Electrospun gelatin/chitosan nanofibers containing curcumin for multifunctional food packaging

Mengxi Dun,Jishui Sun,Yequn Hung,Hixin Jing,Yqin Hu,Jie Png,c,*,Chunhu Wu,c,*

Keywords:Electrospun nanofibers Curcumin NH3 indicator Active and intelligent packaging Antibacterial and antioxidant activities

ABSTRACT Recently,food grade nanofiber-based materials have received growing attentions in food packaging.In this work,novel active and intelligent packaging nanofibers based on gelatin/ chitosan with curcumin (GA/CS/CUR) were developed via electrospinning technique.Effects of the incorporation of CUR content (0.1% -0.3%,m/m) on the microstructure and functional properties of the electrospun nanofibers were investigated.Morphological studies using scanning electron microscopy indicated that loading CUR can affect the average diameter of nanofiber mats,which remained around 160-180 nm.The addition of an appropriate level CUR(0.2%,m/m) led to a stronger intermolecular interaction,and thus enhanced the thermal stability and tensile strength of the obtained nanofibers.Meanwhile,the incorporation of CUR significantly improved antioxidant activity and the antimicrobial activity of GA/CS/CUR nanofibers.Moreover,the sensitivity of nanofibers to ammonia results indicated that GA/CS nanofibers containing 0.2% CUR (GA/CS/CUR II) presented high sensitivity of colorimetric behavior to ammonia (within 3 min).These results suggest GA/CS/CUR II nanofibers has great potential as a multifunctional packaging to protect and monitor the freshness of proteinrich animal foods,such as meat and seafood.

1.Introduction

Recently,there are increasing interest in the development of active and intelligent food packaging films based on food-grade biopolymers and natural bioactive compounds [1,2].Among these films,natural pigment-based colorimetric films have gained considerable attention for non-toxicity,biocompatibility,pH-sensing nature and others [3,4].These pH-sensitive colorimetric films could exhibit visible color changes when reacting with the non-neutral volatile gases generated from deteriorated high protein-content foods,which provide consumers with visual information about the quality and microbial contamination of foods [1,2,5].

Among these natural pigments,curcumin (CUR),a natural lipophilic pigments extracted from turmeric (the rhizomes of the plantCurcuma longa),is very popular colorimetric indicators for intelligent films because of its color variation response to the pH changes [2,6].The mechanism of the CUR color change is due to the diketone groups of CUR is converted into keto-enol form when exposed to alkaline conditions [7,8].Apart from as a colorimetric indicator,CUR has been widely used as natural antibacterial and antioxidant agent in the active food packaging [2,6].As a result,it is an excellent candidate for the design of simultaneously active and intelligent packaging materials.

Electrospinning is a simple,versatile and cost-effective technique for producing sub-micron or nano-scale fibers from natural or synthetic polymers [9,10].Comparing with traditional casting films,electrospun nanofibers have many unique characteristics such as surface-to-volume ratio,nanoporous structure,high porosity,and high absorbance capacity [1,11],which are more responsive to the surrounding variation in acidity/alkality and enable to control the release of incorporated bioactive compounds.Thus,electrospun nanofibers have received great attention for developing food packaging films in the past few years [12,13].Many researchers have demonstrated the feasibility of the application of electrospinning technology in food packaging.According to the report of Zhang et al.and Zhao et al.[14,15],many biopolymers such as polysaccharide,protein,antibacterial materials and high barrier materials have been widely used in electrospinning nanotechnology to develop new food packaging.The electrospun fiber encapsulation could overcome the thermally sensitive and high volatility of the biopolymers [16,17].However,few literature has been reported on electrospun nanofibers containing CUR as intelligent film.For example,Alehosseini et al.prepared electrospun protein fibers to encapsulate CUR,and a green tea extract (GTE) was also incorporated within the formulations to evaluate its impact on the curcumin-loaded fibers;the results show that the zein-based coatings would be more adequate for packaging of high water content food products [18].Yildiz et al.[19]prepared a chitosan/polyethylene/curcumin electrospun nanofiber to monitor chicken freshness;the results show that curcumin loaded nanofiber gave an opportunity to visualize real time monitoring of chicken spoilage.

In this work,the main objective was to develop CUR-loaded electrospun nanofiber by the electrospinning technology,which served as multifunctional food packaging.Gelatin (GA) and chitosan(CS) were chosen as fiber-forming matrixes due to their good fiberforming ability,non-toxicity and biocompatibility [20-25].The effect of the CUR concentrations on morphology,structure,thermal property,antioxidant activity and antibacterial activity of CUR-loaded electrospun nanofiber were investigated.Furthermore,the sensing ability of electrospun nanofiber to NH3was also explored.Finally,we think the nanofiber loaded with a suitable amount of curcumin has great potential in multifunctional packaging to protect and monitor the freshness of protein-rich animal foods,such as meat and seafood.

2.Materials and methods

2.1 Materials

Chitosan (CS,deacetylation degree of 75% -85%,medium molecular weight) and Gelatin (GA) (Type B,Bloom 250,mw～100 kDa) were provided from Sigma-Aldrich Chemical Co.(USA).Curcumin (CUR,from Curcuma longa,purity ≥ 65%,HPLC),cetyltrimethyl ammonium bromide (CTAB) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) were supplied by Sigma-Aldrich Chemical Co.(St.Louis,MO,USA).Other reagents were analytical grade and used without additional purification.

2.2 Preparation of electrospun nanofibers

The preparation method of electrospun nanofiber is based on the report of Yildiz et al.[19].To prepare electrospinning solutions,CS was dissolved in 60% (V/V) acetic acid under vigorous magnetic stirring for 4 h at 45 °C to obtain the CS solution (0.03 g/mL).GA was dissolved in 80% (V/V) acetic acid under vigorous stirring for 4 h at 45 °C to obtain the GA solution (0.25 g/mL).The obtained CS and GA solutions were mixed at a ratio of 3:7 to obtain the CS/GA mixture under vigorous stirring for 1 h.Subsequently,different amounts of CUR (0%,0.1%,0.2% and 0.3%,based on the dry mass of GA and CS) were added into the above obtained mixture,and recorded as GA/CS,GA/CS/CUR I,GA/CS/CUR II and GA/CS/CUR III,respectively.The as-prepared electrospinning solutions (5 mL)were injected into a disposable syringe with a 22G steel needle for electrospinning process at room temperature with a humidity of around 50% .The electrospinning parameters were employed as follows:a constant flow rate of 0.5 mL/h using a syringe pump,a voltage of 20 kV generated by a high voltage power,an appropriate distance of 10 cm from needle tip to a circular rotating drum covered with aluminum foil (35 cm × 20 cm).Finally,the obtained nanofibers with good morphology (smooth and flat surface) were collected for further analysis.

2.3 Surface tension,conductivity,and viscosity properties of spinning solution

Surface tension was measured using a surface tensiometer(Dataphysics DCAT 21),using pendant drop mode.Conductivity measurements were made with Orion Star A322 conductivity meter(Thermo Fisher Scientific Co.,Ltd.,MA,USA).The viscosity properties of the spinning solution were analyzed by an Anton Paar MCR 301 rheometer (Anton Paar Instruments Inc.,Austria) equipped with a standard parallel plate (diameter=50 mm) at 25 °C and at a shear rate of 0.1-1 000 s?1.

2.4 Characterization of electrospun nanofibers

2.4.1 Fourier transform infrared spectroscopy (FT-IR)

The FT-IR spectra of the nanofibers were measured by using a Thermo Nicolette 6700 spectrophotometer (Thermo Fisher Scientific Co.,Ltd.,MA,USA) of 4 000-400 cm-1at a resolution of 4 cm-1.

2.4.2 X-ray diffraction (XRD)

XRD patterns of the nanofibers were characterized using a Bruker AXS D8 Advance X-ray diffractometer (Bruker Inc.,Germany) equipped with Ni-filtered Cu Kα radiation.The machine was operated at 40 kV and 30 mA,at a wavelength of 1.540 56 ? and a nickel monochromator filtering wave.Samples were recorded in the scattering range of 2θ(5°–45°) at a speed of 2°/min at room temperature.

2.4.3 Morphology analysis

The nanofibers were sputtered with a thin layer of gold.Then the nanofibers were observed using a SEM (Japan Electron Optics Laboratory Co.,Ltd,Tokyo,Japan) with an accelerating voltage of 5.0 kV.The nanofibers diameter distribution was statistically measured by ImageJ software (National Institutes of Health,USA)from the SEM images.

2.4.4 Thermogravimetric analysis (TGA)

The thermal stability of the nanofibers was measured using a thermo-gravimetric analyzer (STA409-PC,Netzsch,Germany) from 25 to 600 °C at a 10 °C/min scan rate under a nitrogen gas flow at a flow of 50 mL/min.Th nanofibers was approximately 5.0 mg were weighed in aluminum pans and hermetically sealed.

2.4.5 Mechanical properties

The mechanical properties of the nanofibers were measured by using an AG-IC50kN Texture Analyzer (Shimadzu,Tokyo,Japan).The nanofibers were cut into rectangular shape with 1 cm in width and 5 cm in length and the thicknesses of the fiber mats were measured by using a micrometer (Model MDC-25,Mitutoyo.Tokyo,Japan).The texture analyzer was worked at 10 mm/min.All measurements were tested at least 5 times in parallel.

2.4.6 Antioxidant capacity

The antioxidant capacity of the nanofibers was measured by using DPPH radical scavenging assay according to our previous study [23]with slight modifications.Briefly,20 mg of fiber mats was immersed in 4 mL 50% ethanol aqueous solutions for 1 h.Then 0.5 mL sample solutions (5 mg/mL,50% ethanol aqueous solutions as control)were separately added to 2 mL DPPH ethanol solution (100 μmol/L)at room temperature under dark condition for scavenging reaction of 30 min.The absorbance of solutions was recorded by using an UV–vis spectrophotometer (UV-2600,Shimadzu,Kyoto,Japan)at 517 nm.Finally,the DPPH radical scavenging activity of samples was calculated by the formula.The calculated formula is as follows.

WhereAcis the absorbance of control,Asis the absorbance of the sample.

2.4.7 Antibacterial capacity

The antibacterial capacity of the nanofibers was evaluated against the gram-positiveS.aureusand the gram-negativeE.colibacteria by using agar disk diffusion method with the judgement of the inhibition zones (mm).In brief,the samples were cut into 10 mm disks.Then 300 μL of activated bacterial solution (105colony forming unit CFU/mL) was spread evenly on Luria-Bertani (LB) agar.The obtained 10 mm disks of samples were placed on the above LB agar and incubated at 37 °C for 24 h in biochemical incubator.The diameter of the inhibition zone was recorded.

2.4.8 Sensitivity to volatile ammonia

Sensitivity of the nanofibers to volatile ammonia was evaluated by the method of slight modifications [8].In brief,the fiber mats were exposed for 24 min at a 1 cm distance over a flask poured with 80 mL of ammonia solution (0.8 mol/L),and the color parameters of the nanofibers at different response time were measured by a colorimeter (CS-200,CHNSpec Technology Co.,Ltd.,Hangzhou,China).The total color difference (ΔE) was calculated as follows:

Where ΔL=L*-L0;Δa=a*-a0;Δb=b*-b0;L0,a0andb0are color parameters of the control.

2.5 Statistical analysis

The statistical data were analyzed using Origin 9.0 software(Origin Lab Corporation,USA) and SPSS software (SPSS 25.0 for windows,SPSS Inc.,Chicago,IL,USA).Least significant differences(LSD) multiple comparison tests were used to determine significance of the obtained data (P<0.05).All the statistical data were performed as the mean value ± standard deviation of film.

3.Results and discussion

3.1 Morphology and nanofiber size analysis

SEM was used to investigate morphological changes of GA/CS nanofibers containing different CUR content.The SEM images and diameter distribution of the nanofibers were shown in Fig.1.The obtained electrospun nanofibers were cylindrical morphology with smooth surface,and no obvious changes were observed in the morphological properties of the produced nanofibers upon increasing the concentration of CUR except for the slight increase of diameter.The average nanofiber diameters were between 160 and 180 nm forming a nonwoven mat.The average fiber diameter GA/CS was(160 ± 34.8) nm.With the incorporation of CUR,the average fiber diameter of the samples was (170 ± 34.7) nm (GA/CS/CUR I),(165 ± 32.2) nm (GA/CS/CUR II) and (180 ± 44.9) nm (GA/CS/CUR III),respectively.As shown in Table 1,the addition of CUR can improve the conductivity of spinning solution.However,the conductivity of GA/CS/CUR II is higher than that of GA/CS/CUR III,which may be due to poor aggregation and dispersion when curcumin is added too much.Therefore,the conductivity of GA/CS/CUR CURIII will show a downward trend.Moreover,the hydrogen bond between CUR and GA/CS matrix leads to an increase in the viscosity of the spinning solution and the diameter of the nanofiber [10].Alehosseini et al.[18]showed the incorporation of polyphenols could act as natural cross-linkers for GA and thus increased diameter of electrospun nanofibers.However,there was only a small increase in the diameter of GA/CS/CUR II nanofibers.This may be caused by the highest conductivity of GA/CS/CUR II.Generally,the average diameter of nanofiber could be decreased with the conductivity of spinning solution increase.Because the spinning solution conductivity rising with the addition of CUR and counteracting the enhancement effect of molecular interactions between CUR and GA/CS matrix [24].In addition,as shown in Table 1,the addition of CUR has little effect on the surface tension of electrospinning solution.Therefore,the above results indicated that GA/CS/CUR II had better electrospinning performance than other samples.

Table 1 Properties of spinning solution and the average diameter of nanofiber.

Fig.1 SEM images of diameter distributions of the GA/CS (a),GA/CS/CUR I (b),GA/CS/CUR II (c),GA/CS/CUR III (d) nanofibers.

3.2 FT-IR analysis

The molecular interactions of the samples were characterized by FT-IR spectra.As shown in Fig.2a,the strong peak starting from 3 100 cm-1to 3 600 cm-1with a maximum at 3 425 cm-1in all samples was the characteristic peak for N-H and O-H stretching vibration.The peaks at 2 956,1 649,1 540,1 410,1 157,and 1 080 cm-1corresponding to C-H stretching,C-O stretching,N-H bending amide,C=N stretching,C=O ether stretching,and C-O-C stretching mode,respectively [20].These results confirmed all characteristic bands of GA and CS were observed for spectrum of nanofiber without any new peak,suggesting that no chemical reaction was occurred between the ingredients.Notably,comparing with the GA/CS nanofiber,only a small change in the spectrum shape was detected,which was attributed to the relatively low CUR ratio (0.1% -0.3%,m/m) to polymer and their overlapping peaks.For instance,the intensity and position of O-H stretching band were decreased and shifted to a lower wavenumber when CUR was added into GA/CS nanofiber,which was caused by the addition of CUR affected the formation of hydrogen bonds between GA and CS.Minmin Chen et al.also reported a similar result that the strong hydrogen bonding between Poly (lactide–glycolide) and CS was partially interfered by the presence of CUR [25].Similarly,the bands of C-O stretching and N-H bending decreased and shifted to a higher wavenumber (1 655 cm-1).These results further revealed the formation of intermolecular interactions among CUR,GA and CS.

Fig.2 FT-IR spectra (a) XRD patterns (b) of the electrospun nanofibers.

3.3 XRD analysis

The XRD patterns of the obtained electrospun nanofibers were demonstrated in Fig.2b.All samples presented a broad halo peaks at around at 2θ=15°-28°,due to the amorphous nature of GA and CS,which was in line with previous studies [2,26,27].The addition of CUR to the GA/CS nanofibers did not cause a major change in the XRD pattern,which could be attributed to the low content of CUR in composite matrix.Notably,the crystalline degree of the nanofibers decreased with the increase of CUR,which would be attributed to the formation of new hydrogen bonds among GA,CS and CUR,thereby altering the original crystal structure of GA/CS matrix.Tabli Ghosh et al.also reported similar effects of CUR on the crystal structures of cellulose nanofibers (CNF) and CS [28].

3.4 TGA analysis

Thermal property of nanofibers represented its ability to resist decomposition at high temperature [29].TGA curves of GA/CS with different weight ratio of CUR were presented in Fig.3.The TGA curves of the samples exhibited two main weight loss stages between 25 °C and 600 °C.In brief,the first stage of thermal degradation was 25-200 °C,which was attributed to the evaporation of physically weak and chemically strong bound water [30].The second stage at around 250-600 °C was attributed to thermal degradation of GA and CS [31].As can be seen from Fig.3,the thermal stability were enhanced with increasing CUR (0.1% and 0.2%,m/m),then decreased by the addition of 0.3% CUR The reason may be that a moderate CUR (≤ 0.2%,m/m) can improve the fiber thermal stability through intermolecular hydrogen bonds,while excessive CUR (0.3%,m/m)would hindered their rearrangement of polymeric chains and crystallization during the formation of the fibers [32],altering the original compact structure of GA/CS matrix.Therefore,both GA/CS/CUR II and GA/CS/CUR I have good thermal stability.The thermal stability of GA/CS/CUR III is the worst.This characterization result provide support for the potential application of GA/CS/CUR II in multifunctional food packaging.

Fig.3 TGA curves of the electrospun nanofibers.

3.5 Mechanical properties

The stress-strain curves of the electrospun nanofibrous mats were showed in Fig.4.During the stretching experiment,the mats were broken along the stretching direction with the increasing stretching strength.The breaking strength of the GA/CS electrospun nanofibers mats was (2.38 ± 0.09) MPa.The addition of CUR improved the mechanical properties of the electrospun nanofibrous mats.The GA/CS/CUR II mats showed the highest tensile strength at(2.79 ± 0.17) MPa,suggesting that the GA/CS/CUR II mats may possess relatively good mechanical properties.In addition,the GA/CS/CUR III mats suffered from poor mechanical strength with tensile strength at (2.30 ± 0.22) MPa.This phenomenon may be caused by the high CUR concentration levels.The high CUR content can increase the hydrophobicity of the GA,and as a result,the mechanical performance of the GA/CS/CUR III mats decreased [33].The above results suggested the GA/CS/CUR II mats that may be the appropriate ratio to achieve better mechanical properties.The results indicated that adding CUR in appropriate amount could improve the mechanical properties of nanofiber film.But too much amount of CUR can degrade the mechanical properties of nanofiber film.Therefore,in this study,GA/CS/CUR II is an ideal sample with mechanical properties.

Fig.4 The stress-strain curves of electrospun nanofibers.

3.6 Antioxidant activity

Oxidation reaction has a series of adverse effects on food quality,thus,it is necessary to develop the food packaging material with incorporating natural antioxidants [34].The DPPH radical scavenging activity of the electrospun nanofibrous was presented in Table 2.DPPH radical scavenging activity of the control GA/CS was only(8.95 ± 0.64)%,which might be due to the special groups of GA and CS [35,36].As expected,DPPH radical scavenging activity of the GA/CS electrospun nanofibrous mats was significantly enhanced (P<0.05)with the increase of CUR concentration.The GA/CS/CUR III showed a relatively high DPPH radical scavenging activity of (51.16 ± 0.59)%,an approximately 8 times enhancement in contrast to the control.The improvement in antioxidant capacity could be attributed to good free radical scavenging activity of CUR [2,37].The DPPH radical scavenging rate of GA/CS/CUR II was (41.32 ± 0.60)% .Although the antioxidant capacity of GA/CS/CUR II is worse than that of GA/CS/CUR III,GA/CS/CUR II also shows ideal antioxidant capacity when considering the characterization results of other properties.The DPPH radical scavenging rate of GA/CS/CUR I was only (25.53 ± 0.52)% .The above results showed the antioxidant properties of the GA/CS electrospun nanofibrous mats could be enhanced by incorporating CUR.And with the increase of CUR content,the antioxidant capacity was stronger.

3.7 Antibacterial activity

Antibacterial activity is important for food packaging films since spoilage caused by foodborne pathogens can result in a series of food quality problems [38].The inhibitory effects of electrospun nanofibrous mats against gram-negative (E.coli) and gram-positive(S.aureus) bacteria were illustrated in Table 2.The control GA/CS had very low antibacterial capacity,which was consistent with the previous studies [21,22].As reported in many researches chitosan itself have an antibacterial activity on the tested microorganism [39].Nevertheless,the antibacterial capacity of the GA/CS electrospun nanofibrous mats was significantly enhanced (P<0.05) by the incorporation of CUR.Notably,antibacterial capacity of the electrospun nanofibrous mats enhanced significantly in all strains by increasing the CUR concentration,and the electrospun nanofibrous mats showed a significantly larger inhibition zone for gram-positive(S.aureus) than that of the gram-negative (E.coli).This phenomenon was due to the differences in cell wall structures of these two bacterias [40].Similar enhancements in antibacterial activity were achieved when CUR were incorporated into chitosan-based and gelatin-based films [2,18].GA/CS/CUR III has the highest antibacterial activity in the samples.The antibacterial activity of GA/CS/CUR II is slightly less than GA/CS/CUR III.GA/CS/CUR I has the lower antibacterial activity than the above two samples.These results indicated that GA/CS/CUR electrospun nanofibrous mats may be used as antibacterial packaging materials in food industry to protect food.Moreover,the antibacterial ability of nanofiber film could be increase with the content of curcumin increase.

3.8 Sensitivity of nanofibers to ammonia

Sensitivity of nanofibers to NH3was evaluated to potentially expand their application.The spoilage of most protein-rich animal foods is attributed to microbial contamination,and during spoilage,various organic amines (e.g.ammonia,dimethylamine and trimethylamine) are generated,which changes the pH status of foods [2,7,38].Thus,ammonia can be used to simulate the release of volatile nitrogen compounds protein-rich animal foods during spoilage process.As indicated from Table 3,a rapid color change(yellow→orange-red) occurred within 30 s after exposure to volatile ammonia.Meanwhile,the yellowness degree of GA/CS/CUR II nanofibers gradually deepened after the films were exposed to ammonia for 6 min.The correspondingLandbparameter decreasedsignificantly along with the extension of exposure time,indicating a high sensitivity of colorimetric response.The color variation mechanism of the colorimetric nanofibers in contact with the NH3gas is proposed in Fig.5.The volatilized NH3combined with the H2O in the colorimetric nanofibers to formand OH-.Subsequently,OH-induced the formation of an alkaline environment on the colorimetric nanofibers,which induced the phenolic hydroxyl group transformed to phenolic oxygen anion in the CUR structure [7,8],and thereby caused the color change.Compared with the colorimetric NH3indicator film prepared by biopolymers with natural dyes (response time exceed 3 min) [4,8,41],GA/CS/CUR II nanofibers showed fast response.The high sensitivity of colorimetric behavior was attributed to the hydrophilicity of GA/CS matrixs and the porosity of fibrous mat,which favored the color reaction by providing water molecules and contact sites [1].Hence,GA/CS/CUR II nanofibers could be suitable as pH indicator in intelligent food packaging.

Fig.5 Possible mechanism for the color change of the electrospun nanofibers to NH3.

Table 2 Antioxidant and antibacterial activity of electrospun nanofibrous mats.

Table 3 Color parameters for the GA/CS/CUR II mats response to ammonia.

4.Conclusions

Novel active and intelligent packaging was successfully developed by incorporating CUR into GA/CS matrix through the electrospinning technique.The addition of an appropriate level CUR (0.2%,m/m) had a hydrogen bonding interaction with the GA/CS matrix,as confirmed by FT-IR and XRD analyses,and well dispersed in the electrospun nanofibers obtained from the SEM,which enhanced the thermal stability and tensile strength of the obtained nanofibers.Meanwhile,the incorporation of CUR significantly improved antioxidant activity and the antimicrobial activity of GA/CS/CUR nanofibers.Moreover,GA/CS/CUR nanofibers exhibited high sensitivity of colorimetric behavior to ammonia (within 3 min).Therefore,GA/CS/CUR nanofibers could be used as smart packaging materials to protect and monitor the freshness of protein-rich animal foods,such as meat and seafood.

Conflicts of interest

The authors declare that they have no conflict of interest in the publication of this manuscript.This study is original research that has not been published previously,and not under consideration for publication elsewhere.

Acknowledgements

This work was supported by Distinguished Youth Talent Program of Fujian Agriculture and Forestry University (xjq201912),the National Natural Science Foundation of China (31801616),Scientific Research Foundation of Hainan Tropical Ocean University(RHDRC202117) and Excellent Master Thesis Fund Project of Fujian Agriculture and Forestry University (1122YS01002).