Physical, Mechanical, and Antimicrobial Characteristics of Ethyl cellulose/ Polycaprolactone Electrospun Nanofibers Incorporated to Gallic Acid and Natamycin for Cheese Packaging

Samira Beikzadeh 1,2

Ali Ehsani 2,3✉ Emailehsani@tbzmed.ac.ir

Amir Mohammad Mortazavian 3,4✉ Emailmortazvn@sbmu.ac.ir

Zhian Sheikhi 2,3

Marjan Ghorbani 4

Department of Food science and Nutrition Maragheh University of Medical Sciences Maragheh Iran

Department of Food Science and Technology, Faculty of Nutrition and Food Sciences Tabriz University of Medical Sciences Tabriz Iran

Department of Food Technology, Faculty of Nutrition Sciences and Food Technology/ National Nutrition and Food Technology Research Institute Shahid Beheshti University of Medical Sciences Tehran Iran

Nutrition Research Center Tabriz University of Medical Sciences Tabriz Iran

Samira Beikzadeh¹, Ali Ehsani^2*, Amir Mohammad Mortazavian^3*, Zhian Sheikhi², Marjan Ghorbani⁴

¹ Department of Food science and Nutrition, Maragheh University of Medical Sciences, Maragheh, Iran.

² Department of Food Science and Technology, Faculty of Nutrition and Food Sciences, Tabriz University of Medical Sciences, Tabriz, Iran

³ Department of Food Technology, Faculty of Nutrition Sciences and Food Technology/ National Nutrition and Food Technology Research Institute, Shahid Beheshti University of Medical Sciences, Tehran, Iran.

⁴ Nutrition Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.

Author information

Authors and Affiliations

Department of Food science and Nutrition, Maragheh University of Medical Sciences, Maragheh, Iran.

Samira Beikzadeh

Department of Food Science and Technology, Faculty of Nutrition and Food Sciences, Tabriz University of Medical Sciences, Tabriz, Iran.

Ali Ehsani, Zhian Sheikhi

Department of Food Technology, Faculty of Nutrition Sciences and Food Technology/ National Nutrition and Food Technology Research Institute, Shahid Beheshti University of Medical Sciences, Tehran, Iran.

Amir Mohammad Mortazavian

Nutrition Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.

Marjan Ghorbani

Contributions

Samira Beikzadeh: Writing - review & editing, Writing - original draft, Investigation, Data curation, Conceptualization, Formal analysis, Methodology

Ali Ehsani: review & editing, Writing - original draft

Amir Mohammad Mortazavian: review & editing, Writing - original draft

Zhian Sheikhi: Investigation

Marjan Ghorbani: Conceptualization

Corresponding authors

Ali Ehsani ehsani@tbzmed.ac.ir

Amir Mohammad Mortazavian mortazvn@sbmu.ac.ir

Abstract

Electrospinning is a process for producing nanofibers that can be applied for active food packaging. In this study, polymers of ethyl cellulose (ECL) and polycaprolactone (PCL) in weight ratios of 70:30, 80:20, and 90:10% and natamycin (NAT) (1 and 2% w /w) and gallic acid (GAL) (10 and 30% w/w) were used. Nanofibers were produced after the electrospining process (voltage 16 kV, rate of 1 ml/hour, distance of 15 cm). Then, nanofibers were examined for morphological, FTIR, thermal, mechanical, antioxidant, water contact angle, and antifungal properties. The results showed that ECL90/PCL10, ECL80/PCL20, and ECL70/PCL30 had an average diameter of 1250.86 ± 18 nm, 1174 ± 21.4 nm, 754.73 ± 23 nm. ECL and PCL in a ratio of 70 to 30 were selected and combined with NAT and GAL. The average diameter of the produced nanofibers was between 779.32- 924.63 nm. Also, with the addition of NAT and GAL, the thermal stability, mechanical, and antifungal properties of ECL/PCL were increased. The addition of 2% NAT with 10% or 30% GAL completely prevented the growth of both molds. Cheese samples packed with nanofibers containing NAT and GAL in all treatments had a lower increase in mold and yeast during the period than the control sample. Also, the results of the sensory evaluation score did not show a significant difference between the packaged samples.

Keywords:

Polycaprolactone

Gallic acid

Ethyl cellulose

Natamycin

Cheese

Introduction

Fungi, as one of the main food spoilers, can make food consumption undesirable for humans due to reduced nutritional value and mycotoxin production (1). Mold (especially Aspergillus and Penicillium species) growth is one of the common problems during production, ripening, curing, and storage in the refrigerator until consumption (2, 3).

Natamycin (Pimaricin) is produced by Streptomyces species and is classified as a polyene macrolide fungicide group (4). It is applied as an antimicrobial food component for surface protection in various sausages and cheeses (Regulation 1333/2008/EC). NAT is effective in comparison with other fungicides, and most fungi are destroyed in the amount (0.5-6 µg NAT/mL) (4, 5). However, NAT can be less effective when added directly to food due to its reaction with other food components (6). Therefore, incorporating NAT into different polymers can protect and control the release and amount of this compound on the food surface.

GAL ( 3,4,5-trihydroxybenzoic acid) and its derivatives such as catechin and tannin can be found in various plants, wine, tea, cereals, citrus fruits, and berries (7). Due to this compound's antioxidant and antimicrobial properties, it is used as an additive in various food and pharmaceutical industries. However, its bitterness and astringency have limited its use in the food industry. Also, this compound is sensitive to light, oxygen, pH, and temperature (8, 9). Encapsulation can be used to prevent oxidation, increase stability, and mask the unpleasant taste of gallic acid [10]. Common encapsulation methods include: lyophilization and cocrystallization, liposome entrapment, coacervation, extrusion, spray cooling/chilling, and spray drying (8).

Nowadays, the use of novel technologies in encapsulation has caused toxic solvents and high temperatures not to be applied. For several reasons, the electrospinning method is suitable for encapsulating food and pharmaceutical compounds. One of the reasons, is the production of nanofibers at room temperature, which prevents the destruction of active compounds. With this method, it is possible to form nano-sized capsules. Also, limiting the size of nanofibers from micron to nano reduces the release rate of target materials, and due to the increase in the surface of nanofibers, this release can be controlled (3, 10). In addition, limited surface area slows down the release of active substances. As the size of fibers goes from micron to nano-size, it offers a very large surface area for controlled release (3, 11, 12). In the electrospinning process, synthetic and natural polymers, can be used to produce nanofibers (3). Today, environmental pollution due to the disposal of packaging materials wastes, especially synthetic polymers, which have a long life cycle in nature, is one of the major concerns worldwide. One solution to reduce the volume of packaging waste is to use biopolymers in packaging (13–15). Among them, ethyl cellulose is one of the most important and widely used cellulose ether derivatives. It is hydrophobic and resistant to salt and alkali (3). One of the most important economica and practical methods to produce components with favorable mechanical and thermal characteristics is mixing various polymers (3, 16). PCL is considered a polymer that can be compatible with other polymers. This compound consists of 6 carbons and has hydroxyl end connections. The only drawback of this polymer is its low melting point (60°C). To overcome this problem, this polymer can be combined with other polymers (17).

In this work, the ECL/PCL nanofibers at various levels of NAT and GAL were produced. The

formed electrospun nanofibers' properties were analyzed.

Experimental Methods

Required Materials

Fungi were attained from “Iranian research organization for science and technology”. Polymers containing PCL and ECL as well as other substances such as NAT, and GAL (Sigma-Aldrich) were achieved.

Production Steps of Electrospinning Solutions

About 12.5 w/v% of ECL and PCL were obtained in 50/50 of ethanol/chloroform solvent and solutions with ratios of 90:10, 80:20, and 70:30 were prepared (25°C). At the end, ECL/PCL:70:30 was selected, and NAT (1,2, and 5 wt%) and GAL (10 and 30 wt%) were added to nanofiber solution (18).

Electrospinning Operation

The electrospinning operation was done with a voltage of 16 kV, rate of 1 ml/hour, distance of 15 cm, and temperature of 25 ± 2°C, and a humidity of 30 ± 1%. The produced fibers were collected on the collector (18).

Characteristics of Produced Nanofibers

Scanning Electron Microscopy

SEM (26 kV) was applied to measure the microstructure of nanofibers. Also, NIS Elements 0.3 image analysis software was used to evaluate the fiber diameter.

Fourier Transforms Infrared

Spectrometer (Tensor27, Ettlingen, Germany) was used for the FTIR test. Interferograms were obtained in the spectral range (1000–4500 cm^− 1).

Antioxidant Activity

DPPH radical scavenging method evaluates antioxidant activities (19). Nanofibers (100 mg) and water (2 ml) were stirred for this test. Then, the prepared solution (1 ml) with DPPH solution (0.2 ml) were dissolved. After incubating the prepared samples at 25°C for 30 minutes, their absorption at 517 nm was read using a spectrophotometer. In the end, the scavenging activity equation was applied to calculate the antioxidant activities.

Water Contact Angle (WCA)

Water (a drop about 5 ml diameter) was poured on the nanofibers photographed, and analyzed (20).

Mechanical Properties

To measure the mechanical properties, the fibers were cut into dumbbell shapes (0.6 cm × 9 cm) and fixed between grips (speed 1.0 mm/min) (21).

Thermogravimetric Analysis (TGA)

For TGA test, approximately (15 mg) nanofibers were heated under a nitrogen atmosphere (10°C/min) and brought to 600°C by thermogravimetric analyzer.

Antifungal Properties

To evaluate the antimicrobial activity, Aspergillus niger (PTCC5012) and Penicillium notatum (PTCC5014) strains were cultured in potato dextrose agar media. After preparing the strain suspension (10⁶ spores/ml), the suspension (1 ml) and PDA (99 ml) was homogenized. The nanofibers were cut into sizes (1×1 cm), sterilized with ultraviolet rays (1 hour) and placed on plates and incubated for 5 days, and the diameter of the zone was indicated (22).

UF cheese packaging

According to the results obtained from examining the morphological, thermographic, mechanical and antifungal properties of the produced nanofibers, nanofibers with optimal properties were used for packaging UF cheese. For this purpose, after the production of UF cheese, aluminum foils coated with nanofibers were used for packaging. Aluminum foils coated with nanofibers including nanofibers without preservatives and aluminum foils coated with nanofibers containing preservatives were sterilized by ultraviolet rays (UV) for 40 minutes. Then the produced cheese was cut into 5 × 25 × 30 mm dimensions and after being sterilized by UV for 20 minutes, the cheese samples were packed with aluminum foils.

Desired tests on packaged cheeses

Microbial analysis of produced cheeses

Counting of mold and yeast was done using Sabourd dextrose agar culture medium and by deep culture method with 0.01 dilution under cold storage conditions during storage days.

Sensory analysis of manufactured cheeses

The samples were evaluated by 30 evaluators. The cheese samples were taken out of the refrigerator one hour before the test to be at the same temperature as the outside environment and were numbered by a table of random numbers. The samples were given to the evaluator along with a special evaluation questionnaire and were evaluated based on texture, taste, smell and overall acceptance. The evaluation of the samples was done using a 5-point hedonic scale.

Statistical Evaluation

The results from the data obtained with three repetitions were determined as mean ± standard deviation. Statistical differences between values were determined with SPSS and one-way analysis of variance (ANOVA). Also, Duncan's statistical analysis was used to confirm the significance level (p < 0.05).

Results and Discussion

Scanning Electron Microscopy

Figure 1

Figure 2

SEM results and the mean diameter distribution of prepared fibers of ECL/PCL polymers are shown in Fig. 1. ECL90 /PCL10, ECL80/PCL20, and ECL70/PCL30 had an average diameter of 1250.86 ± 18 nm, 1174 ± 21.4 nm, and 754.73 ± 23 respectively. For ECL nanofibers, Wang et al. (2020) reported an diameter of 649 nm (23). Liu et al. (2018) reported a spindle-like structure with an average value of 413 nm (24). Although, PCL nanofibers with an average value of 419 nm were indicated. Also, as shown in Fig. 2, nanofibers ECL70/PCL30/NAT1/GAL10, ECL70/PCL30/NAT2/GAL10, ECL70/PCL30/NAT1/GAL30, and ECL70/PCL30/NAT2/GAL30 had an average diameter of 779.32 ± 35 nm, 886.27 ± 13.5 nm, 805.45 ± 12 nm, and 924.63 ± 19 nm. The highest mean diameter was obtained for ECL70/PCL30 /NAT2/ GAL30. In general, NAT and GAL enhanced the fiber diameters. Neo et al. (2013) reported that adding GAL to the zein solution increased the fiber diameters. The solution viscosity is enhanced with the presence of GAL and thus increases the molecular entanglement numbers, which leads to a diameter enhancement (12). Also, according to the reports of Veras et al. (2020), the presence of natamycin in the poly-caprolactone electrospinning solution, enhanced the fiber diameters; with the tripling of the amount of natamycin compared to the first sample, the diameter of the produced nanofibers increased by 64% (25). These results were consistent with our findings.

Fig. 1

Fiber diameter distributions (ECL90/PCL10 (a), ECL80/PCL20 (b), and ECL70/PCL30 (c): NAT: Natamycin, GAL: Gallic acid, ECL: Ethyl cellulose, and PCL: Polycaprolactone).

Fig. 2

SEM results and average nanofiber diameter of ECL70/PCL30/NAT1/GAL10 (d), ECL70/PCL30/NAT2/GAL10 (e), ECL70/PCL30/NAT1/GAL30 (f), and ECL70/PCL30/NAT2/GAL30 (g): NAT: Natamycin, GAL: Gallic acid, ECL: Ethyl cellulose, and PCL: Polycaprolactone.

Fourier Transforms Infrared (FTIR)

Table 1 and Fig. 3 indicate the wavenumber of specified peaks of nanofibers. For ECL nanofiber, the peaks at 2879, 3450–3741, 2621–2742, and 1249 cm^− 1 could belong to –CH₃ link vibration, O–H vibration, CH₂ and CH stretches, and C–O–C link, respectively (3). For PCL, the peaks at 2889–3030 cm^− 1, 1743 cm^− 1, 1375 cm^− 1, and 1047–1257 cm^− 1 could be due to CH2 stretching vibrations, CO connection vibration, CC connection vibrations, and C–O–C connection vibration, respectively (3). For NAT, the presence of peaks in the range of 1100–1750 was related to the C = O bond. In the case of GAL, the peaks at 3490–3748 cm^− 1corresponded to the O–H stretching vibration. The peaks at 1520–1710 cm^− 1 were related to stretching vibrations of the C–C/C–H aromatic groups. Also, the 1193–1411 cm^− 1 peaks were related to the C–O stretching vibration. Changes in the ECL spectrum were obtained due to the addition of PCL to ECL. The peaks at 2870 and 3450 cm^− 1 changed to higher wavelengths. A new peak at 2504 cm^− 1 was created. In addition, with the addition of NAT and GAL, changes in the ECL70/PCL30 spectrum were obtained. Novel peaks at 3380, 3220, and 3114 cm^− 1 were created. The peak at 2804 cm^− 1 changed to lower wavelengths. Veras et al. (2020) reported that mixing NAT with PCL had no significant difference in the nanofiber spectrums, which can result from the lack of chemical linkage between polymers and antifungal compounds (25).

Table 1

Types of nanofibers, connections, and wavenumber of specified peaks of nanofibers
Nanofiber types	Connection	Wavenumber (1/cm)
ECL	–CH₃ connection vibration	2879
	O–H vibrations	3450–3741
	CH₂ and CH stretches	2621–2742
	C–O–C connection	1249
PCL	CH₂ stretching vibration	2889–3030
	CO connection vibration	1743
	CC connection vibration	1375
	C–O–C connection vibration	1047–1257
NAT	C = O connection	1100–1750
GAL	O–H stretching vibration	3490–3748
	C–C/C–H stretching vibration	1520–1710
	C–O stretching vibration	1193–1411

Fig. 3

FTIR results of various nanofibers: PCL: Polycaprolactone, ECL: Ethyl cellulose, NAT: Natamycin, GAL: Gallic acid.

Table 1

Figure 3

Mechanical Properties

The mechanical evaluation of fibers containing NAT and GAL are reported in Table 2. Thickness (0.112 ± 0.007), tensile strength (8.22 ± 0.4 MPa), and elongation at break (4.49 ± 0.3%) were obtained for ECL70/PCL30 nanofibers. ECL70/PCL30 had higher mechanical values than ECL 90/PCL10 and ECL80/PCL20. The tensile strength parameter was increased due to novel connection formation between the polymers (24), and FTIR results also indicated this phenomenon. The addition of NAT and GAL also increased the mechanical properties of formed nanofibers.

Table 2

Mechanical properties of nanofibers
Sample	Thickness (mm)	Tensile strength (MPa)	Elongation at break (%)
ECL90/PCL10	0.108 ± 0.005 ^b	5.93 ± 0.6 ^d	2.66 ± 0.2 ^c
ECL80/PCL20	0.109 ± 0.001 ^b	6.78 ± 0.4 ^c	3.12 ± 0.4 ^c
ECL70/PCL30	0.112 ± 0.007 ^b	8.22 ± 0.4 ^b	4.49 ± 0.3 ^b
ECL70/PCL30/NAT/GAL	0.124 ± 0.003 ^a	9.12 ± 0.5 ^a	6.32 ± 0.4 ^a
Mean ± Standard deviation

Table 2

Water Contact Angle (WCA)

WCA analysis was performed to check the surface wettability of the formed nanofibers, and the results are shown in Fig. 4. The WCA analysis of ECL90/PCL10, ECL80/PCL20, and ECL70/PCL30 were attained as 49.03^◦, 54.84^◦, and 59.77^◦, respectively. As the amount of PCL increased, the WCA content improved. This property could be attributed to the hydrogen connection between the ECL and PCL (3). Also adding NAT and GAL to ECL 70/PCL30 nanofiber enhanced the WCA. Shen et al. (2021) reported that with increasing the weight of encapsulated natamycin, formed gelatin/zein/polyurethane nanofiber WCA increased due to the hydrophobic property of natamycin (26).

Fig. 4

The water contact angles of formed nanofibers: ECL: Ethyl cellulose, PCL: Poly caprolactone, NAT: Natamycin, GAL: Gallic acid.

Figure 4

Thermogravimetric Analysis (TGA)

The results of thermogravimetric analysis of formed nanofibers containing natamycin and gallic acid are displayed in Fig. 5. ECL had a decomposition temperature of about 310.56°C. However, ECL/PCL and ECL/PCL /NAT/GAL nanofibers had decomposition temperatures of about 315°C and 325.3°C. Therefore, the thermal stability of the produced fibers enhanced with the incorporation of NAT and GAL to ECL nanofiber,. Also, the difference between the two graphs (ECL and PCL) and the graph (ECL/PCL/NAT/GAL) indicates the presence of NAT and GAL compounds. Neo et al. (2013) reported that the mixture of GAL with zein increased the decomposition temperature of formed nanofibers (12).

Fig. 5

TGA curves of the ECL70/PCL30, and ECL70/PCL30/NAT/GAL: ECL: Ethyl cellulose, PCL: Polycaprolactone, NAT: Natamycin, and GAL: Gallic acid.

Figure 5

Antioxidant Activity Measurements

The antioxidant property of the nanofibers in 80% ethanol aqueous solutions is indicated in Fig. 6. GAL decreases radicals by performing as a/an hydrogen or electron atom donor (27). ECL70/PCL30 electrospun nanofibers incorporated with 10 and 30% GAL indicated an antioxidant value of about 62.38 ± 3.5% and 78.23 ± 2.9%, respectively. In contrast, the neat ECL70/PCL30 nanofiber indicated 4.12 ± 1.8% antioxidant activity. Neo et al. (2013) reported that the zein nanofiber containing GAL had antioxidant properties ranging from 58–89%.

Fig. 6

Antioxidant properties of electrospun nanofibers

Figure 6

Antifungal Activity

The results of the antifungal activity of nanofibers containing NAT and GAL are shown in Table 3 and Fig. 7. Control samples (ECL70/PCL 30) and samples containing 1% NAT and 10 and 30% GAL did not indicate antifungal activity against the growth of Aspergillus niger and Penicillium notatum. Whereas, nanofibers containing 2% NAT prevented fungal growth. ECL/PCL /NAT/GAL nanofibers also had lower antimicrobial activity against Aspergillus niger compared to Penicillium notatum. In general, the results indicated that the antifungal properties of nanofibers enhance with increasing the amount of NAT. The results also showed that adding 2% NAT with 10% or 30% GAL completely prevented the growth of both molds.

Table 3

The colony diameter of Aspergillus niger and Penicillium notatum
Nanofiber samples	Colony Diameter (mm)
Nanofiber samples	Aspergillus niger	Penicillium notatum
ECL70/PCL30	38 ± 1.4	36 ± 1.6
ECL70/PCL30/NAT1/GAL10	32 ± 0.9	25 ± 1.1
ECL70/PCL30/NAT2/GAL10	0	0
ECL70/PCL30/NAT5/GAL10	0	0
ECL70/PCL30/NAT1/GAL30	18 ± 0.7	12 ± 1.3
ECL70/PCL30/NAT2/GAL30	0	0
ECL70/PCL30/NAT5/GAL30	0	0

Fig. 7

The antifungal properties (ECL70/PCL30 (control), ECL70/PCL30/NAT1/GAL10 (S1), ECL70/PCL30/NAT2/GAL10 (S2), ECL70/PCL30/NAT1/GAL30 (S3), and ECL70/PCL30/NAT2/GAL30 (S4) nanofibers. ECL: Ethyl cellulose, PCL: Polycaprolactone, NAT: Natamycin, GAL: Gallic acid

Veras et al. (2020) reported that the inhibition zone of PCL nanofibers containing NAT in percentages of 1 to 4% was about 6.6 to 24 mm against Aspergillus flavus and Penicillium citrinum molds. With increasing the amount of NAT, the antifungal properties of nanofibers increased (22).

Table 3

Figure 7

Mold and yeast analysis of cheeses packaged by nanofibers

Changes in the amount of mold and yeast in cheeses packed with nanofibers are shown in the Table 4. In all treatments, there was an increase in the amount of mold and yeast during the period, and the increase in the control sample (the sample packed with nanofibers without NAT and GAL) was higher than the other samples.

Table 4

Mold and yeast levels Log (CFU g^− 1) of cheeses packaged with nanofibers during 56 days at 4°C
Packaging type	Storage period (days)
Packaging type	1	14	28	56
Without packaging	5.16 ± 0.11 ^aC	6.29 ± 0.43 ^aB	7.55 ± 0.54 ^aA	-
ECL70/PCL30	5.09 ± 0.26 ^aC	5.33 ± 0.47 ^bBC	5.98 ± 0.29 ^aAB	6.28 ± 0.61 ^aA
ECL70/PCL30/NAT2/GAL10	5.12 ± 0.09 ^aC	5.28 ± 0.16 ^bBC	5.78 ± 0.24 ^aAB	6.03 ± 0.27 ^aA
ECL70/PCL30/NAT2/GAL30	5.04 ± 0.17 ^aB	5.15 ± 0.46 ^bB	5.69 ± 0.15 ^aA	5.97 ± 0.35 ^aA
Values with different lowercase letters are significantly different according to Duncan's test at the level (p < 0.05) in each day between the treatments and with different capital letters on the maintenance days. (mean of three repetitions ± standard deviation)

Fajardo et al. (2010) packaged semi-hard cheese (Saloio cheese) with chitosan and chitosan coatings containing natamycin for 37 days at 4°C. Microbial analysis of cheese samples showed that the amount of mold and yeast was found in the range of 4.53–6.06 Log (CFU g^− 1). The shelf life for Salvio cheese is about 50 days, but after 37 days, the growth of fungus on the surface of the cheese led to the end of the microbial analysis of the cheese (28). These findings are consistent with our results. Also, Jalilzadeh et al. (2020) used whey protein containing natamycin to package ultra-refined cheese, and the results showed that the amount of mold and yeast in the control sample was higher than the coated samples in 28 days (29).

Table 4

Overall acceptance of cheeses packaged by nanofibers:

Overall acceptance of cheese samples packed with nanofibers during storage in the refrigerator are shown in the Table 5. The results of the sensory evaluation score for packaged cheese samples did not show a significant difference between samples packaged with natamycin-free nanofibers, nanofibers containing 2% NAT and 10% GAL, and nanofibers containing 2% NAT and 30% GAL. The control sample (sample without packaging) had a lower score in terms of overall acceptance.

Table 5

Sensory evaluation score of cheeses packaged with nanofibers
Overall acceptance	Storage time (days)
Overall acceptance	1	14	28
Without packaging	4.0 ± 0.18 ^aA	3.8 ± 0.35 ^aA	3.6 ± 0.16 ^bA
ECL70/PCL30	3.8 ± 0.14 ^aA	4.0 ± 0.15 ^aA	4.1 ± 0.37 ^abA
ECL70/PCL30/NAT2/GAL10	4.2 ± 0.17 ^aA	3.9 ± 0.39 ^aA	4.2 ± 0.32 ^aA
ECL70/PCL30/NAT2/GAL30	3.9 ± 0.36 ^aA	4.0 ± 0.10 ^aA	4.0 ± 0.29 ^abA
Values with different lowercase letters are significantly different according to Duncan's test at the level (p < 0.05) in each day between treatments and with different uppercase letters on maintenance days. (mean of three repetitions ± standard deviation)

Nottagh et al. (2019) used chitosan containing natamycin to pack ultra-refined cheese for 6 weeks. The highest score of taste, smell and overall acceptance was related to the packaged samples in the third week compared to the control sample (without packaging)(30).

Table 5

Conclusion

This study investigated the antioxidant, antifungal, morphological, and thermal properties of ECL/PCL nanofibers containing NAT and GAL. The results revealed that ECL70/PCL30 had the desired morphological and mechanical properties compared to the ECL90/PCL10 and ECL80/PCL20. Also, the mixing NAT with GAL increased the average diameter of fibers, mechanical characteristics, WCA, and thermal stability. Electrospun nanofibers ECL70/PCL30 containing 30% GAL had the highest antioxidant contents. In addition, ECL70/PCL30/NAT2/GAL10 and ECL70/PCL30/NAT2/GAL30 nanofibers completely prevented the growth of Aspergillus niger and Penicillium notatum. Therefore, ECL/PCL nanofibers incorporated in 2% NAT and 10% GAL can be used to package of products sensitive to fungal spoilage, such as cheese (instead of using NAT in the formulation of cheese production).

Acknowledgment

This research can be introduced with ethics code (IR.SBMU.RETECH.REC.1400.950) from the Student Research Committee, Shahid Beheshti University of Medical Sciences, Tehran, Iran.

We also appreciate the “Student Research Committee” in Shahid Beheshti University of Medical Sciences for their financial support of this study.

Funding

This work was financially supported by the “Student Research Committee” in Shahid Beheshti University of Medical Sciences (IR.SBMU.RETECH.REC.1400.950).

Author Contribution

1: Writing - review & editing, Writing - original draft, Investigation, Data curation, Conceptualization, Formal analysis, Methodology2: Review & editing, Writing - original draft3:Review & editing, Writing - original draft4: Investigation5: Conceptualization

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