.The retention rate of GEO in the film matrix was higher,and the AG-SA bilayer film containing GEO exhibited better bacteriostatic effect on the three tested microorganisms.2.Effects of EO types on the structure-activity analysis of AG-SA bilayer film’s antibacterial propertiesEOs（ginger,lemon,mint,sweet orange,bergamot,grapefruit EO,5%v/v）significantly enhanced the blocking ability of the film against visible and ultraviolet light by reducing the surface brightness and increasing the transparency of the film;and reduced the water vapor permeability and oxygen permeability by generating a porous structure in film matrix.Due to the hydrophobic properties of EOs,the water content,water solubility,and swelling properties of AG-SA films decreased.In addition,significant changes in thermal stability,microscopic morphology and crystal structure of films were observed with the incorporation of EOs,and GEO could better maintain the thermal properties and enhance the crystal characteristics of AG-SA film,along with the more uniform distribution in the inner layer film matrix.Meanwhile,the types and contents of the main compounds of EOs were proved to be the main factors that cause the differences in physical performance,bacteriostatic properties,and structures of AG-SA antibacterial films,as well as the distribution characteristics of EOs in film matrix.Besides,the overall performance of AG-SA film containing GEO was better could be mainly related to the unsaturated structures presented in monocyclic sesquiterpene（containing83.91%sesquiterpenes,of which 71.01%are monocyclic sesquiterpenes）that riched in GEO was more likely to form a p-πconjugated structure with the glycosidic bond presented in the molecular structure of agar and sodium alginate,in addition to the H-bond interaction between GEO and the AG-SA film matrix,which is beneficial to the structural stability of GEO in film matrix and the slow release of GEO from AG-SA films.3.Effects of GEO amount on structure,properties and food preservation effect of AG-SA antibacterial filmFirst,the optical profiles,barrier properties,dissolution properties,and bacteriostatic properties of AG-SA antibacterial films containing 1%～5%GEO were evaluated,along with their microstructures characterized and the distribution properties of GEO in the film matrix observed.The results showed that the water content,water solubility and swelling ratio decreased gradually with the increase of GEO amount;while the thickness,light blocking properties and growth inhibition of E.coli,S.aureus and B.subtilis of films increased.The WVP and OP of the film decreased gradually with GEO content increased to 4%v/v,whereas became larger than the control film when up to 5%v/v.The porous structure and the number of oil droplets in the inner layer film matrix became more obvious with the increase of GEO amount,also the distribution of oil droplets in film matrix was more dispersed and more uniform.Secondly,taking the PE bag and the AG-SA film without GEO as the reference,the effect of the AG-SA antibacterial film containing 1%-5%GEO on the comprehensive quality change of beef refrigerated at 4°C was investigated.Meanwhile,the microbial growth of beef that refrigerated to the 18th day was compared between the AG-SA film and the AG-SA coating both containing 5%v/v GEO,so as to preliminarily investigate the application effect of this two active packaging methods.It was found that the addition of GEO to the AG-SA film could effectively inhibit the growth of representative microorganisms during beef refrigeration,the addition of GEO can effectively inhibit the growth and reproduction of representative microorganisms during beef refrigeration,delaying the lipid oxidation of beef by reducing the PV value,TBARS value,and TVB-N value,reducing the degradation of beef protein and the production of amines in meat samples,as well as inhibiting the generation of meat-like odor/stink,maintaining the elasticity,hardness and chewiness of beef better,thereby prolonging the shelf life of beef;and this fresh-keeping effect was overall positively correlated with the amount of GEO added to the film matrix less than 4%v/v,while the AG-SA antibacterial film with 4%v/v and 5%v/v GEO amount showed similar effect on preservation quality,which might be explained by the fact that the molecular groups provided by 4%v/v GEO that can interact with the molecular chains groups（such as-OH,-COOH）of SA and AG used for fabrication of AG-SA antimicrobial film at a fixed amount reached maximum.The AG-SA antibacterial coating containing GEO showed a remarkable effect on the low temperature preservation of beef and grass fish.In addition,the growth inhibition of tested bacteria during beef refrigeration of films and emulsion coatings containing 5%v/v GEO differs,with antimicrobial coatings showing stronger growth inhibition to fungi and psychrophilic bacteria,while antimircobial films exhibited more obvious inhibition towards bacteria（E.coli,S.aureus,etc.）.4.Release characteristics and kinetic behavior of GEO in AG-SA antibacterial filmThe impregnation release kinetics and the unilateral release kinetics of AG-SA bilayer antibacterial film containing 5%v/v GEO was studied,and the release behavior of EOs was fitted by using 7 mathematical models.The results showed that the impregnation release（except n-hexane）and unilateral release of GEO in the simulated solution exhibited the rapid release at first with following a stable and sustained release,and the release rate of GEO in impregnation release process in the simulated solution was higher than that of unilateral release.In addition,for the impregnation release,the cumulative release of GEO in AG-SA antibacterial film reached the release equilibrium following descending order:95%ethanol>distilled water>50%ethanol>10%ethanol>n-hexane.The cumulative release in the unilateral release process is 95%ethanol>n-hexane.Furthermore,the release behavior of GEO fitted by different models showed that the Peppas-Sahlin model could better simulate and explain the GEO released from AG-SA antimicrobial film toward five food simulants in impregnation release and unilateral release,followed by Peleg model;and based on the overall results of the correlation coefficient R²from each model showed that the main mechanism of GEO release from AG-SA antibacterial membrane was Fickian diffusion.The obtained results lay a good foundation for further revealing the main mechanism of（ginger）EO migration from the film matrix and diffusion and release to the packaging-food interface in a specific use environment（specific temperature,humidity,etc.）,providing a research and scientific basis for the practical application of seaweed polysaccharid-based slow-release film,which is conducive to promoting the high-value development and comprehensive utilization of seaweed.