Ecient Eco-Friendly Nano-Extracted Gelatin as Biodegradable Packaging Material

Extracted gelatin from the waste of fresh and grilled chicken skin was used to prepare lms as a biodegradable packaging material from solutions of various gelatin concentrations using a casting approach. The thermal behavior of extracted gelatins was investigated by differential scanning calorimetric. The particle size and zeta potential of dispersed nanoparticles of gelatins were measured by dynamic light scattering. The surface area of lyophilized gelatin nanoparticles was calculated from the adsorption of N 2 gas. Mechanical properties, water vapor permeability (WVP), and oil uptake (OU) of all manufactured lms were studied. Tensile strength values signicantly increased for lms manufactured from both gelatin sources when the concentration increased from 4 % to 6 % up to 5.1 MPa. The elongation of waste skin gelatin-based lms was higher than waste grilled skin gelatin (WG)-based lms using 4 % and 8 % concentrations up to 57 %. Films manufactured from WG had signicantly lower WVP than waste skin gelatin (WS) analogous at a 4 % gelatin concentration. The WVP of lms manufactured from gelatin signicantly increased as gelatin concentration increased where OU showed higher oil resistance for lms manufactured from WS up to 91 % using 4 % gelatin concentration. The morphological structure of the gelatin lm was investigated with scanning electron microscopy (SEM). A homogenized and smooth lm surface was observed. The percentage of heavy metal was examined by inductively coupled plasma (ICP). The results of this study showed that the lms manufactured using higher concentrations of gelatin possessed promising mechanical properties, good barrier properties, and high safety as a recommended biopolymer packaging material for food contact and pharmaceutical applications.


Introduction
Packaging of food and beverage is one of the largest upward industries within the plastic, glass, and paper packaging [1]. The conservatively plastic industries of packaging materials face great challenges such as sustainability, environmental problems, and the dwindling of fossil fuel-based feedstocks [2]. Biopolymers sources could replace or reduce commonly plastics production which are disbursed industrially, mainly for applications in the food packaging sector [3]. Recently, natural biopolymers gained great advantages as renewable in several types, biodegradable in the environment or edible [4] biopolymers can form polymer lms with a wide range of natural polymers (chitosan, starch, carrageenans, pectin, gelatin, collagen, whey protein, corn, etc.) [5]. (eira et al., 2011) One of the main industries for supplying the meat and protein needs for the customers is poultry [7].
The production rate is expanding with high rates [8]. Large quantities of solid wastes are produced as a by-product. For example, the waste for the slaughter weight of chicken is 33 % [9]. Many efforts are going to exploit these wastes and convert them to high-value-added products. Gelatin is one of the most addvalue products from protein waste especially the slaughter of chicken. Gelatin was extracted from leather solid waste for potential application in chemical sand-xation [10]. Bone can be used as a source to produce gelatin through the biocatalysis process [11]. Collagen is a special type of extracted protein from raw trimming waste of tannery for many applications like tissue engineering and the pharmaceutical industry [12]. Gelatin is selected as an interesting biodegradable material due to the semi-crystalline behavior of hydrated gelatin and low melting temperature [13]. The high density for the dynamic hydrogen bonding of amide linkages provides unique mechanical properties. Gelatin is known as an extracted protein through a precise hydrolysis process of the insoluble brous of collagen [14]. Skin and bones produced as waste during animal slaughtering and processing are the main sources of collagen [15]. Therefore, gelatin can be considered a low-cost biopolymer with excellent functional lm-forming properties [16].
The effect of plasticizers on the thermal and functional properties of gelatin had been studied [17], but less attention has focused on the effect of utilizing various ingredient concentrations and their subsequent impact on the properties of such lms. The study of nanoparticle formation of technical grade of gelatin as applied packaging lm is one of the novel characters during this research.
As shown in the previous state of the art, most of the recent researches are aimed to extract gelation as protein for various applications with clear de ciency to used nanotechnology. For that, this work was modi ed cheap and easy preparation method as a novel waste reduction technique combined nanotechnology add-value product. This new trend in waste assessment was targeted to produce nanogelatin and evaluate its enhancement in one of the vital application sectors as novel packaging nanolm.
The objective of this study was to prepare nanoparticles of extracted gelatins from waste chicken skin.
Two types of gelatin were extracted waste skin gelatin (WS) and waste grilled skin gelatin (WG). The size, charge, and surface area of gelatin nanoparticles were evaluated. Additionally, the mechanical properties, water vapor permeability, and oil permeability properties of gelatin lms manufactured using various ingredient concentrations from waste chicken skin fresh or grilled were evaluated.

Materials
The fresh chicken skin was obtained from a local company. Grilled chicken waste was purchased from Food manufacturing factories. Glycerol used as plasticizer and glutaraldehyde as crosslinker were purchased from Sigma Aldrich, Germany. Sodium hydroxide and hydrochloric acid were used as received without pre-puri cation.

Chicken skin preparation
The chilled chicken skin was kept in a refrigerator (4°C) overnight. After that, waste fresh skin was washed with a water stream to remove any impurities, the skin was cut into small parts and lyophilizedried for 72 hrs. Completely dry skin was milled and then defatted according to (AOAC, 2006

Composition of gelatin
The ash, moisture, and fat content of gelatins were characterized according to the methods described by AOAC [19]. The content of protein was measured as a function of total nitrogen content by the Kjeldahl method [19]. The value of nitrogen in gelatin protein can be calculated by multiplying the determined value with factor 5.5. The extraction e ciency of gelatin was monitored based on the dry weight of fresh skin according to the following formula:

Synthesis of gelatin nanoparticles
The double desolvation technique was used for the preparation of gelatin nanoparticles [20] with our vital modi cations. 6.1 wt.% gelatin solution was prepared by dissolving 3.05 g of gelatin (WS or WG) in 50 mL Milli-Q water with warming condition with magnetic stirring. The desolvation step was carried out by adding 60 mL of acetone to the gelatin solution. This was followed by re-desolvation in Milli-Q water and lyophilizing the precipitated gelatin fraction. A 2% gelatin solution was obtained via dissolving 0.2 g of the lyophilized gelatin (0.2 g) in 20 mL of Milli-Q water at 50°C. The dropwise addition of acetone under continuous stirring at 800 rpm for 12 hours was leading to the formation of nanoparticles. The puri ed nanoparticles were prepared through three-step centrifugation at 32,000 rpm for 15 min and redispersed in acetone/water (30/70 mixture). The resulted dispersion of nanoparticles in Milli-Q water was kept in the refrigerator.
The nanoparticles were analyzed for the mean diameter and zeta potential of the waste gelatins (WS and WG) particles at 170°, by dynamic light scattering (DLS) (NICOMP 380 ZLS, PSS, Santa Barbara, CA, USA). The dispersed solution was lyophilized and stored as a powder to use for surface area measurement.

Differential scanning calorimetry
Differential scanning calorimetry DSC131 evo (SETARAM Inc., France) was used to perform the differential scanning calorimeter analysis. The instrument was calibrated using the standards (Mercury, Indium, Tin, Lead, Zinc, and Aluminum). Nitrogen and Helium were used as the purging gases. The test was programmed including the heating zone from 25°C to 400°C with a heating rate of 10°C / min. The samples were weighted in an Aluminum crucible 30 ul and introduced to the DSC. The thermogram results were processed using (CALISTO Data processing software v.149).

Film preparation
WG and WS based on gelatin lms were prepared by solution casting employing concentrations that ranged from 4 % to 8 %. Glycerol as plasticizer (0.4 w/w) and glutaraldehyde as crosslinker (2.5 w/w) were added based on gelatin content. The lms were cast in Te on plates and dried for 24 hours at 80-85 % relative humidity and 35°C. After drying, lms were peeled off from the Te on plates and cut into test specimens. The prepared lm was characterized according to scheme 1.

Mechanical properties
The mechanical properties are should be taken into consideration to meet the desired processing and applications demands. Mechanical properties of casted lms were evaluated; tensile strength (TS), and elongation at break (E) of lms were measured by Zwick/RoellZ020 instruments, (Ulm, Germany) according to the ASTM-D412.

Measurement of oil uptake and water vapor permeability (WVP)
The control to permeate the gases, vapors is one of the important issues for exible packages. That is very critical for some products that need speci c barrier protection.

Gelatin Composition and bloom strength of gelatin gels
The composition of (waste skin gelatin) WS and (waste grilled skin gelatin) WG are tabulated in Table 1.
The protein content, moisture and ash content were 81.37 %, 9.96 and 0.41 % respectively for the lyophilized chicken skin gelatin. The evaluation of the difference in the composition of the extracted gelatin from chicken skin fresh (WS) and grilled (WG) is necessary to understand the properties of the prepared lms. The gel strength of WS is higher than WG 28.2 % probably due to the thermal treatment of WG through the grilling process. The gel strength of extracted gelatin from sh or chicken gives 181 and 263, respectively [24]. In addition, horse mackerel gelatin shows bloom strength 280 [25] and 177 g [11]. The low value of sh gelatin may be due to hydroxyproline content being relatively low in sh skin [26]. The hydroxyproline and proline can form hydrogen bonding with free water and triple-helix of gelatin structure [15]. Also, the bloom strength of gelatin is affected by many factors like chemical handling of gelatin, type, form, concentration, and source of gelatin, besides the thermal history of the extracted gelatin [11].
In addition, the value of bloom strength is directly proportional to the gelation point, melting, and gelation time of the extracted gelatin.

Differential Scanning Calorimetry
One of the most popular tools to investigate thermal behaviors in food ingredients such as gelatin is Differential scanning calorimetry (DSC). Recently, endothermic T m of bovine gelatin was characterized during the thermal investigation of extracted gelatin [27]. This is can be attributed to the thermal treatment of chicken skin through the grilling process.

Particle size and zeta potential
The particle size of WS and WG was measured according to Intensity-weight with uniform bell shape Gaussian distribution. The double desolvation process can be produced unique gelatin particles in nanoscale size.
The particle size distributions with unimodal form are illustrated in Fig. 2. Moreover, the zeta potential at low voltage was measured in an aqueous solution (pH 7). Over many measurements, the average zeta potential value was calculated as shown in Fig. 2. the stability of gelatin particles through eleven measurements is perfect and nearly the same. As well known, the unique relationship between volume/surface area of creating nanomaterials will be generated a novel character [29]. the prepared nano-gelatin with particle size less than 80 nm was shown a promising enhancement in physical properties of prepared nono-lm. The zeta potential can give a clue for homogeneity and well dispersion of gelatin solution. This is directly related to the lm performance and stability of collided particles during the casting process with a proper zeta value in the moderated stable range from 10 to 40 mV [30]. Particle size and zeta potential are vitally characterized by nanomaterials especially gelatin dispersed in an aqueous medium.

Mechanical properties
The mechanical properties of gelatin lms were presented in Table 3. The tensile strength (TS) of WG and WS-based lms were not signi cantly different from each other. This was also observed regardless of the gelatin concentrations used. However, TS values for lms, manufactured from both sources of gelatin used in this study, signi cantly increased when the concentration increased from 4 % to 6 %. No further signi cant increases occurred when gelatin concentration increased from 6 % to 8 %. Means in the same column for 5 replicated samples with signi cant difference (P < 0.05) TS was unaffected by the gelatin source. TS for lms containing 8 % gelatin concentration manufactured using WS presented greater lm strength than lms manufactured from WG. This also happened to be the lm with the greatest TS (20.42 N) of all lms tested. The concentration of gelatin used also signi cantly affected TS, whereby the higher the concentration of gelatin used, the greater was TS.
No signi cant differences were observed for Elongation (E) values from lms manufactured from both sources of gelatin and at gelatin concentrations between 4 % and 6 %. However, at an 8 % gelatin concentration, E values for lms derived from WG had greater E than analogous lms manufactured from WS. However, when gelatin concentrations ranged from 4 % to 6 %, E properties decreased, irrespective of gelatin species origin.

Water vapor permeability (WVP)
WVP values are indicated in Figure 3. WG and WS gelatin behaved differently from each other concerning WVP. This is maybe revealed to heat treatment of chicken skin within the grilling process. WG-based lms containing a gelatin concentration of 4 % had lower WVP values when compared to WS-derived gelatin lm equivalents. WVP values for both WG and WS-derived gelatin lms had similar values by using 6 % and 8 % gelatin concentrations were used to manufacture lms. The lms manufactured from WG showed no signi cant differences from each other for WVP as the concentration of gelatin used to produce the lms increased. However, lms manufactured from WS showed increased resistance to WVP as the gelatin concentration increased from 4 % to 6 %, but not at any other concentration used.
WS skin with 4 % concentration possessed the lowest WVP value, below 60 g·mm/kPa·d·m 2 . it was noted that as the gelatin concentration increased in lms, so too did the requirement for greater plasticizer addition. This consequently led to an increase in WVP, but this was especially apparent for lms manufactured from WS. It is well recognized that the addition of plasticizer to a protein-based lm mixed solution causes the protein network to become less dense and more permeable [31], however, the plasticizing effect noted in this study is interesting in that it appears to affect the same protein source in different ways, depending on the differences associated with the protein-based on its species origin.

Oil uptake (OU)
OU of lms manufactured from WG-and WS and used at a 4 % concentration showed no signi cant differences ( Figure 4). The lms manufactured using both 6 % and 8 % gelatin showed signi cant differences in oil uptake by both lms which resulted in differences in weight gain. The lms manufactured from WS had more resistance to oil uptake. In terms of gelatin concentration, no signi cant differences were observed within each species-de ned lm type for OU.

Topographic investigation of gelatin lms
The surface morphologies of the prepared lms were characterized utilizing scanning electron microscopy (SEM). This helps to understand the effect of chicken waste sources (i.e. fresh or grilled skin).
It can be observed from the SEM images shown in Fig. 5 that the lms exhibited uniform surface morphology. Gelatin-based fresh chicken skin wastes lm was presence more homogenized and smoother than that based on grilled chicken skin wastes. This is maybe due to some impurities that remain in gelatin lms based on WG or thermal degradation for the polymer chains during the grilling process.
3.8. Study the overall migration of chemical substances from the prepared lms The migration of any chemical substances from the prepared lms has been studied according to the EU Regulation Nr. 10/2011. The stimulants were selected carefully to represent different food natures. As shown in Table 4, the prepared lms showed highly acceptable migration limits. The overall migration (OM) from the WG and WS gelatin lms were ranged from 0 up to 0.3 mg/dm 2 . The regulation limits the accepted level up to 10 mg/dm 2 . lead, mercury, chrome, aluminum, arsenic, and antimony. The levels of migrated heavy metals in different simulants were in the accepted levels as shown in Table 5 [32]. The highly acceptable levels of overall migration and the traces of the heavy metal ensure the prepared gelatin lms are appropriate as a foodcontact layer.

Conclusions
Overall, it was shown in this study that gelatin formed very functional lms which possessed some very good mechanical and barrier properties. Film properties, as affected by ingredient concentration, were interesting, but more so were the differences that emerged when gelatin was utilized from different animal sources for lm manufacture and compared. The originality of bio-waste recycling especially gelatin is a global issue. For that, this research has deep originality by preparation add value products from waste with eco-friendly behavior as e cient packaging lm. Moreover, this research article is discussing two novels missed research points: the effect of utilizing various ingredient concentrations and their subsequent impact on the properties of such lms and the study of nanoparticle formation of technical grade of gelatin as applied packaging lm Gelatin was extracted from grilled and fresh chicken skin with a low-cost process. Biodegradable, cheap, and eco-friendly lms based on the extracted gelatin have been fabricated. The effect of various ingredients and gelatin concentration were studied. The prepared lms were fully evaluated as practical packaging substrates. The evaluation was achieved by investigation of mechanical properties, water vapor, and oil uptake, and migration of any undesirable substances.
Declarations Figure 1 DSC thermograms of WS and WG extracted gelatins Page 17/20 Oil uptake test of WG and WS gelatin lms Figure 5 Surface morphological structure of a) gelatin lm from WS and b) gelatin lm from WG.