Providing Nanofiber Membrane of Nylon-6 Master-batch, Ag, Chitosan, Rosemary, and Investigating the Anti-oxidant and Antimicrobial effect for Packaging in Food Industries
الموضوعات :
1 - Department Chemical and Engineering, Shahreza Branch, Islamic Azad University, Shahreza, Iran.
الکلمات المفتاحية: Antioxidant, Antibacterial, Nanofibers, Glucose oxidase, Nylon. Silver Master,
ملخص المقالة :
In the present research, nylon 6-silver (NS)/ Chitosan (CS)/Rosemary master-batch nanofibers was provided in order to be used in packaging food. Morphology and structure of the nanofibers was investigated using the Scanning Electron Microscope (SEM) and Fourier-transform infrared spectroscopy (FTIR). The antibacterial activity of synthetic nanofibers was evaluated for E. Coli and S. Aureus bacteria. The antioxidant activity of oxidant glucose of glucose oxidase and Rosemary as well as the function of the provided membranes in packaging and maintaining strawberry was investigated. Morphology and diameter of synthetic nanofibers were determined with SEM images. According to the results of the carried out investigations on the deterrent effect of silver on the activity of glucose oxidase of 70/30 NS-CS sample was selected as the optimum for subsequent studies, and based on the results, the highest enzyme activity (79.5%) with 0.05 grams of glucose oxidase was obtained. The antibacterial test of the membranes showed the perfect antibacterial activity of such membranes against the negative and gram-positive and gram-negative bacteria. In addition, the FTIR spectroscopy approved the presence of glucose oxidase, silver, and Rosemary in synthetic membranes.
Providing Nanofiber Membrane of Nylon-6 Master-batch, Ag, Chitosan, Rosemary, and Investigating the Anti-oxidant and Antimicrobial effect for Packaging in Food Industries
Abstract:
In the present research, nylon 6-silver (NS)/ Chitosan (CS)/Rosemary master-batch nanofibers was provided in order to be used in packaging food. Morphology and structure of the nanofibers was investigated using the Scanning Electron Microscope (SEM) and Fourier-transform infrared spectroscopy (FTIR). The antibacterial activity of synthetic nanofibers was evaluated for E. Coli and S. Aureus bacteria. The antioxidant activity of oxidant glucose of glucose oxidase and Rosemary as well as the function of the provided membranes in packaging and maintaining strawberry was investigated. Morphology and diameter of synthetic nanofibers were determined with SEM images. According to the results of the carried out investigations on the deterrent effect of silver on the activity of glucose oxidase of 70/30 NS-CS sample was selected as the optimum for subsequent studies, and based on the results, the highest enzyme activity (79.5%) with 0.05 grams of glucose oxidase was obtained. The antibacterial test of the membranes showed the perfect antibacterial activity of such membranes against the negative and gram-positive and gram-negative bacteria. In addition, the FTIR spectroscopy approved the presence of glucose oxidase, silver, and Rosemary in synthetic membranes.
1-Introduction
One of the most important industrial sector of all countries which is related to food security is food industries. Due to lack of food and population growth, development of this sector seems necessary. Using modern technologies in this sector is a new approach that can be noticed. Convergence of nanotechnology and food science leads to the appearance of many capabilities, which has resulted in around 200 large companies all around the world making massive investments in this field and introducing some products to the market. Social changes, globalization, and the need to take hard safety measures have increased the pressure for producing new packaging systems that are able to transport food and can be tracked in the food supply chain. Hence, nanostructure or microstructure materials on metal basis are included in food contact polymers in order to raise mechanical properties, barrier properties and prevent the destructions caused by light. Moreover, the heavy materials with antibacterial effects are used in the form of salts, oxides and colloids, complexes like silver zeolite or as its nanoparticles. Such nanoparticles are added so as to preserve and conserve food and save the surfaces from the pollutions of industrial environments. Other related features of them in active packaging is their capability to oxidize ethylene or oxygen inhibition which can be used to increase food's shelf life [1]. A lot of driving forces have been created for innovation in food packaging technology, and active and smart technologies have been developed as a result of such driving forces [2].
In a research entitled "Investigation of Antioxidant Effect of Rosemary Plant on Preserving Food", Hosseini et al. (2011) took Rosemary extract using Ethanol solvent and Ethanol device in order to make use of Rosemary as a natural antioxidant. Next, in order to investigate the preservative effects of Rosemary extract, they added some of it to oil and kept it for 30 days in a certain place along with some oil without antioxidant. The results proved that Rosemary extract caused the oxidation of oil in 30 days, and the oil containing the extract was more efficient. Therefore, Rosemary extract has been used for the first time as a preservative in the current research [3]. Salari and Mansouri Najand (2013).
2-Experiences
The materials and equipment of nylon-silver master-batch have been provided by New Persian Polymer Company in Iran. chitosan, acid acetic, and glucose oxidase were provided Merck in Germany. Rosemary plant has been taken from Kerman, Iran (Table 1).
Table 1: Chemical Compound of Rosemary Extract Collected from Kerman in Iran
Percentage | Compound Name | Number | Percentage | Compound Name | Number |
18/7 | α-Pinen | 11 | 0.2 | α-Thujene | 1 |
0.8 | Verbenone | 12 | 5.19 | Camphene | 2 |
0.04 | Myrcene | 13 | 0.34 | B- Pinen | 3 |
21.3 | 1,8 cineole | 14 | 4.6 | Limonene | 4 |
0.1 | P-Cymenene | 15 | 0.1 | Trephine | 5 |
0.3 | Camphonelal | 16 | 12.33 | Linalool | 6 |
0.2 | Cis-Verbenole | 17 | 12.9 | Camphor | 7 |
4.86 | Borneol | 18 | 0.1 | Iso-Pinocamphone | 8 |
2.21 | Verbenone | 19 | 1.2 | Myrtenole | 9 |
|
|
| 0.1 | α- Humulene | 10 |
3- Methodology
3-1- Rosemary Extraction
Fresh leaves of Rosemary were provided and dried somewhere far from sunlight. The dried leaves were ground with an electric mill and dissolved an Erlenmeyer steilized in methanol. Then, extraction was done using the soxhlet extractor. After Rosemary was extracted with methanol, it came to separating the solvent from the extract, which is called concentration. The solvent can be separated from the extract in the form of vapor by heating. In order to prevent energy loss and the risks of high temperature as well as to prevent the produced compounds from blending, vacuum distillation devices like Rotary can be used. This way, the solvent can be separated from the extract in 30-40 degrees centigrade.
3-2- Producing Nylon/Silver- Chitosan Nanofibers Containing Rosemary Extract and Glucose Oxidase
Firstly, two solutions were provided one of which was 8 weight/volume percent of nylon-silver (NS) master-batch in acid citric and the other one was 2 weight/volume percent of chitosan (CS) in acid citric. Next, electrospinning solutions were provided from the combination of various proportions of 80/20, 70/30, 60/40, 50/50, 40/60, 30/70, and 20/80 of nylon/silver and chitosan polymers solutions (NS/CS). In order to obtain a homogeneous and uniform mixture, combination was carried out in 12 hours at room temperature using a mechanical mixer. Electrospinning operations were conducted under constant voltage of 18 kilowatts using Gamma High Voltage ResearchRR60 device on an aluminum layer as a collector. The distance between the needle and the collector was set on 16 centimeters and the supply rate of the polymer solution was set on 1.2 millimeters per hour. Among different electrospun nanofibers of NS/CS, the optimum proportion of two except were selected based on the fibers' morphology and their uniformity and then, the electrospinning solution with the optimum value was provided and after the total combination of both polymers, different values of glucose oxidase (GOx) (0.01, 0.03, 0.05, and 0.07 grams) were added to the solution and mixed for two hours. Afterward, the solutions with abovementioned conditions were electrospun. Finally, 1.5 percent of rosemary extract was added to the optimum solution (containing GOx), and electrospinning operations were done with the conditions above. It should be noted that in order to completely remove the solvent, all provided nanofibers were put in an oven at 70 degrees centigrade for 3 hours in different stages.
3-3- Fourier-transform infrared spectroscopy (FTIR)
FTIR spectroscopy of nanofibers was used using Fourier-transform infrared spectroscopy (Thermo Nicolet NEXUS 870 FTIR from Nicolet Instrument Corp., USA) to identify functional groups.
3-4- Scanning Electron Microscope (SEM)
Morphology of the surface of all nanofibers was investigated using the scanning electron microscope (SEM, LEO1455VP, and ENGLAND).
3-5- Antibacterial Test
Two bacteria, i.e. Staphylococcus aureus (S. Aureus) of American Culture Collection No.6538 type as a gram-positive bacterium and Escherichia Coli of American Culture Collection No.11303 type have been cultivated in Luria – Bertania (LB) medium at 37 degrees and density of 2×〖10〗^5 cells/ml. Then, these bacteria were used for antibacterial test. The antibacterial activity of NS-CS (60/40), NS-CS-R (60/40/3%), NS-CS-GOx (60/40/0.05), NS-CS-GOx-R (60/40//0.05/3%) membranes was investigated using colony count method (100 milliliters of dilute bacterial suspension was produced and rapidly placed in LB growth medium). The plates were put in an incubator at 37 degrees centigrade for 24 hours and finally, the colonies were counted. it is noteworthy that NS-CS was selected as the control sample. The growth control area and the number of colonies were determined across three frequencies.
3-6- Glucose Oxidase Activity Determination
Glucose oxidase activity was measured through Carmine method [12].milliliters of glucose oxidase of 1 milligram per milliliter and 4 milliliters of glucose oxidase solution of 0.2 moles per liter were poured into a test tube at 37 degrees centigrade for 10 minutes to make them react. The enzyme solution was the substrate. Acetic acid- acetate sodium was used as the medium and Indigo carmine was used as the calibration. The test tube was placed in the boiling water path for 30 minutes, and the absorption rate was measured using distilled water at wavelength of 615 nanometers. The activity of each glucose oxidase unit (U/ml) has been defined as the amount of glucose oxidase that 1 milligram of hydrogen peroxide has produced in one minute. 〖GO〗_xactivity for the provided sample (U/mg) has been obtained through equation 1 as follows:
4- Analysis
4-1-Nanofibers Morphology
Figure 1 shows SEM images of NS-CS nanofibers with different proportions of NS and CS. It should be noted that spinning solutions with 20/80 and 30/70 (NS-CS) proportions do not have fiber formation ability due to high percentage of chitosan. As it is evident, the samples containing 60 and 50 percent of CS have some defects in their structures. Such defects in nanofibers are evident in uneven diameters and beads presence.
Fig. 1: SEM images of NS-CS 70-percent samples of chitosan (A), 60-percent of chitosan (B), 50-percent of chitosan (C), 40-percent of chitosan (D), 30-percent of chitosan (E), 20-percent of chitosan (F).
Figure 2 shows SEM images of 70/30 proportions of (NS-CS) containing0.01, 0.03, 0.05, and 0.07 grams of glucose oxidase. According to the images, as the amount of glucose oxidase has increased, the rate of unevenness and defects of nanofibers have risen as well.
Fig. 2: SEM images of 70/30 samples containing 0.01 grams of 〖GO〗_x (NS-CS) (A), 0.03 grams of 〖GO〗_x (B), 0.05 grams of 〖GO〗_x (C), 0.07 grams of 〖GO〗_x (D)
4-2- Silver's Deterrent Effect on Glucose Oxidase
Since glucose oxidase is perceived as an enzyme, it can naturally display its effect under certain circumstances such as pH and presence of different materials. One of the deterrent materials in glucose oxidase activity is heavy metals and silver [14]. Therefore, the samples with proportions of 40/60, 50/50, 60/40, 70/30, and 80/20 (NS-CS) containing 0.03 grams of glucose oxidase were tested in order to investigate the activity of this enzyme, (Figure 3 & Table 1).
Figure 3. Glucose oxidase activity regarding the amount of silver in nanofibers
Table 2. Glucose oxidase activity regarding the amount of silver in nanofibers.
%GOx activity (U/mg of preparation) | GOx activity (U/ml of sample solution) | Nanofibers (NS-CS) |
63.2 | 139.04 | 40/60 |
60.4 | 132.88 | 50/50 |
58.8 | 129.136 | 60/40 |
55.5 | 122.1 | 70/30 |
38.8 | 85.36 | 80/20 |
Generally, the mechanism of glucose oxidase preservative effect can be illustrated as follows in Figure 4 [15].
Fig. 4. Two probable mechanisms for reaction [117]
Figure 5 and Table 3 show different amounts of glucose oxidase (0.01, 0.03, 0.05, and 0.07 grams) in nanofibers 70/30 (NS-CS). Based on the results, as the amount of enzyme in nanofibers increased, the preservative capability of nanofibers increased up to 0.05 grams of enzyme, and in higher amounts, saturation of the solution caused the activity of glucose oxidase to decrease. As a result, the amount of 0.05 grams of glucose oxidase with the most enzyme activity (79.5%) was selected as the optimum.
Fig. 5. Glucose oxidase activity regarding the amount of silver in nanofibers.
Table 3. Effect of Different Glucose oxidase Doses in Nano-Fibers 70/30 (NS-CS)
%GOx activity (U/mg of preparation) | GOx activity (U/ml of sample solution) | Gluxacoxidase (mg) |
42.7 | 95.76 | 10 |
55.5 | 124.47 | 30 |
79.5 | 178.30 | 50 |
68.7 | 154.08 | 70 |
4-3- Antibacterial Test
As shown in Figure 6, the size of NS-CS control area for S. Aureus is stronger than E.coli. Electrospinning membranes of NS-CS-R, NS-CS-GOx, and NS-CS-GOx-R have the ability to remove the cells of both bacteria. As rosemary was added to the electrospun membrane, the antibacterial activity increased despite the presence of cinede 1.8, α- pinen, camphor, and [16-17] verbenone compounds with antimicrobial properties. Three phases of cooperation, opposition, and intensifier1 might occur between these compounds; each of these phases is effective on antimicrobial property of the essence.
Figure 6. Antibacterial test for various electrolyzed membranes
4-4- FTIR Analysis
FTIR spectrum of various samples of electrospun membranes have been demonstrated in Figure 7. In NS-CS curve, the stretching and bending vibrations of NH (in the initial amine) appeared at 3423 and 1546 Cm-1, respectively. Moreover, the stretching vibrations related to C=O groups appeared at 1698 Cm-1. The absorption band at 1643 Cm-1 belongs to stretching vibrations of NH (the second amine) [20]. The stretching and bending vibrations of CH groups (including CH3, CH2, and CH) appeared at 2922 Cm-1 and 1307-1456 Cm-1 range, respectively. After rosemary was put into nanofibers, the absorption band widened at 3423 Cm-1 and its intensity increased, which is because of the presence hydroxyl groups of rosemary and overlap of OH and NH groups. In addition, the absorption band at 958 Cm-1 belongs to stretching vibrations of OH groups in carboxylic acid. In the sample spectrum of NS-CS-GOx-R, intensity of the absorption band rose at 3313 Cm-1 due to amino and hydroxyl groups placement in nanofibers. The appeared absorption band at 1039 Cm-1 belongs to vibrations of cysteine groups in glucose oxidase. Also, intensity of the absorption band increased at 1542 and 1698 Cm-1, which is because of the presence of more amino and carboxyl groups in nanofibers structure. Amino and carboxyl groups in nanofibers structure.
Fig. 7 The FTIR spectra of different electrospinning membranes
4-5- The Function of Electrospun Nanofibers in Packaging Food.
The strawberry is a very sensitive fruit that quickly spoils and is normally stored at cold temperature. Therefore, in order to investigate the efficiency of different obtained nanofibers, they were used in packaging strawberry. Initially, the of glucose oxidase activity of all four types of packaging were examined, and the results were presented in Figure 8 and Table 4. The results revealed that the package made by NS-CS-GOx-R nanofibers had the most activity (90.1%) and highest preservative effect.
Fig. 8. Glucose oxidase activity of different strawberry packages
Table 4. Glucose oxidase activity of different strawberry packets
%GOx activity (U/mg of preparation) | GOx activity (U/ml of sample solution) | Packages |
12.9 | 28.38 | Normal |
65.3 | 143.66 | NS-CS-R |
79.5 | 178.30 | NS-CS-GOx |
90.1 | 198.22 | NS-CS-R-GOx |
Strawberries were packed in different packaging for 15 days at 21 ° C and at some time intervals, weight loss and apparent caries were studied On the 15th day, the weight of the control sample (typical packaging) was 43.1%, while the decrease in samples packed with Ns-Cs-Go, Nc-Cs-R, Ns-Cs-R-Go was %23.8, %20.3, %5.73 respectively.
In addition, as shown in Figure 9, the control sample has lost its gloss over time and has been rotted and moldy. Which is probably due to the destruction of the cell wall and, consequently, loss of water due to the breakdown of the cell [21]. while samples packed with nanofibers are later decayed and the sample packed with Ns-Cs-R-Go remains on the 15th day without mildew.
The results suggest that nanofibers are a good option for storing strawberries. Generally, anti-bacterial nanofibers can not only prolong the storage time but also maintain their appearance, taste and health during storage.
Fig. 9. Cosmetic Changes in Strawberry at 21 degrees (a) Control (Normal Packaging), (b) NS-CS-R- Packaging, (c) NS-CS-GOx Packaging, (d) NS-CS-R-GOx Packaging.
5- Conclusion
Composite membranes of NS-CS-R-GOx with antibacterial properties against two gram-positive and gram-negative bacteria were produced using electrospinning method. Glucose oxidase and rosemary were successfully placed in composite nanofibers. The chemical compound of nanofibers was confirmed by FTIR. The results of rosemary's presence were confirmed as the 3423 Cm-1absorption band widened and its intensity increased, which shows the overlap of hydroxyl groups of rosemary, OH and NH and bending vibrations of OHJ groups of carboxylic acid at 958 Cm-1. Moreover, the presence of glucose oxidase was confirmed as the 1039 Cm-1 absorption band of cysteine groups vibrations appeared. SEM images revealed that the best sample in terms of uniform morphology of the surface and nanofibers' diameter was 70/30 (NS-CS) sample. After different amounts of glucose oxidase were added to the optimal sample of NS-CS, it was observed that with an increase in the amount of glucose oxidase, some non-uniformities and defects were caused in nanofibers. In addition, SEM images after rosemary was added demonstrated that the presence of rosemary did not cause noticeable changes in nanofibers morphology. Regarding the fact that the activity of glucose oxidase is affected by some factors such as pH and the presence of various materials, the effect of silver on antioxidant activity of glucose oxidase in different nanofibers was investigated. The results reflected that antioxidant activity decreased as the amount of silver increased, and the optimal amount of silver is 70/30 (NS-CS) sample with 0.05 grams of glucose oxidase due to the fact that rosemary has antioxidant activity and the main purpose of the current study is the function of such membranes in packaging food. In order to investigate the efficiency of different synthetic nanofibers, we used the membranes to package strawberries, and the results showed that NS-CS-R-GOx could be used for preserving food with its appearance, taste, and color kept unchanged and its shelf life increased as a result of this membrane's great antibacterial and antioxidant property and its fragrance because of the presence of rosemary.
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