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chitosan nanoparticles embedded gelatin nanofibers for food packaging against Listeria monocytogenes and Staphylococcus aureus on cheese
Food Packaging and Shelf Life 19 (2019) 86–93
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Moringa oil/chitosan nanoparticles embedded gelatin nanofibers for food packaging against Listeria monocytogenes and Staphylococcus aureus on cheese Lin Lin, Yulei Gu, Haiying Cui
T
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School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Moringa oil Chitosan nanoparticles Electrospun nanofibers Cheese Food packaging
The current study aims to prepare moringa oil-loaded chitosan nanoparticles (MO@CNPs) and fabricate MO@CNPs embedded gelatin nanofibers for biocontrol of Listeria monocytogenes and Staphylococcus aureus on cheese. The optimal MO@CNPs were prepared by the ionic crosslinking method with the concentration of moringa oil at 20 mg/mL and chitosan at 3.0 mg/mL. The nanoparticle exhibited desirable particle size, PDI and zeta potential. Furthermore, the optimal concentration of MO@CNPs embedded in gelatin nanofibers was found to be 9.0 mg/mL after the determination of nanofiber physical properties. The results of SEM and AFM confirmed that the nanofibers were prepared successfully and achieved uniform diameter at 142.5 nm. The release rate of moringa oil from the nanoparticles declined due to the encapsulation of nanofibers. For the application on cheese, MO@CNPs nanofibers possessed high antibacterial activity against L. monocytogenes and S. aureus at 4 °C and 25 °C for 10 days, without any effect on the sensory quality of cheese. As a result, MO@CNPs nanofibers could be a promising active food packaging material for food preservation.
1. Introduction Cheese is particularly popular in our daily life due to its intrinsic nutritional ingredients, such as proteins, fat, vitamins and inorganic salts (Felicio et al., 2016). Nevertheless, cheese has been found to be susceptible to pathogenic bacterial growth whether in processing or storage stages. The pathogenic microorganisms including Listeria monocytogenes (L. monocytogenes) and Staphylococcus aureus (S. aureus) have received great attention in food industry (Lee, Cappato, Corassin, Cruz, & Oliveira, 2015). These two pathogenic bacteria could be transferred to human and bring about a high risk for immunocompromised people, pregnant women, newborns and the elderly (Muhteremuyar et al., 2018). In order to minimize the incidence of disease caused by L. monocytogenes and S. aureus, natural antibacterial agents have been utilized in food industry (Cui, Ma, Li, & Lin, 2016). Essential oils (EOs) have captured increasing attention because of their safety and high efficiency properties. Moringa oil is extracted from Moringa oleifera which is a fast-growing softwood tree found in the Middle East areas, African and Asian countries (Leone et al., 2016). Recent studies displayed that moringa oil became popular because of its healthful properties, excellent oxidative stability and high antimicrobial activity (Singh,
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Singla, Upadhyay, & Singh, 2017; Zhong et al., 2018). Unfortunately, moringa oil is hard to inhibit the growth of bacteria for a long period on account of its volatility and sensitivity to light, air and high temperature (Cui, Bai, Rashed, & Lin, 2017; Cui, Yuan, Li, & Lin, 2017). Therefore, for increasing stability and prolonging action time of moringa oil, nanoencapsulation technology was employed to embed moringa oil and prevent loss of active compositions. Chitosan, a polysaccharide obtained by partial deacetylation of chitin, has been extensively used as drug-loaded nanoparticles on account of its biological intermiscibility and biodegradable properties (Liang et al., 2017). Furthermore, chitosan nanoparticles have special strengths, including slow and controlled drug release rate, enhanced drug absorption and minimal drug side effects (Elgadir et al., 2015). However, the spraying of nanoparticles on the surface of food would lead to diffusion and instability. In consequence, the electrospun nanofibers have been introduced to immobilize chitosan nanoparticles on the surface of food. Electrospinning, an economic and promising technique, has been applied to fabricate nanofibers with large specific surface area, high mechanical strength and high drug encapsulation efficiency (Lin, Dai, & Cui, 2017). In food fields, gelatin-based nanofibers have been widely employed due to its biodegradability, nontoxicity and bioactivity
Corresponding author. E-mail address: [email protected] (H. Cui).
https://doi.org/10.1016/j.fpsl.2018.12.005 Received 10 August 2018; Received in revised form 22 November 2018; Accepted 13 December 2018 2214-2894/ © 2018 Elsevier Ltd. All rights reserved.
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number of residual bacteria was determined at 0, 0.5, 1, 2, 4 and 8 h. Transmission Electron Microscopy (TEM) (Model-JEM-2010HR, Hitachi, Tokyo, Japan) was carried out to survey the integrity and morphological structure of L. monocytogenes and S. aureus after treatment of MO.
(Ramos, Valdés, Beltrán, & Garrigós, 2016). It has been reported that electrospun nanofibers could achieve a long-term efficiency against microbial growth on food when it was utilized as food packaging material (Lin, Gu, Li, Vittayapadung, & Cui, 2018; Lin, Liao, Duraiarasan, & Cui, 2018; Sotelo-Boyás, Correa-Pacheco, Bautista-Baños, & CoronaRangel, 2017). Hence, the coating of cheese surfaces with moringa oil/ chitosan nanoparticles embedded gelatin nanofibers would be a creative strategy to prevent microbial contamination. Based on the above-mentioned reasons, this study aims to increase the stability and prolong action time of moringa oil by loading in chitosan nanoparticles. Afterwards, electrospinning technique was put into use to carry out the application of moringa oil/chitosan nanoparticles in food packaging purposes. The antibacterial activity of the nanofibers against L. monocytogenes and S. aureus on cheese was also investigated, along with surface color and sensory evaluation.
2.4. Preparation and characterization of MO@CNPs 2.4.1. Preparation of MO@CNPs The process of MO@CNPs (MO/chitosan nanoparticles) preparation could be divided into two parts, namely the preparation of oil-in-water (o/w) type emulsion and ionic gelation of chitosan and STPP (Ragelle et al., 2015). In the beginning, MO (concentration at 2MBC) was dissolved in Tween-80 (1:1, v/v). Tween-80, not only serves as a nonionic surfactant for the configuration of o/w type emulsion, but also employs to speed up the dissolving of MO. Different concentrations of chitosan solutions (1.0, 2.0, 3.0, 4.0, 5.0, mg/mL) were ready by dissolving chitosan powder in acetic acid solution (1%, v/v) and stirred for 2 h. Then, the MO/Tween-80 was added dropwise into chitosan solutions to form the coarse emulsion and stirred for 6 h to obtain homogeneous solutions. Subsequently, STPP solutions (0.25, 0.50, 0.75, 1.00, 1.25, mg/mL) were added gradually to MO/chitosan emulsion solutions under stirring continuously at room temperature for 24 h, respectively (Pant & Negi, 2018). The ratio of chitosan and STPP was 4:1. All the homogenous solutions were filtered using a 0.22 μm filter membrane. At last, the self-assembled MO@CNPs were obtained after centrifuging at 6952 × g for 15 min, then suspended in sterile water and stored at 4 °C. Five MO@CNPs solutions were prepared including MO@CNPs-I (1.0 mg/mL chitosan), MO@CNPs-II (2.0 mg/mL chitosan), MO@CNPsIII (3.0 mg/mL chitosan), MO@CNPs-IV (4.0 mg/mL chitosan), MO@CNPs-V (5.0 mg/mL chitosan).
2. Materials and methods 2.1. Materials and bacterial culture The moringa oil (MO) was purchased from J.E International (Caussols Plateau, France). Chitosan (85% deacylated) and sodium tripolyphosphate (STPP) were bought from Sigma-Aldrich Chemical Co. (St. Louis, Mo, USA). Gelatin (type B from porcine skin) was obtained from Delong Glue Co., Ltd. (Shanghai, China). Acetic acid (> 99.7% purity) and Tween 80 were bought from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). The fresh hard cheese (Cheshire cheddar cheese; protein 23.7%, fat 31.3%, moisture content 38.2%) was bought from the Metro supermarket. L. monocytogenes ATCC 19115 and S. aureus ATCC 25923 were bought from China General Microbiological Culture Collection Center (Beijing, China), and stored with liquid paraffin wax at 4 °C. These two strains were cultured with shaking at 37 °C for 48 h.
2.4.2. Particle size, PDI, zeta potential and encapsulation efficiency of MO@CNPs The particle size, polydispersity index (PDI) and zeta potential of MO@CNPs were determined by a dynamic light scattering with a Zetasizer Nano instrument (Nano ZS90, Malvern Instruments, Worcester, UK). Encapsulation efficiency was detected by GC–MS.
2.2. Chemical compositions of MO The chemical compositions of MO were detected by GC–MS (Agilent 6890 N/5973 N, Agilent Technologies, USA) acting in electron-impact ionization (EI) mode with a mass scan range from m/z 33 to 500 at 70 eV A fused silica capillary column (30 m × 0.25 mm × 0.25 μm) was used for separation. And helium was utilized as carrier gas. Temperatures of injector and detector were adjusted to 250 °C and 280 °C, respectively. The initial temperature of the column was 50 °C for 1 min, followed by raising to 200 °C at a rate of 10 °C/min and maintained for 2 min; after that, it was raised to 220 °C at 5 °C/min and held there for 10 min (Saini, Shetty, & Giridhar, 2014). Chemical compositions were identified on the basis of fragmentation patterns and retention times and compared with authentic standards and NIST library.
2.4.3. Fourier transform infrared spectroscopy (FTIR) analysis of MO@CNPs FTIR spectra of MO@CNPs and pure components in a wavenumber of 400-4000 cm−1 were recorded using a Nicolet 6550 FTIR Spectrometer (Thermo, Electron, Massachusetts, US). Each sample was scanned 32 times using the transmission mode. 2.5. Preparation of spinning solution First of all, gelatin (25%, w/v) was dissolved in acetic acid solution (20%, v/v) under magnetic stirring to prepare pure gelatin solution. Subsequently, the MO@CNPs were added into gelatin solutions to obtain different concentrations at 3.0, 6.0, 9.0, 12.0 and 15.0 mg/mL. The gelatin-MO@CNPs spinning solutions were stirred for 6 h to acquire homogeneous solutions and then proceeded to electrospinning process.
2.3. Evaluation of antibacterial activity of MO The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were determined by the broth dilution method. Different concentrations of MO (0.625, 1.25, 2.5, 5, 10 and 20 mg/mL) were prepared in nutrient broth with activated L. monocytogenes and S. aureus (105-6 CFU/mL). All the tubes were incubated at 37 °C for 48 h. MIC was regarded as the lowest concentration of MO that inhibited visible growth. MBC was considered as the lowest concentration of MO that lessens the viability of the initial bacteria inoculum by ≥ 99.9%. Time-kill curve analysis of MO was performed using the plate colony counting method. MO was diluted in phosphate buffer solution (PBS, pH 7.2) containing L. monocytogenes and S. aureus cell suspension (105-6 CFU/mL) to obtain concentrations of 1/2 MIC, MIC, 2 MIC and incubated at 37 °C. The samples without MO were set as control. The
2.6. Preparation of electrospun nanofibers The electrospinning apparatus (SNAN-01, Electrospinning Setup, MECC Co., Ltd., Fukuoka, Japan) was employed to prepare electrospun nanofibers. In the first place, the homogeneous gelatin-MO@CNPs spinning solutions were transferred into a glass syringe with a needle tip of 0.50 mm inner diameter, respectively. The applied voltage was 23.0 kV, the flow rate was turned to 0.2 mL/h. Nanofibers were collected on the aluminized paper (20 cm × 20 cm), which was placed at 15 cm vertical distance to the needle tip (Cui, Bai et al., 2017, 2017b). 87
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2.7. Evaluation of nanofiber physical properties
2.8. Scanning electron microscopy (SEM)
2.7.1. Thickness The thickness of nanofibers was measured by an electronic digital micrometer (Guangdong, China) with the sensitivity of 0.001 mm. At least 10 random spots of nanofibers were chosen for analysis. Obtained data was used for further analysis of water vapor permeability and mechanical properties.
The microstructure of nanofibers was investigated by scanning electron microscopy (SEM, JSM-7001 F, JEOL, Tokyo, Japan). Samples were spraying with gold using a K450X sputter coater (Emitech, England) (Ziuzina, Han, Cullen, & Bourke, 2015). Gold coated samples were visualized on electron microscope in BSE mode. The diameters of the nanofibers were measured by the image analysis software NIS Elements 0.3.
2.7.2. Moisture content To determine the moisture content of nanofibers, nanofibers were weighed after prepared. After that, the nanofibers were drying at 110 °C until a constant weight was acquired. The moisture content (%) was calculated by the following Eq. (1):
Moisture content=
M0 − M1 × 100% M0
2.9. Atomic force microscopy (AFM) The surface roughness of gelatin nanofibers and MO@CNPs nanofibers was surveyed by Atomic Force Microscopy (AFM, Agilent 5500, Agilent Technologies, USA). AFM Images were further analyzed with NanoScope Analysis (version 1.70) and switched into 3-Dimensional images.
(1)
Where M0 represents for the mass of initial sample (mg), M1 is the mass of dried sample (mg) (Aguirreloredo, Rodríguezhernández, Moralessánchez, Gómezaldapa, & Velazquez, 2016).
2.10. Release rate of MO from MO@CNPs nanofibers The release rates of MO from MO@CNPs nanofibers at different temperatures (4 °C and 25 °C) for 60 days were tested by determining the total and free amount of MO. Both MO@CNPs and MO@CNPs nanofibers were dispersed in ethanol and sonicated at 200 W for 2 h and then detected by GC–MS. The date was recorded each 5 days. The release rate was calculated by the following Eq. (6):
2.7.3. Water solubility Nanofibers (2 cm × 2 cm) were drying at 110 °C to obtain constant weight. Followed by, nanofibers were immersed in distilled water for 15 min under shaking condition. Undissolved nanofiber pieces were taken out and dried at 110 °C to achieve a constant weight. The water solubility (%) was calculated by the Eq. (2):
W − W1 Water solubility= 0 × 100% W0
RR(%) = (2)
2.7.4. Water vapor permeability (WVP) Nanofiber samples were fastened on the top of permeation cups containing distilled water. The permeation cups were placed in a drying cabinet to maintain a relative humidity (RH) of 40% and temperature of 25 °C. Afterwards, the cups were weighed per day for 10 days. The WVP was figured out through Eq. (3):
Δm ∙ X A∙ Δt ∙ ΔP
2.11. The application of MO@CNPs nanofibers on cheese Fresh cheese samples (30 mm × 20 mm × 10 mm) were inoculated with L. monocytogenes and S. aureus to obtain the bacterial concentration of approximately 103 CFU/g. MO@CNPs nanofibers and blank aluminized papers were sterilized by UV irradiation for 30 min. Subsequently, the inoculated cheese samples were wrapped with MO@CNPs nanofibers (Fig.1). Inoculated and blank aluminized paper wrapped samples were acted as control. All samples were packaged in sterile bags and stored at 4 °C and 25 °C for 10 days. Microbial analysis was performed by plate-colony counting method (Lin, Gu et al., 2018, 2018b). The cells were cultured in Listeria Chromogenic Medium (Huankai Microbial Technology Co., Ltd. Guangzhou, China) and Staphylococcus Selective Agar (Qingdao Hope Bio-Technology Co., Ltd. Shandong, China) and observed after 24 h.
(3)
Where Δm/ΔtΔm/Δt is weight of moisture loss per unit of time (g/h), X is thickness of nanofiber (mm), A is nanofiber area exposed to moisture transfer (2.0096 × 10−4 m2), ΔPΔP is the differential water vapor partial pressure across the nanofiber (1.753 kPa) (Bounos et al., 2017) 2.7.5. Mechanical properties According to ASTM standard method D882-00 (ASTM, 2001), the tensile strength (TS, MPa) and elongation at break (EAB, %) of nanofibers were measured by TA-XT2i Texture Analyzer (Stable Microsystems, UK). Nanofibers were cut into 20 mm × 100 mm rectangles. The initial separation distance was 30 mm. Tensile load was adjusted at 10 g and rectangles were stretched at a constant speed of 10 mm/s. TS was calculated according to the following Eq. (4):
TS=
F L×X
2.12. Surface color and sensory evaluation Fresh cheese samples (30 mm × 20 mm × 10 mm) without bacteria were wrapped with MO@CNPs nanofibers. Samples wrapped with blank aluminized paper were set as control. All the samples were stored at 4 °C and 25 °C for 4 days. A Chromatic meter (Color Quest XE, HunterLab Co., USA) was employed to measure surface color of cheese samples in darkness. Xenon lamp was used as illuminant. The measuring aperture size of the chromatic meter was 19.0 mm and the standard observer angle was 10°. Results were shown as of L* (luminosity), a* (red-green index) and b* (yellow-blue index). Sensory characteristics of cheese samples include color, smell, taste and overall acceptability. Sensory evaluation was tested by 30 untrained panelists. Cheese quality was evaluated by 7-point hedonic scale from 1 (extremely dislike) to 7 (extremely like).
(4)
Where F is the axial tensile force (N), X is nanofiber thickness (mm), L is the nanofiber width (mm). EAB was calculated as the following Eq. (5):
EAB=
H − H0 × 100% H0
(6)
where RR is the release rate (%), wR (mg/mL) represents the amount of MO released from nanoparticles and nanofibers, and wT (mg/mL) is the total amount of MO in nanoparticles and nanofibers (Cui, Bai, Li, Liu, & Lin, 2018).
Where W0 and W1 stand for the weight of initial and final samples (mg).
WVP=
WR × 100% WT
(5)
Where H0 is the initial separation distance and H is the distance at breakage of nanofibers. 88
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Fig. 1. Schematic of electrospinning for MO@CNPs nanofibers.
the bacteria were incomplete, distorted and became rough after treated by MO as shown in Fig. 2C and F. The cell membrane was stripped from the cytoplasm, resulting in the leakage of cell contents. In brief, the results revealed that MO could cause irreversible destructions in L. monocytogenes and S. aureus. This current finding was consistent with the results demonstrated by Marrufo et al. (2013).
2.13. Statistical analysis All experiments were repeated three times. The results were presented as means ± standard deviation (SD). All values were applied to check statistically differences using SPSS (version 22.0; IBM Corp., Armonk, NY). One-way analysis of variance (ANOVA) and the Bonferroni statistical test were introduced to confirm the level of significance, and p < 0.05 was regarded as significant.
3.3. Characterization of MO@CNPs 3. Results and discussion
3.3.1. Particle size, PDI, zeta potential and encapsulation efficiency of MO@CNPs In order to obtain MO@CNPs with good performance, properties of MO@CNPs with different concentration of chitosan (1.0, 2.0, 3.0, 4.0 and 5.0 mg/mL) were determined and given in Table 2. The initial concentration of MO was 20 mg/mL in each nanoparticle. From MO@CNPs-I to MO@CNPs-V, the average particle size of the five nanoparticles ranged from 94.3 ± 2.1 to 246.1 ± 6.3 nm with the increase of chitosan concentration. It has been reported that nanoparticles with smaller particle size (< 200 nm) could be beneficial to the formation of nanofibers during electrospinning process (Esmaeili & Haseli, 2017). The values of PDI reflect the width of particle size distribution, and low PDI value (< 0.3) demonstrates that the nanoparticles have a uniform and narrow size distribution (Tan et al., 2013). In Table 2, the PDI of MO@CNPs-I, MO@CNPS-II and MO@CNPs-III achieved smaller values, which was 0.139 ± 0.017, 0.162 ± 0.021 and 0.227 ± 0.026, respectively. In addition, the PDI values of MO@CNPsIV and MO@CNPs-V both increased to 0.325 ± 0.031 and 0.432 ± 0.029. The zeta potential is an index indicating the stability of nanoparticles systems. In general, high zeta potential (< -30 mV and > + 30 mV) could maintain a stable system due to the repelling force between particles (Lu et al., 2014). The zeta potential values of MO@CNPs ranged from 17.2 ± 1.5 to 45.1 ± 4.2 mV. The results revealed that MO@CNPs-III to MO@CNPs-V were much stable. In addition, the encapsulation efficiency of MO@CNPs-III was the highest (41.3 ± 0.5%). As a consequence, the nanoparticles with small and uniform particle size, high zeta potential and high encapsulation efficiency could be considered as a stale system. Based on the above-mentioned results, the optimal concentration of chitosan was 3.0 mg/mL, and MO@CNPs-III was chosen for further study.
3.1. Chemical compositions of MO The chemical compositions of MO analyzed by GC–MS were shown in Table 1. Generally, 13 compounds were observed to account for 77.86% of MO composition, including fatty acids, alkanes, aldehydes, ketones, etc. Palmitic acid (38.67%) was the most abundant composition found in MO. Phytol (6.18%) and ethyl palmitic (4.92%) were the second and third major compositions. 3.2. Antibacterial activity of MO In Fig.2A, both MIC and MBC of MO against L. monocytogenes were 10 mg/mL. Meanwhile, the MIC was 5 mg/mL, and MBC was 10 mg/mL for S. aureus (Fig. 2D). The time-kill curve analysis was evaluated and exhibited inhibitory effect against L. monocytogenes and S. aureus with the increase concentration of MO. From the time-kill curve of 2 MIC, population of bacteria decreased constantly and 93.61% (Fig. 2A) and 99.999% (Fig. 2D) reduction were found after MO treated for 8 h. TEM analysis was also demonstrated the satisfactory antibacterial activity of MO. In Fig. 2B and E, the untreated L. monocytogenes and S. aureus appeared with a regular, intact and smooth cell structure. Conversely, Table 1 Chemical compositions of MO. Compound
Proportion (%)
Compound
Proportion (%)
Palmitic acid Phytol Ethyl palmitate Hexadecanal Heptacosane Pentacosane Nonacosane
38.67 6.18 4.92 4.74 4.35 3.86 3.13
Methyl palmitate Ethyl linolenate Methyl linolenate Nonadecane Ethyl linoleate β-Ionone Total
2.93 2.19 2.07 1.89 1.57 1.36 77.86
89
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Fig. 2. (A) Time-kill curve of MO against L. monocytogenes; TEM images of L. monocytogenes before (B) and after (C) treated by MO. (D) Time-kill curve of MO against S. aureus; TEM images of S. aureus before (E) and after (F) treated by MO.
3.3.2. FTIR analysis of MO@CNPs In order to characterize the formation of nanoparticles systems and analyze the interactions between components, the FTIR spectra of MO, chitosan, STPP and MO@CNPs were recorded. As shown in Fig. 3, the absorbance characteristic peaks of MO at wavenumber of 1172 cm−1 and 1713 cm−1 represented skeletal vibration of –C–O–C and –C = O2. In addition, the peaks of MO at 2850 cm−1 and 2923 cm−1 were attributed to the CeH stretching vibration of aliphatic CH2 bonds (Bhutada, Jadhav, Pinjari, Nemade, & Jain, 2016). As for chitosan, the absorbance characteristic peaks at the wavenumber of 2871 cm−1 (eCeH), 1592 cm−1 (eNeH) and 1084 cm−1 (eCeOeC) were observed (Branca et al., 2016). Simultaneously, the characteristic peak of STPP appeared at 912 cm−1 which correspond to the stretching P]O. For MO@CNPs, the peaks at 912 cm−1, 1100 cm-1, 1721 cm-1, 2854 cm1 and 2923 cm-1 were found to be similar with the absorption peaks of MO, chitosan and STPP. The results revealed that MO@CNPs were prepared precisely and entrapped MO successfully. 3.4. Physical properties of MO@CNPs nanofibers The thickness of gelatin nanofibers and MO@CNPs nanofibers was determined to know the effect of loading different concentrations of MO@CNPs in gelatin nanofibers. In Table 3, the thickness of gelatin nanofibers was 0.102 ± 0.004 mm. Compared with gelatin nanofibers, average thickness of MO@CNPs nanofibers ranged from 0.104 ± 0.003 to 0.124 ± 0.003 mm with the proportion of MO@CNPs increased from 3.0 to 15.0 mg/mL. The reason could be attributed to the encapsulation of MO@CNPs in the nanofibers.
Fig. 3. FTIR spectrum of MO, chitosan, STPP and MO@CNPs.
Simultaneously, the moisture content decreased from 16.24 ± 0.12% to 11.02 ± 0.24% and water solubility declined from 88.54 ± 0.17% to 81.59 ± 0.20%. The results demonstrated that the
Table 2 The particle size, PDI, zeta potential and encapsulation efficiency of MO@CNPs. CNPs
MO (mg/mL)
Chitosan (mg/mL)
Particle size (nm)
PDI
MO@CNPs-I MO@CNPs-II MO@CNPs-III MO@CNPs-IV MO@CNPs-V
20 20 20 20 20
1.0 2.0 3.0 4.0 5.0
94.3 ± 2.1 117.5 ± 3.2 143.8 ± 4.6 208.7 ± 4.2 246.1 ± 6.3
0.139 0.162 0.227 0.325 0.432
90
± ± ± ± ±
0.017 0.021 0.026 0.031 0.029
Zeta potential (mV)
Encapsulation efficiency (%)
17.2 25.9 32.5 38.4 45.1
27.5 34.1 41.3 32.8 24.5
± ± ± ± ±
1.5 2.1 1.8 3.6 4.2
± ± ± ± ±
0.6 0.3 0.5 0.9 0.5
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Table 3 The physical properties of MO@CNPs nanofibers. Parameters
MO@CNPs (mg/mL) 0
Thickness (mm) Moisture content (%) Water solubility (%) WVP (g mm/m2 h kPa) Tensile strength (MPa) Elongation at break (%)
3.0 a
0.102 ± 0.004 16.24 ± 0.12a 88.54 ± 0.17a 0.49 ± 0.03a 0.63 ± 0.28a 57.43 ± 2.31a
0.104 ± 0.003 15.74 ± 0.19a 88.06 ± 0.16a 0.46 ± 0.02a 0.76 ± 0.31a 54.57 ± 1.96a
6.0 a
9.0
0.109 ± 0.005 14.87 ± 0.14b 87.21 ± 0.19a 0.42 ± 0.04b 0.98 ± 0.29b 52.18 ± 2.62b
a
0.113 ± 0.002 13.42 ± 0.17c 86.13 ± 0.15b 0.36 ± 0.05c 1.24 ± 0.34c 47.61 ± 2.84c
12.0 b
0.118 ± 0.004 12.29 ± 0.23d 84.87 ± 0.23c 0.28 ± 0.06c 1.13 ± 0.36c 48.92 ± 3.07c
15.0 b
0.124 ± 0.003c 11.02 ± 0.24e 81.59 ± 0.20d 0.21 ± 0.08d 1.02 ± 0.33b 49.47 ± 2.69b
with approximate fiber diameter of 85.7 nm. As shown in Fig. 4D, AFM image of MO@CNPs nanofibers was found to have a smooth surface. Besides, some small bumps were observed on the image, which could be clearly seen in 3-Dimensional image (Fig. 4d). Because of the small bumps, the height of nanofibers achieved about 147 nm. As a result, the small bumps should be the MO@CNPs. The results confirmed further that MO@CNPs were loaded into gelatin nanofibers, which is consist with the result of SEM.
incorporation of MO@CNPs could decrease the hydrophilicity of the nanofibers. Meanwhile, WVP of gelatin nanofibers also decreased from 0.49 ± 0.03 to 0.21 ± 0.08 g mm/m2 h kPa. WVP is a major parameter of nanofibers which related to the moisture transition between the food and environment (Bourlieu, Guillard, Vallès-Pamiès, Guilbert, & Gontard, 2009). Therefore, materials with low WVP are suitable to wrap food products and prolong shelf life. As shown in Table 3, tensile strength (TS, MPa) and elongation at break (EAB, %) of nanofibers were also evaluated. For gelatin nanofiber, TS value was 0.63 ± 0.28 MPa and EAB value was 57.43 ± 2.31%. The results demonstrated that gelatin nanofiber was less resistant to breakage (TS) and more deformable (EAB). With increasing the concentration of MO@CNPs, the TS values of nanofibers increased first and then decreased. The maximum TS value was 1.24 ± 0.34 MPa when the MO@CNPs concentration was 9.0 mg/mL. As a result, the addition of MO@CNPs could enhance the tensile strength of nanofibers to some degree, and higher proportion may be not always improving tensile strength. Based on the above analysis, the 9 mg/mL MO@CNPs in gelatin nanofibers was chosen for further experiments.
3.6. Release rate of MO from MO@CNPs nanofibers The release rate of MO from MO@CNPs and MO@CNPs nanofibers were investigated during 60 days storage at 4 °C (Fig. 5A) and 25 °C (Fig. 5B). The total release rate of MO from MO@CNPs reached 31.73% and 54.57% at 4 °C and 25 °C, respectively. It is obvious that the release rate of MO increased with the increase of temperature. Moreover, the release rate of MO from MO@CNPs nanofibers was 19.61% and 35.19% under different temperature. The release efficiency was lower than from MO@CNPs because of the encapsulation of gelatin nanofibers. That is to say that the electrospun nanofibers could delay the release the essential oil and extend the action time of MO@CNPs. The finding was agreement with the results of Cui et al. (2018) that the release rate of clove oil from nanofibers was reduced.
3.5. Morphology and surface characterizations by SEM and AFM SEM images of gelatin nanofibers and MO@CNPs nanofibers were presented in Fig. 4. Generally, the SEM images of gelatin nanofibers (Fig. 4A) and MO@CNPs nanofibers (Fig. 4B) were smooth and stable and showed no obvious differences. However, the average diameter of the gelatin nanofibers was 92 nm (Fig. 4a) and MO@CNPs (Fig. 4b) was 142.5 nm. The increase of average diameter indicated that MO@CNPs were loaded in gelatin nanofibers successfully. The surface and 3-Dimensional AFM images of nanofibers were given in Fig. 4. In Fig. 4C, the gelatin nanofibers had a smooth surface
3.7. The application of MO@CNPs nanofibers on cheese The application of MO@CNPs nanofibers against L. monocytogenes and S. aureus on cheese under 4 °C and 25 °C was evaluated and the results were given in Fig. 6A-D. In general, bacteria on cheese stored at 4 °C grew slower than those on cheese at 25 °C. The population of L. monocytogenes and S. aureus in control group at 25 °C increased dramatically, achieving 7.82 Log CFU/g (Fig. 6B) and 7.58 Log CFU/g
Fig. 4. SEM images and fiber diameter distributions with average diameters of gelatin nanofibers (A), (a) and MO@CNPs nanofibers (B), (b); Surface and 3Dimensional AFM images of gelatin nanofibers (C), (c) and MO@CNPs nanofibers (D), (d). 91
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Fig. 5. The release rate of MO from MO@CNPs nanofibers stored at 4 °C (A) and 25 °C (B) for 60 days.
values between control samples and MO@CNPs nanofibers wrapped samples at 4 °C after 4-days storage. However, the surface color of cheese samples in control group decreased significantly at 25 °C for 4 days while the MO@CNPs nanofibers wrapped samples showed minor undulation. Similarly, the sensory attributes of cheese samples were almost unaffected after MO@CNPs nanofibers wrapped at 4 °C and 25 °C for 4 days. Therefore, MO@CNPs nanofibers can not only prolong the shelf life of cheese, but also keep its sensory quality.
(Fig. 6D), respectively. Conversely, the population of L. monocytogenes and S. aureus only reached 3.11 and 2.2 Log CFU/g at 25 °C, which could be attributed to the addition of MO@CNPs in the nanofibers. The similar results were also found in the samples wrapped by MO@CNPs nanofibers at 4 °C. The population reduction of MO@CNPs nanofibers treated samples reached 78.63% (Fig. 6A) and 98.67% (Fig. 6C) for 10 days storage at 4 °C. These results demonstrated that MO@CNPs nanofibers exhibited excellent antibacterial activity against L. monocytogenes and S. aureus on cheese. The finding of the current study was consistent with the results found by Wen et al. (2016) which showed that polyvinyl alcohol/cinnamon essential oil/β-cyclodextrin nanofibers has satisfactory antibacterial activity and extend shelf life of strawberry. Hence, MO@CNPs-embedded gelatin nanofibers will have a broad prospect for food packaging and preservation.
4. Conclusion In order to improve the stability and prolong action time of MO, MO was loaded into chitosan nanoparticles. The particle size, PDI, zeta potential and encapsulation efficiency of the optimal MO@CNPs were 143.8 ± 4.6 nm, 0.227 ± 0.026, 32.5 ± 1.8 mV and 41.3 ± 0.5%, respectively. The MO@CNPs nanofibers were fabricated successfully with MO@CNPs at 9.0 mg/mL. The nanofibers exhibited excellent physicochemical properties. Besides, the nanofibers possessed high antibacterial effect against L. monocytogenes and S. aureus on cheese at 4 °C and 25 °C and negligible impact on the surface color and sensory
3.8. Surface color and sensory evaluation The surface color values and sensory evaluation of cheese samples wrapped by MO@CNPs nanofibers stored at 4 °C and 25 °C were presented in Table 4. No significant differences were found in L*, a* and b*
Fig. 6. Antibacterial activity of MO@CNPs nanofibers against L. monocytogenes at 4 °C (A) and 25 °C (B). Antibacterial activity of MO@CNPs nanofibers against S. aureus at 4 °C (C) and 25 °C (D). 92
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Table 4 Effect of MO@CNPs nanofibers on the quality of cheese after 4-days storage at 4 °C and 25 °C. Temperature 4 °C
25 °C
Parameters L* a* b* Color Smell Taste Overall acceptability L* a* b* Color Smell Taste Overall acceptability
Fresh cheese (0 d) a
72.75 ± 0.12 0.78 ± 0.09a 20.15 ± 0.16a 3.52 ± 0.08a 3.46 ± 0.08a 3.41 ± 0.11a 3.47 ± 0.13a 72.51 ± 0.15a 0.79 ± 0.12a 19.75 ± 0.14a 3.41 ± 0.10a 3.46 ± 0.12a 3.48 ± 0.13a 3.45 ± 0.15a
quality of cheese during 4 days storage. Hence, the MO@CNPs embedded gelatin nanofiber was a promising active food packaging material for further food application.
Control (4 d)
MO@CNPs nanofiber (4 d) a
70.57 ± 0.17 0.75 ± 0.15b 18.52 ± 0.21a 3.46 ± 0.13a 3.41 ± 0.14a 3.40 ± 0.19a 3.42 ± 0.17a 67.31 ± 0.21b 0.73 ± 0.18b 17.19 ± 0.19b 3.31 ± 0.13b 3.29 ± 0.11b 3.27 ± 0.14b 3.28 ± 0.16b
71.15 ± 0.14a 0.76 ± 0.12a 19.07 ± 0.15a 3.49 ± 0.12a 3.45 ± 0.11a 3.43 ± 0.17a 3.46 ± 0.15a 70. 23 ± 0.12a 0.76 ± 0.14a 19.32 ± 0.18a 3. 38 ± 0.08a 3.41 ± 0.09a 3.42 ± 0.15a 3.40 ± 0.13a
on biofilms formed by Staphylococcus aureus and Listeria monocytogenes isolated from dairy plants. Journal of Dairy Science, 99, 1–7. Leone, A., Spada, A., Battezzati, A., Schiraldi, A., Aristil, J., & Bertoli, S. (2016). Moringa oleifera seeds and oil: Characteristics and uses for human health. International Journal of Molecular Sciences, 17, 2141–2155. Liang, J., Yan, H., Puligundla, P., Gao, X., Zhou, Y., & Wan, X. (2017). Applications of chitosan nanoparticles to enhance absorption and bioavailability of tea polyphenols: A review. Food Hydrocolloids, 69, 286–292. Lin, L., Dai, Y., & Cui, H. (2017). Antibacterial poly(ethylene oxide) electrospun nanofibers containing cinnamon essential oil/beta-cyclodextrin proteoliposomes. Carbohydrate Polymers, 178, 131–140. Lin, L., Gu, Y., Li, C., Vittayapadung, S., & Cui, H. (2018). Antibacterial mechanism of εpoly-lysine against Listeria monocytogenes and its application on cheese. Food Control, 91, 76–84. Lin, L., Liao, X., Duraiarasan, S., & Cui, H. (2018). Preparation of ε-polylysine/chitosan nanofibers for food packaging against Salmonella on chicken. Food Packaging and Shelf Life, 17, 134–141. Lu, Q., Lu, P. M., Piao, J. H., Xu, X. L., Chen, J., Zhu, L., et al. (2014). Preparation and physicochemical characteristics of an allicin nanoliposome and its release behavior. LWT-Food Science and Technology, 57, 686–695. Marrufo, T., Nazzaro, F., Mancini, E., Fratianni, F., Coppola, R., De, M. L., et al. (2013). Chemical composition and biological activity of the essential oil from leaves of Moringa oleifera Lam cultivated in Mozambique. Molecules, 18, 10989–11000. Muhteremuyar, M., Ciolacu, L., Wagner, K. H., Wagner, M., Schmitzesser, S., & Stessl, B. (2018). New aspects on Listeria monocytogenes ST5-ECVI predominance in a heavily contaminated cheese processing environment. Frontiers in Microbiology, 9, 1–14. Pant, A., & Negi, J. S. (2018). Novel controlled ionic gelation strategy for chitosan nanoparticles preparation using TPP-β-CD inclusion complex. European Journal of Pharmaceutical Sciences, 112, 180–185. Ragelle, H., Colombo, S., Pourcelle, V., Vanvarenberg, K., Vandermeulen, G., Bouzin, C., et al. (2015). Intracellular siRNA delivery dynamics of integrin-targeted, PEGylated chitosan-poly(ethylene imine) hybrid nanoparticles: A mechanistic insight. Journal of Controlled Release, 211, 1–9. Ramos, M., Valdés, A., Beltrán, A., & Garrigós, M. (2016). Gelatin-based films and coatings for food packaging applications. Coatings, 6, 41–61. Saini, R. K., Shetty, N. P., & Giridhar, P. (2014). GC-FID/MS analysis of fatty acids in Indian cultivars of Moringa oleifera: Potential sources of PUFA. Journal of the American Oil Chemists Society, 91, 1029–1034. Singh, Y., Singla, A., Upadhyay, A., & Singh, A. K. (2017). Sustainability of moringa-oil based biodiesel blended lubricant. Energy Sources, 39, 313–319. Sotelo-Boyás, M. E., Correa-Pacheco, Z. N., Bautista-Baños, S., & Corona-Rangel, M. L. (2017). Physicochemical characterization of chitosan nanoparticles and nanocapsules incorporated with lime essential oil and their antibacterial activity against foodborne pathogens. LWT - Food Science and Technology, 77, 15–20. Tan, C., Xia, S., Xue, J., Xie, J., Feng, B., & Zhang, X. (2013). Liposomes as vehicles for lutein: Preparation, stability, liposomal membrane dynamics, and structure. Journal of Agricultural & Food Chemistry, 61, 8175–8184. Wen, P., Zhu, D. H., Wu, H., Zong, M. H., Jing, Y. R., & Han, S. Y. (2016). Encapsulation of cinnamon essential oil in electrospun nanofibrous film for active food packaging. Food Control, 59, 366–376. Zhong, J., Wang, Y., Yang, R., Liu, X., Yang, Q., & Qin, X. (2018). The application of ultrasound and microwave to increase oil extraction from Moringa oleifera seeds. Industrial Crops & Products, 120, 1–10. Ziuzina, D., Han, L., Cullen, P. J., & Bourke, P. (2015). Cold plasma inactivation of internalised bacteria and biofilms for Salmonella enterica serovar Typhimurium, Listeria monocytogenes and Escherichia coli. International Journal of Food Microbiology, 210, 53–61.
Acknowledgements This research project was financially supported by Natural Science Foundation of Jiangsu Province (Grant no. BK20170070), Jiangsu Province Foundation for talents of six key industries (Grant no. NY-013) and Jiangsu University Research Fund (Grant no. 11JDG050). References Aguirreloredo, R. Y., Rodríguezhernández, A. I., Moralessánchez, E., Gómezaldapa, C. A., & Velazquez, G. (2016). Effect of equilibrium moisture content on barrier, mechanical and thermal properties of chitosan films. Food Chemistry, 196, 560–566. ASTM (2001). D882-00 standard test methods for tensile properties of thin plastic sheeting. Annual book of ASTM standards. Philadelphia: American Society for Testing and Materials. Bhutada, P. R., Jadhav, A. J., Pinjari, D. V., Nemade, P. R., & Jain, R. D. (2016). Solvent assisted extraction of oil from Moringa oleifera Lam seeds. Industrial Crops & Products, 82, 74–80. Bounos, G., Andrikopoulos, K. S., Moschopoulou, H., Lainioti, G. C., Roilo, D., Checchetto, R., et al. (2017). Enhancing water vapor permeability in mixed matrix polypropylene membranes through carbon nanotubes dispersion. Journal of Membrane Science, 524, 576–584. Bourlieu, C., Guillard, V., Vallès-Pamiès, B., Guilbert, S., & Gontard, N. (2009). Edible moisture barriers: How to assess of their potential and limits in food products shelflife extension. Critical Reviews in Food Science and Nutrition, 49, 474–499. Branca, C., D’Angelo, G., Crupi, C., Khouzami, K., Rifici, S., Ruello, G., et al. (2016). Role of the OH and NH vibrational groups in polysaccharide-nanocomposite interactions: A FTIR-ATR study on chitosan and chitosan/clay films. Polymer, 99, 614–622. Cui, H., Bai, M., Li, C., Liu, R., & Lin, L. (2018). Fabrication of chitosan nanofibers containing tea tree oil liposomes against Salmonella spp. in chicken. LWT - Food Science and Technology, 96, 671–678. Cui, H., Ma, C., Li, C., & Lin, L. (2016). Enhancing the antibacterial activity of thyme oil against Salmonella on eggshell by plasma-assisted process. Food Control, 70, 183–190. Cui, H., Bai, M., Rashed, M., & Lin, L. (2017). The antibacterial activity of clove oil/ chitosan nanoparticles embedded gelatin nanofibers against Escherichia coli O157: H7 biofilms on cucumber. International Journal of Food Microbiology, 266, 69–78. Cui, H., Yuan, L., Li, W., & Lin, L. (2017). Antioxidant property of SiO2-eugenol liposome loaded nanofibrous membranes on beef. Food Packaging & Shelf Life, 11, 49–57. Elgadir, M. A., Uddin, M. S., Ferdosh, S., Adam, A., Chowdhury, A. J. K., & Sarker, M. Z. I. (2015). Impact of chitosan composites and chitosan nanoparticle composites on various drug delivery systems: A review. Journal of Food & Drug Analysis, 23, 619–629. Esmaeili, A., & Haseli, M. (2017). Electrospinning of thermoplastic carboxymethyl cellulose/poly(ethylene oxide) nanofibers for use in drug-release systems. Materials Science & Engineering C, 77, 1117–1127. Felicio, T. L., Esmerino, E. A., Vidal, V. A. S., Cappato, L. P., Garcia, R. K. A., Cavalcanti, R. N., et al. (2016). Physico-chemical changes during storage and sensory acceptance of low sodium probiotic Minas cheese added with arginine. Food Chemistry, 196, 628–637. Lee, S., Cappato, L., Corassin, C., Cruz, A., & Oliveira, C. (2015). Effect of peracetic acid
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Moringa oil/chitosan nanoparticles embedded gelatin nanofibers for food packaging against Listeria monocytogenes and Staphylococcus aureus on cheese Lin Lin, Yulei Gu, Haiying Cui
T
⁎
School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Moringa oil Chitosan nanoparticles Electrospun nanofibers Cheese Food packaging
The current study aims to prepare moringa oil-loaded chitosan nanoparticles (MO@CNPs) and fabricate MO@CNPs embedded gelatin nanofibers for biocontrol of Listeria monocytogenes and Staphylococcus aureus on cheese. The optimal MO@CNPs were prepared by the ionic crosslinking method with the concentration of moringa oil at 20 mg/mL and chitosan at 3.0 mg/mL. The nanoparticle exhibited desirable particle size, PDI and zeta potential. Furthermore, the optimal concentration of MO@CNPs embedded in gelatin nanofibers was found to be 9.0 mg/mL after the determination of nanofiber physical properties. The results of SEM and AFM confirmed that the nanofibers were prepared successfully and achieved uniform diameter at 142.5 nm. The release rate of moringa oil from the nanoparticles declined due to the encapsulation of nanofibers. For the application on cheese, MO@CNPs nanofibers possessed high antibacterial activity against L. monocytogenes and S. aureus at 4 °C and 25 °C for 10 days, without any effect on the sensory quality of cheese. As a result, MO@CNPs nanofibers could be a promising active food packaging material for food preservation.
1. Introduction Cheese is particularly popular in our daily life due to its intrinsic nutritional ingredients, such as proteins, fat, vitamins and inorganic salts (Felicio et al., 2016). Nevertheless, cheese has been found to be susceptible to pathogenic bacterial growth whether in processing or storage stages. The pathogenic microorganisms including Listeria monocytogenes (L. monocytogenes) and Staphylococcus aureus (S. aureus) have received great attention in food industry (Lee, Cappato, Corassin, Cruz, & Oliveira, 2015). These two pathogenic bacteria could be transferred to human and bring about a high risk for immunocompromised people, pregnant women, newborns and the elderly (Muhteremuyar et al., 2018). In order to minimize the incidence of disease caused by L. monocytogenes and S. aureus, natural antibacterial agents have been utilized in food industry (Cui, Ma, Li, & Lin, 2016). Essential oils (EOs) have captured increasing attention because of their safety and high efficiency properties. Moringa oil is extracted from Moringa oleifera which is a fast-growing softwood tree found in the Middle East areas, African and Asian countries (Leone et al., 2016). Recent studies displayed that moringa oil became popular because of its healthful properties, excellent oxidative stability and high antimicrobial activity (Singh,
⁎
Singla, Upadhyay, & Singh, 2017; Zhong et al., 2018). Unfortunately, moringa oil is hard to inhibit the growth of bacteria for a long period on account of its volatility and sensitivity to light, air and high temperature (Cui, Bai, Rashed, & Lin, 2017; Cui, Yuan, Li, & Lin, 2017). Therefore, for increasing stability and prolonging action time of moringa oil, nanoencapsulation technology was employed to embed moringa oil and prevent loss of active compositions. Chitosan, a polysaccharide obtained by partial deacetylation of chitin, has been extensively used as drug-loaded nanoparticles on account of its biological intermiscibility and biodegradable properties (Liang et al., 2017). Furthermore, chitosan nanoparticles have special strengths, including slow and controlled drug release rate, enhanced drug absorption and minimal drug side effects (Elgadir et al., 2015). However, the spraying of nanoparticles on the surface of food would lead to diffusion and instability. In consequence, the electrospun nanofibers have been introduced to immobilize chitosan nanoparticles on the surface of food. Electrospinning, an economic and promising technique, has been applied to fabricate nanofibers with large specific surface area, high mechanical strength and high drug encapsulation efficiency (Lin, Dai, & Cui, 2017). In food fields, gelatin-based nanofibers have been widely employed due to its biodegradability, nontoxicity and bioactivity
Corresponding author. E-mail address: [email protected] (H. Cui).
https://doi.org/10.1016/j.fpsl.2018.12.005 Received 10 August 2018; Received in revised form 22 November 2018; Accepted 13 December 2018 2214-2894/ © 2018 Elsevier Ltd. All rights reserved.
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number of residual bacteria was determined at 0, 0.5, 1, 2, 4 and 8 h. Transmission Electron Microscopy (TEM) (Model-JEM-2010HR, Hitachi, Tokyo, Japan) was carried out to survey the integrity and morphological structure of L. monocytogenes and S. aureus after treatment of MO.
(Ramos, Valdés, Beltrán, & Garrigós, 2016). It has been reported that electrospun nanofibers could achieve a long-term efficiency against microbial growth on food when it was utilized as food packaging material (Lin, Gu, Li, Vittayapadung, & Cui, 2018; Lin, Liao, Duraiarasan, & Cui, 2018; Sotelo-Boyás, Correa-Pacheco, Bautista-Baños, & CoronaRangel, 2017). Hence, the coating of cheese surfaces with moringa oil/ chitosan nanoparticles embedded gelatin nanofibers would be a creative strategy to prevent microbial contamination. Based on the above-mentioned reasons, this study aims to increase the stability and prolong action time of moringa oil by loading in chitosan nanoparticles. Afterwards, electrospinning technique was put into use to carry out the application of moringa oil/chitosan nanoparticles in food packaging purposes. The antibacterial activity of the nanofibers against L. monocytogenes and S. aureus on cheese was also investigated, along with surface color and sensory evaluation.
2.4. Preparation and characterization of MO@CNPs 2.4.1. Preparation of MO@CNPs The process of MO@CNPs (MO/chitosan nanoparticles) preparation could be divided into two parts, namely the preparation of oil-in-water (o/w) type emulsion and ionic gelation of chitosan and STPP (Ragelle et al., 2015). In the beginning, MO (concentration at 2MBC) was dissolved in Tween-80 (1:1, v/v). Tween-80, not only serves as a nonionic surfactant for the configuration of o/w type emulsion, but also employs to speed up the dissolving of MO. Different concentrations of chitosan solutions (1.0, 2.0, 3.0, 4.0, 5.0, mg/mL) were ready by dissolving chitosan powder in acetic acid solution (1%, v/v) and stirred for 2 h. Then, the MO/Tween-80 was added dropwise into chitosan solutions to form the coarse emulsion and stirred for 6 h to obtain homogeneous solutions. Subsequently, STPP solutions (0.25, 0.50, 0.75, 1.00, 1.25, mg/mL) were added gradually to MO/chitosan emulsion solutions under stirring continuously at room temperature for 24 h, respectively (Pant & Negi, 2018). The ratio of chitosan and STPP was 4:1. All the homogenous solutions were filtered using a 0.22 μm filter membrane. At last, the self-assembled MO@CNPs were obtained after centrifuging at 6952 × g for 15 min, then suspended in sterile water and stored at 4 °C. Five MO@CNPs solutions were prepared including MO@CNPs-I (1.0 mg/mL chitosan), MO@CNPs-II (2.0 mg/mL chitosan), MO@CNPsIII (3.0 mg/mL chitosan), MO@CNPs-IV (4.0 mg/mL chitosan), MO@CNPs-V (5.0 mg/mL chitosan).
2. Materials and methods 2.1. Materials and bacterial culture The moringa oil (MO) was purchased from J.E International (Caussols Plateau, France). Chitosan (85% deacylated) and sodium tripolyphosphate (STPP) were bought from Sigma-Aldrich Chemical Co. (St. Louis, Mo, USA). Gelatin (type B from porcine skin) was obtained from Delong Glue Co., Ltd. (Shanghai, China). Acetic acid (> 99.7% purity) and Tween 80 were bought from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). The fresh hard cheese (Cheshire cheddar cheese; protein 23.7%, fat 31.3%, moisture content 38.2%) was bought from the Metro supermarket. L. monocytogenes ATCC 19115 and S. aureus ATCC 25923 were bought from China General Microbiological Culture Collection Center (Beijing, China), and stored with liquid paraffin wax at 4 °C. These two strains were cultured with shaking at 37 °C for 48 h.
2.4.2. Particle size, PDI, zeta potential and encapsulation efficiency of MO@CNPs The particle size, polydispersity index (PDI) and zeta potential of MO@CNPs were determined by a dynamic light scattering with a Zetasizer Nano instrument (Nano ZS90, Malvern Instruments, Worcester, UK). Encapsulation efficiency was detected by GC–MS.
2.2. Chemical compositions of MO The chemical compositions of MO were detected by GC–MS (Agilent 6890 N/5973 N, Agilent Technologies, USA) acting in electron-impact ionization (EI) mode with a mass scan range from m/z 33 to 500 at 70 eV A fused silica capillary column (30 m × 0.25 mm × 0.25 μm) was used for separation. And helium was utilized as carrier gas. Temperatures of injector and detector were adjusted to 250 °C and 280 °C, respectively. The initial temperature of the column was 50 °C for 1 min, followed by raising to 200 °C at a rate of 10 °C/min and maintained for 2 min; after that, it was raised to 220 °C at 5 °C/min and held there for 10 min (Saini, Shetty, & Giridhar, 2014). Chemical compositions were identified on the basis of fragmentation patterns and retention times and compared with authentic standards and NIST library.
2.4.3. Fourier transform infrared spectroscopy (FTIR) analysis of MO@CNPs FTIR spectra of MO@CNPs and pure components in a wavenumber of 400-4000 cm−1 were recorded using a Nicolet 6550 FTIR Spectrometer (Thermo, Electron, Massachusetts, US). Each sample was scanned 32 times using the transmission mode. 2.5. Preparation of spinning solution First of all, gelatin (25%, w/v) was dissolved in acetic acid solution (20%, v/v) under magnetic stirring to prepare pure gelatin solution. Subsequently, the MO@CNPs were added into gelatin solutions to obtain different concentrations at 3.0, 6.0, 9.0, 12.0 and 15.0 mg/mL. The gelatin-MO@CNPs spinning solutions were stirred for 6 h to acquire homogeneous solutions and then proceeded to electrospinning process.
2.3. Evaluation of antibacterial activity of MO The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were determined by the broth dilution method. Different concentrations of MO (0.625, 1.25, 2.5, 5, 10 and 20 mg/mL) were prepared in nutrient broth with activated L. monocytogenes and S. aureus (105-6 CFU/mL). All the tubes were incubated at 37 °C for 48 h. MIC was regarded as the lowest concentration of MO that inhibited visible growth. MBC was considered as the lowest concentration of MO that lessens the viability of the initial bacteria inoculum by ≥ 99.9%. Time-kill curve analysis of MO was performed using the plate colony counting method. MO was diluted in phosphate buffer solution (PBS, pH 7.2) containing L. monocytogenes and S. aureus cell suspension (105-6 CFU/mL) to obtain concentrations of 1/2 MIC, MIC, 2 MIC and incubated at 37 °C. The samples without MO were set as control. The
2.6. Preparation of electrospun nanofibers The electrospinning apparatus (SNAN-01, Electrospinning Setup, MECC Co., Ltd., Fukuoka, Japan) was employed to prepare electrospun nanofibers. In the first place, the homogeneous gelatin-MO@CNPs spinning solutions were transferred into a glass syringe with a needle tip of 0.50 mm inner diameter, respectively. The applied voltage was 23.0 kV, the flow rate was turned to 0.2 mL/h. Nanofibers were collected on the aluminized paper (20 cm × 20 cm), which was placed at 15 cm vertical distance to the needle tip (Cui, Bai et al., 2017, 2017b). 87
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2.7. Evaluation of nanofiber physical properties
2.8. Scanning electron microscopy (SEM)
2.7.1. Thickness The thickness of nanofibers was measured by an electronic digital micrometer (Guangdong, China) with the sensitivity of 0.001 mm. At least 10 random spots of nanofibers were chosen for analysis. Obtained data was used for further analysis of water vapor permeability and mechanical properties.
The microstructure of nanofibers was investigated by scanning electron microscopy (SEM, JSM-7001 F, JEOL, Tokyo, Japan). Samples were spraying with gold using a K450X sputter coater (Emitech, England) (Ziuzina, Han, Cullen, & Bourke, 2015). Gold coated samples were visualized on electron microscope in BSE mode. The diameters of the nanofibers were measured by the image analysis software NIS Elements 0.3.
2.7.2. Moisture content To determine the moisture content of nanofibers, nanofibers were weighed after prepared. After that, the nanofibers were drying at 110 °C until a constant weight was acquired. The moisture content (%) was calculated by the following Eq. (1):
Moisture content=
M0 − M1 × 100% M0
2.9. Atomic force microscopy (AFM) The surface roughness of gelatin nanofibers and MO@CNPs nanofibers was surveyed by Atomic Force Microscopy (AFM, Agilent 5500, Agilent Technologies, USA). AFM Images were further analyzed with NanoScope Analysis (version 1.70) and switched into 3-Dimensional images.
(1)
Where M0 represents for the mass of initial sample (mg), M1 is the mass of dried sample (mg) (Aguirreloredo, Rodríguezhernández, Moralessánchez, Gómezaldapa, & Velazquez, 2016).
2.10. Release rate of MO from MO@CNPs nanofibers The release rates of MO from MO@CNPs nanofibers at different temperatures (4 °C and 25 °C) for 60 days were tested by determining the total and free amount of MO. Both MO@CNPs and MO@CNPs nanofibers were dispersed in ethanol and sonicated at 200 W for 2 h and then detected by GC–MS. The date was recorded each 5 days. The release rate was calculated by the following Eq. (6):
2.7.3. Water solubility Nanofibers (2 cm × 2 cm) were drying at 110 °C to obtain constant weight. Followed by, nanofibers were immersed in distilled water for 15 min under shaking condition. Undissolved nanofiber pieces were taken out and dried at 110 °C to achieve a constant weight. The water solubility (%) was calculated by the Eq. (2):
W − W1 Water solubility= 0 × 100% W0
RR(%) = (2)
2.7.4. Water vapor permeability (WVP) Nanofiber samples were fastened on the top of permeation cups containing distilled water. The permeation cups were placed in a drying cabinet to maintain a relative humidity (RH) of 40% and temperature of 25 °C. Afterwards, the cups were weighed per day for 10 days. The WVP was figured out through Eq. (3):
Δm ∙ X A∙ Δt ∙ ΔP
2.11. The application of MO@CNPs nanofibers on cheese Fresh cheese samples (30 mm × 20 mm × 10 mm) were inoculated with L. monocytogenes and S. aureus to obtain the bacterial concentration of approximately 103 CFU/g. MO@CNPs nanofibers and blank aluminized papers were sterilized by UV irradiation for 30 min. Subsequently, the inoculated cheese samples were wrapped with MO@CNPs nanofibers (Fig.1). Inoculated and blank aluminized paper wrapped samples were acted as control. All samples were packaged in sterile bags and stored at 4 °C and 25 °C for 10 days. Microbial analysis was performed by plate-colony counting method (Lin, Gu et al., 2018, 2018b). The cells were cultured in Listeria Chromogenic Medium (Huankai Microbial Technology Co., Ltd. Guangzhou, China) and Staphylococcus Selective Agar (Qingdao Hope Bio-Technology Co., Ltd. Shandong, China) and observed after 24 h.
(3)
Where Δm/ΔtΔm/Δt is weight of moisture loss per unit of time (g/h), X is thickness of nanofiber (mm), A is nanofiber area exposed to moisture transfer (2.0096 × 10−4 m2), ΔPΔP is the differential water vapor partial pressure across the nanofiber (1.753 kPa) (Bounos et al., 2017) 2.7.5. Mechanical properties According to ASTM standard method D882-00 (ASTM, 2001), the tensile strength (TS, MPa) and elongation at break (EAB, %) of nanofibers were measured by TA-XT2i Texture Analyzer (Stable Microsystems, UK). Nanofibers were cut into 20 mm × 100 mm rectangles. The initial separation distance was 30 mm. Tensile load was adjusted at 10 g and rectangles were stretched at a constant speed of 10 mm/s. TS was calculated according to the following Eq. (4):
TS=
F L×X
2.12. Surface color and sensory evaluation Fresh cheese samples (30 mm × 20 mm × 10 mm) without bacteria were wrapped with MO@CNPs nanofibers. Samples wrapped with blank aluminized paper were set as control. All the samples were stored at 4 °C and 25 °C for 4 days. A Chromatic meter (Color Quest XE, HunterLab Co., USA) was employed to measure surface color of cheese samples in darkness. Xenon lamp was used as illuminant. The measuring aperture size of the chromatic meter was 19.0 mm and the standard observer angle was 10°. Results were shown as of L* (luminosity), a* (red-green index) and b* (yellow-blue index). Sensory characteristics of cheese samples include color, smell, taste and overall acceptability. Sensory evaluation was tested by 30 untrained panelists. Cheese quality was evaluated by 7-point hedonic scale from 1 (extremely dislike) to 7 (extremely like).
(4)
Where F is the axial tensile force (N), X is nanofiber thickness (mm), L is the nanofiber width (mm). EAB was calculated as the following Eq. (5):
EAB=
H − H0 × 100% H0
(6)
where RR is the release rate (%), wR (mg/mL) represents the amount of MO released from nanoparticles and nanofibers, and wT (mg/mL) is the total amount of MO in nanoparticles and nanofibers (Cui, Bai, Li, Liu, & Lin, 2018).
Where W0 and W1 stand for the weight of initial and final samples (mg).
WVP=
WR × 100% WT
(5)
Where H0 is the initial separation distance and H is the distance at breakage of nanofibers. 88
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Fig. 1. Schematic of electrospinning for MO@CNPs nanofibers.
the bacteria were incomplete, distorted and became rough after treated by MO as shown in Fig. 2C and F. The cell membrane was stripped from the cytoplasm, resulting in the leakage of cell contents. In brief, the results revealed that MO could cause irreversible destructions in L. monocytogenes and S. aureus. This current finding was consistent with the results demonstrated by Marrufo et al. (2013).
2.13. Statistical analysis All experiments were repeated three times. The results were presented as means ± standard deviation (SD). All values were applied to check statistically differences using SPSS (version 22.0; IBM Corp., Armonk, NY). One-way analysis of variance (ANOVA) and the Bonferroni statistical test were introduced to confirm the level of significance, and p < 0.05 was regarded as significant.
3.3. Characterization of MO@CNPs 3. Results and discussion
3.3.1. Particle size, PDI, zeta potential and encapsulation efficiency of MO@CNPs In order to obtain MO@CNPs with good performance, properties of MO@CNPs with different concentration of chitosan (1.0, 2.0, 3.0, 4.0 and 5.0 mg/mL) were determined and given in Table 2. The initial concentration of MO was 20 mg/mL in each nanoparticle. From MO@CNPs-I to MO@CNPs-V, the average particle size of the five nanoparticles ranged from 94.3 ± 2.1 to 246.1 ± 6.3 nm with the increase of chitosan concentration. It has been reported that nanoparticles with smaller particle size (< 200 nm) could be beneficial to the formation of nanofibers during electrospinning process (Esmaeili & Haseli, 2017). The values of PDI reflect the width of particle size distribution, and low PDI value (< 0.3) demonstrates that the nanoparticles have a uniform and narrow size distribution (Tan et al., 2013). In Table 2, the PDI of MO@CNPs-I, MO@CNPS-II and MO@CNPs-III achieved smaller values, which was 0.139 ± 0.017, 0.162 ± 0.021 and 0.227 ± 0.026, respectively. In addition, the PDI values of MO@CNPsIV and MO@CNPs-V both increased to 0.325 ± 0.031 and 0.432 ± 0.029. The zeta potential is an index indicating the stability of nanoparticles systems. In general, high zeta potential (< -30 mV and > + 30 mV) could maintain a stable system due to the repelling force between particles (Lu et al., 2014). The zeta potential values of MO@CNPs ranged from 17.2 ± 1.5 to 45.1 ± 4.2 mV. The results revealed that MO@CNPs-III to MO@CNPs-V were much stable. In addition, the encapsulation efficiency of MO@CNPs-III was the highest (41.3 ± 0.5%). As a consequence, the nanoparticles with small and uniform particle size, high zeta potential and high encapsulation efficiency could be considered as a stale system. Based on the above-mentioned results, the optimal concentration of chitosan was 3.0 mg/mL, and MO@CNPs-III was chosen for further study.
3.1. Chemical compositions of MO The chemical compositions of MO analyzed by GC–MS were shown in Table 1. Generally, 13 compounds were observed to account for 77.86% of MO composition, including fatty acids, alkanes, aldehydes, ketones, etc. Palmitic acid (38.67%) was the most abundant composition found in MO. Phytol (6.18%) and ethyl palmitic (4.92%) were the second and third major compositions. 3.2. Antibacterial activity of MO In Fig.2A, both MIC and MBC of MO against L. monocytogenes were 10 mg/mL. Meanwhile, the MIC was 5 mg/mL, and MBC was 10 mg/mL for S. aureus (Fig. 2D). The time-kill curve analysis was evaluated and exhibited inhibitory effect against L. monocytogenes and S. aureus with the increase concentration of MO. From the time-kill curve of 2 MIC, population of bacteria decreased constantly and 93.61% (Fig. 2A) and 99.999% (Fig. 2D) reduction were found after MO treated for 8 h. TEM analysis was also demonstrated the satisfactory antibacterial activity of MO. In Fig. 2B and E, the untreated L. monocytogenes and S. aureus appeared with a regular, intact and smooth cell structure. Conversely, Table 1 Chemical compositions of MO. Compound
Proportion (%)
Compound
Proportion (%)
Palmitic acid Phytol Ethyl palmitate Hexadecanal Heptacosane Pentacosane Nonacosane
38.67 6.18 4.92 4.74 4.35 3.86 3.13
Methyl palmitate Ethyl linolenate Methyl linolenate Nonadecane Ethyl linoleate β-Ionone Total
2.93 2.19 2.07 1.89 1.57 1.36 77.86
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Fig. 2. (A) Time-kill curve of MO against L. monocytogenes; TEM images of L. monocytogenes before (B) and after (C) treated by MO. (D) Time-kill curve of MO against S. aureus; TEM images of S. aureus before (E) and after (F) treated by MO.
3.3.2. FTIR analysis of MO@CNPs In order to characterize the formation of nanoparticles systems and analyze the interactions between components, the FTIR spectra of MO, chitosan, STPP and MO@CNPs were recorded. As shown in Fig. 3, the absorbance characteristic peaks of MO at wavenumber of 1172 cm−1 and 1713 cm−1 represented skeletal vibration of –C–O–C and –C = O2. In addition, the peaks of MO at 2850 cm−1 and 2923 cm−1 were attributed to the CeH stretching vibration of aliphatic CH2 bonds (Bhutada, Jadhav, Pinjari, Nemade, & Jain, 2016). As for chitosan, the absorbance characteristic peaks at the wavenumber of 2871 cm−1 (eCeH), 1592 cm−1 (eNeH) and 1084 cm−1 (eCeOeC) were observed (Branca et al., 2016). Simultaneously, the characteristic peak of STPP appeared at 912 cm−1 which correspond to the stretching P]O. For MO@CNPs, the peaks at 912 cm−1, 1100 cm-1, 1721 cm-1, 2854 cm1 and 2923 cm-1 were found to be similar with the absorption peaks of MO, chitosan and STPP. The results revealed that MO@CNPs were prepared precisely and entrapped MO successfully. 3.4. Physical properties of MO@CNPs nanofibers The thickness of gelatin nanofibers and MO@CNPs nanofibers was determined to know the effect of loading different concentrations of MO@CNPs in gelatin nanofibers. In Table 3, the thickness of gelatin nanofibers was 0.102 ± 0.004 mm. Compared with gelatin nanofibers, average thickness of MO@CNPs nanofibers ranged from 0.104 ± 0.003 to 0.124 ± 0.003 mm with the proportion of MO@CNPs increased from 3.0 to 15.0 mg/mL. The reason could be attributed to the encapsulation of MO@CNPs in the nanofibers.
Fig. 3. FTIR spectrum of MO, chitosan, STPP and MO@CNPs.
Simultaneously, the moisture content decreased from 16.24 ± 0.12% to 11.02 ± 0.24% and water solubility declined from 88.54 ± 0.17% to 81.59 ± 0.20%. The results demonstrated that the
Table 2 The particle size, PDI, zeta potential and encapsulation efficiency of MO@CNPs. CNPs
MO (mg/mL)
Chitosan (mg/mL)
Particle size (nm)
PDI
MO@CNPs-I MO@CNPs-II MO@CNPs-III MO@CNPs-IV MO@CNPs-V
20 20 20 20 20
1.0 2.0 3.0 4.0 5.0
94.3 ± 2.1 117.5 ± 3.2 143.8 ± 4.6 208.7 ± 4.2 246.1 ± 6.3
0.139 0.162 0.227 0.325 0.432
90
± ± ± ± ±
0.017 0.021 0.026 0.031 0.029
Zeta potential (mV)
Encapsulation efficiency (%)
17.2 25.9 32.5 38.4 45.1
27.5 34.1 41.3 32.8 24.5
± ± ± ± ±
1.5 2.1 1.8 3.6 4.2
± ± ± ± ±
0.6 0.3 0.5 0.9 0.5
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Table 3 The physical properties of MO@CNPs nanofibers. Parameters
MO@CNPs (mg/mL) 0
Thickness (mm) Moisture content (%) Water solubility (%) WVP (g mm/m2 h kPa) Tensile strength (MPa) Elongation at break (%)
3.0 a
0.102 ± 0.004 16.24 ± 0.12a 88.54 ± 0.17a 0.49 ± 0.03a 0.63 ± 0.28a 57.43 ± 2.31a
0.104 ± 0.003 15.74 ± 0.19a 88.06 ± 0.16a 0.46 ± 0.02a 0.76 ± 0.31a 54.57 ± 1.96a
6.0 a
9.0
0.109 ± 0.005 14.87 ± 0.14b 87.21 ± 0.19a 0.42 ± 0.04b 0.98 ± 0.29b 52.18 ± 2.62b
a
0.113 ± 0.002 13.42 ± 0.17c 86.13 ± 0.15b 0.36 ± 0.05c 1.24 ± 0.34c 47.61 ± 2.84c
12.0 b
0.118 ± 0.004 12.29 ± 0.23d 84.87 ± 0.23c 0.28 ± 0.06c 1.13 ± 0.36c 48.92 ± 3.07c
15.0 b
0.124 ± 0.003c 11.02 ± 0.24e 81.59 ± 0.20d 0.21 ± 0.08d 1.02 ± 0.33b 49.47 ± 2.69b
with approximate fiber diameter of 85.7 nm. As shown in Fig. 4D, AFM image of MO@CNPs nanofibers was found to have a smooth surface. Besides, some small bumps were observed on the image, which could be clearly seen in 3-Dimensional image (Fig. 4d). Because of the small bumps, the height of nanofibers achieved about 147 nm. As a result, the small bumps should be the MO@CNPs. The results confirmed further that MO@CNPs were loaded into gelatin nanofibers, which is consist with the result of SEM.
incorporation of MO@CNPs could decrease the hydrophilicity of the nanofibers. Meanwhile, WVP of gelatin nanofibers also decreased from 0.49 ± 0.03 to 0.21 ± 0.08 g mm/m2 h kPa. WVP is a major parameter of nanofibers which related to the moisture transition between the food and environment (Bourlieu, Guillard, Vallès-Pamiès, Guilbert, & Gontard, 2009). Therefore, materials with low WVP are suitable to wrap food products and prolong shelf life. As shown in Table 3, tensile strength (TS, MPa) and elongation at break (EAB, %) of nanofibers were also evaluated. For gelatin nanofiber, TS value was 0.63 ± 0.28 MPa and EAB value was 57.43 ± 2.31%. The results demonstrated that gelatin nanofiber was less resistant to breakage (TS) and more deformable (EAB). With increasing the concentration of MO@CNPs, the TS values of nanofibers increased first and then decreased. The maximum TS value was 1.24 ± 0.34 MPa when the MO@CNPs concentration was 9.0 mg/mL. As a result, the addition of MO@CNPs could enhance the tensile strength of nanofibers to some degree, and higher proportion may be not always improving tensile strength. Based on the above analysis, the 9 mg/mL MO@CNPs in gelatin nanofibers was chosen for further experiments.
3.6. Release rate of MO from MO@CNPs nanofibers The release rate of MO from MO@CNPs and MO@CNPs nanofibers were investigated during 60 days storage at 4 °C (Fig. 5A) and 25 °C (Fig. 5B). The total release rate of MO from MO@CNPs reached 31.73% and 54.57% at 4 °C and 25 °C, respectively. It is obvious that the release rate of MO increased with the increase of temperature. Moreover, the release rate of MO from MO@CNPs nanofibers was 19.61% and 35.19% under different temperature. The release efficiency was lower than from MO@CNPs because of the encapsulation of gelatin nanofibers. That is to say that the electrospun nanofibers could delay the release the essential oil and extend the action time of MO@CNPs. The finding was agreement with the results of Cui et al. (2018) that the release rate of clove oil from nanofibers was reduced.
3.5. Morphology and surface characterizations by SEM and AFM SEM images of gelatin nanofibers and MO@CNPs nanofibers were presented in Fig. 4. Generally, the SEM images of gelatin nanofibers (Fig. 4A) and MO@CNPs nanofibers (Fig. 4B) were smooth and stable and showed no obvious differences. However, the average diameter of the gelatin nanofibers was 92 nm (Fig. 4a) and MO@CNPs (Fig. 4b) was 142.5 nm. The increase of average diameter indicated that MO@CNPs were loaded in gelatin nanofibers successfully. The surface and 3-Dimensional AFM images of nanofibers were given in Fig. 4. In Fig. 4C, the gelatin nanofibers had a smooth surface
3.7. The application of MO@CNPs nanofibers on cheese The application of MO@CNPs nanofibers against L. monocytogenes and S. aureus on cheese under 4 °C and 25 °C was evaluated and the results were given in Fig. 6A-D. In general, bacteria on cheese stored at 4 °C grew slower than those on cheese at 25 °C. The population of L. monocytogenes and S. aureus in control group at 25 °C increased dramatically, achieving 7.82 Log CFU/g (Fig. 6B) and 7.58 Log CFU/g
Fig. 4. SEM images and fiber diameter distributions with average diameters of gelatin nanofibers (A), (a) and MO@CNPs nanofibers (B), (b); Surface and 3Dimensional AFM images of gelatin nanofibers (C), (c) and MO@CNPs nanofibers (D), (d). 91
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Fig. 5. The release rate of MO from MO@CNPs nanofibers stored at 4 °C (A) and 25 °C (B) for 60 days.
values between control samples and MO@CNPs nanofibers wrapped samples at 4 °C after 4-days storage. However, the surface color of cheese samples in control group decreased significantly at 25 °C for 4 days while the MO@CNPs nanofibers wrapped samples showed minor undulation. Similarly, the sensory attributes of cheese samples were almost unaffected after MO@CNPs nanofibers wrapped at 4 °C and 25 °C for 4 days. Therefore, MO@CNPs nanofibers can not only prolong the shelf life of cheese, but also keep its sensory quality.
(Fig. 6D), respectively. Conversely, the population of L. monocytogenes and S. aureus only reached 3.11 and 2.2 Log CFU/g at 25 °C, which could be attributed to the addition of MO@CNPs in the nanofibers. The similar results were also found in the samples wrapped by MO@CNPs nanofibers at 4 °C. The population reduction of MO@CNPs nanofibers treated samples reached 78.63% (Fig. 6A) and 98.67% (Fig. 6C) for 10 days storage at 4 °C. These results demonstrated that MO@CNPs nanofibers exhibited excellent antibacterial activity against L. monocytogenes and S. aureus on cheese. The finding of the current study was consistent with the results found by Wen et al. (2016) which showed that polyvinyl alcohol/cinnamon essential oil/β-cyclodextrin nanofibers has satisfactory antibacterial activity and extend shelf life of strawberry. Hence, MO@CNPs-embedded gelatin nanofibers will have a broad prospect for food packaging and preservation.
4. Conclusion In order to improve the stability and prolong action time of MO, MO was loaded into chitosan nanoparticles. The particle size, PDI, zeta potential and encapsulation efficiency of the optimal MO@CNPs were 143.8 ± 4.6 nm, 0.227 ± 0.026, 32.5 ± 1.8 mV and 41.3 ± 0.5%, respectively. The MO@CNPs nanofibers were fabricated successfully with MO@CNPs at 9.0 mg/mL. The nanofibers exhibited excellent physicochemical properties. Besides, the nanofibers possessed high antibacterial effect against L. monocytogenes and S. aureus on cheese at 4 °C and 25 °C and negligible impact on the surface color and sensory
3.8. Surface color and sensory evaluation The surface color values and sensory evaluation of cheese samples wrapped by MO@CNPs nanofibers stored at 4 °C and 25 °C were presented in Table 4. No significant differences were found in L*, a* and b*
Fig. 6. Antibacterial activity of MO@CNPs nanofibers against L. monocytogenes at 4 °C (A) and 25 °C (B). Antibacterial activity of MO@CNPs nanofibers against S. aureus at 4 °C (C) and 25 °C (D). 92
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Table 4 Effect of MO@CNPs nanofibers on the quality of cheese after 4-days storage at 4 °C and 25 °C. Temperature 4 °C
25 °C
Parameters L* a* b* Color Smell Taste Overall acceptability L* a* b* Color Smell Taste Overall acceptability
Fresh cheese (0 d) a
72.75 ± 0.12 0.78 ± 0.09a 20.15 ± 0.16a 3.52 ± 0.08a 3.46 ± 0.08a 3.41 ± 0.11a 3.47 ± 0.13a 72.51 ± 0.15a 0.79 ± 0.12a 19.75 ± 0.14a 3.41 ± 0.10a 3.46 ± 0.12a 3.48 ± 0.13a 3.45 ± 0.15a
quality of cheese during 4 days storage. Hence, the MO@CNPs embedded gelatin nanofiber was a promising active food packaging material for further food application.
Control (4 d)
MO@CNPs nanofiber (4 d) a
70.57 ± 0.17 0.75 ± 0.15b 18.52 ± 0.21a 3.46 ± 0.13a 3.41 ± 0.14a 3.40 ± 0.19a 3.42 ± 0.17a 67.31 ± 0.21b 0.73 ± 0.18b 17.19 ± 0.19b 3.31 ± 0.13b 3.29 ± 0.11b 3.27 ± 0.14b 3.28 ± 0.16b
71.15 ± 0.14a 0.76 ± 0.12a 19.07 ± 0.15a 3.49 ± 0.12a 3.45 ± 0.11a 3.43 ± 0.17a 3.46 ± 0.15a 70. 23 ± 0.12a 0.76 ± 0.14a 19.32 ± 0.18a 3. 38 ± 0.08a 3.41 ± 0.09a 3.42 ± 0.15a 3.40 ± 0.13a
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