Design of Gelatin Pouches for the Preservation of Flaxseed Oil during Storage

: Flaxseed oil (FO) is composed mainly of polyunsaturated fatty acids that are very sensitive to oxidation induced by oxygen, temperature, and light. To overcome this problem, gelatin-based ﬁlms containing glucose are crosslinked via the Maillard reaction (MR) at 120 ◦ C and used for the conception of pouches in which the FO was packaged. The prepared pouches, as well as the oil alone, are incubated at 50 ◦ C for 21 days. The results show that the peroxide index of the oil stored in the gelatin-based pouches is almost stable and decreases on the 21st day. The speciﬁc extinction coe ﬃ cients prove a more pronounced degradation of the non-package oil (control). In addition, the results of thiobarbituric acid reactive substances (TBARs) test reveal higher values in the control, with a tendency to continuously increase up until the 21st day. However, the oil stored in the pouches reveals less TBARs content, decreasing with oxidation time. The non-heated glucose-supplemented pouches showed the best results, suggesting a moderate and gradual development of the MR at 50 ◦ C. The application of pouches based on gelatin ﬁlms seems to be an e ﬀ ective and interesting tool for protecting FO against lipid oxidation, acting as perfectly biodegradable and sustainable containers for small doses. and 34.59 mL equivalent MDA/g of oil, respectively, for the positive and negative controls. The continuous increase of TBARs content could be due to the availability of oxygen in FO stored in the open bottle (negative control) and the lower oxygen barrier property of the closed bottle (positive control) as compared to gelatin pouches. These findings indicate the protective effect of gelatin pouches in retarding the oxidation of flaxseed oil.


Introduction
Flaxseed oil (FO) is rich in polyunsaturated fatty acids (PUFA), namely α-linolenic acid (18:3 n-3), which is especially important for human health [1]. In addition, this vegetable oil is known to have therapeutic potential as an anti-inflammatory and anti-hypertensive agent, as well as possessing high bioavailability and solubility of lipophilic compounds for efficient drug delivery [2,3]. However, flaxseed oil is extremely susceptible to oxidative rancidity because of the presence of double bonds in the main chain of the unsaturated fatty acid molecules, which represent more than 75% of the oil [4]. These properties lead to a major problem during storage and represent a limitation for application in functional foods [3].
Indeed, the process of lipid oxidation is initiated by light radiation and atmospheric oxygen and is maintained through the generation of free radicals during the consequent chain reaction [5], where hydroperoxides are formed as a result. As they are unstable, they decompose relatively quickly into aldehydes, ketones, alcohols, acids, esters, or hydrocarbons [6]. These latter, secondary oxidative products induce the characteristic off-flavor of oxidized oils, leading therefore to a loss in nutritional

Oxygen Transfer Through the Films
The oxygen transfer was assessed using a manometric method with a commercial permeability testing apparatus (Brugger, Type GDP-C, Brugger Feinmechanik GmbH, Munich, Germany). The film sample of the 144 cm 2 surface area was placed between the top and bottom part of the permeation cell. The exposed area to the transfer of oxygen was 95.1 cm 2 . Prior to each test, the bottom part of the permeation cell was out-gassed under a primary vacuum (<2 hPa). During testing, the top part was continuously flushed with the tested gas (O 2 ). In the bottom chamber, the pressure increase was monitored over time. The permeation transfer rate was calculated from the steady state and then expressed in cm 3 /m 2 .d. Temperature was maintained (25 ± 0.5 • C) using a water bath (Julabo F32). The oxygen permeation measurements were conducted at 0% relative humidity because the oil is considered as a dry product.

Packaging of Flaxseed Oil (FO) in Gelatin Pouches
Gelatin-based films either containing or not containing glucose, heated or not at 120 • C (FG 25 • C, FG 120 • C, FG-Glc 25 • C, and FG-Glc 120 • C), were used for the preparation of the pouches. To this end, aforementioned films were first folded in half and then thermally welded on 2 sides (the sealing temperature was about 120-130 • C) in order to form pouches. This was done using a CONSERVAIR ® machine (heat seal machine). Then, FO (10 mL) was transferred into the pouches which were subsequently welded thermally on the fourth side. Furthermore, two controls were prepared. The positive control contained FO in a closed bottle, while the negative control contained FO in an open bottle. The prepared pouches, as well as the positive and negative controls, were incubated in darkness at 50 • C for 21 days as an accelerated test ( Figure 1). Oil samples were periodically sampled in order to monitor the FO thermo-oxidation kinetics.

Peroxide Value (PV)
The peroxide value (PV) was determined according to the method of Uluata et al. [17]. An oil sample of 0.2 g was added to a mixed solvent of 1.6 mL of isooctane/2-propanol (3:1 v/v) and then vigorously vortexed (10 s, 3 times). Then, 0.2 mL of the upper extract was carefully removed and blended with a mixed solvent of 2.8 mL of methanol/1-butanol mixture (2:1, v/v). Subsequently, 15 µL of Fe 2+ solution (freshly prepared from the supernatant of a mixture of equal volume of 0.132 M BaCl 2 and 0.144 M FeSO 4 in 0.4 M HCl) and 15 µL of 3.94 M ammonium thiocyanate were added. The final mixture (3 mL) was vortexed and kept at room temperature in the dark for 20 min. The absorbance of the mixture was measured at 510 nm with a UV spectrophotometer (Model V-530, Jasco, Nantes, France). Peroxide values were calculated with a standard curve of cumenehydroperoxide at concentrations ranging from 0 to 0.2 mg/3 mL. The results were expressed in terms of mmoles of cumene hydroperoxide equivalent per g of oil.

Quality Changes of FO Packaged in Gelatin Pouches
The peroxide value (PV) was determined according to the method of Uluata et al. [17]. An oil sample of 0.2 g was added to a mixed solvent of 1.6 mL of isooctane/2-propanol (3:1 v/v) and then vigorously vortexed (10 s, 3 times). Then, 0.2 mL of the upper extract was carefully removed and blended with a mixed solvent of 2.8 mL of methanol/1-butanol mixture (2:1, v/v). Subsequently, 15 μL of Fe 2+ solution (freshly prepared from the supernatant of a mixture of equal volume of 0.132 M BaCl2 and 0.144 M FeSO4 in 0.4 M HCl) and15 μL of 3.94 M ammonium thiocyanate were added. The final mixture (3 mL) was vortexed and kept at room temperature in the dark for 20 min. The absorbance of the mixture was measured at 510 nm with a UV spectrophotometer (Model V-530, Jasco, Nantes, France). Peroxide values were calculated with a standard curve of cumenehydroperoxide at concentrations ranging from 0 to 0.2 mg/3 mL. The results were expressed in terms of mmoles of cumene hydroperoxide equivalent per g of oil.

Concentration of Thiobarbituric Acid Reactive Substances (TBARs)
Thiobarbituricacid reactive substance (TBARs) values were determined with the modified method of Qiu et al. [18]. A TBA reagent was prepared by dissolving 0.375 g of TBA, 1.76 mL of 12 M HCl and 15 g of trichloroacetic acid into 82.9 mL of distilled water. An oil sample of 0.2 mL was incorporated into the 3.8 mL TBA reagent in a test tube with slight shaking and then placed in a

Concentration of Thiobarbituric Acid Reactive Substances (TBARs)
Thiobarbituricacid reactive substance (TBARs) values were determined with the modified method of Qiu et al. [18]. A TBA reagent was prepared by dissolving 0.375 g of TBA, 1.76 mL of 12 M HCl and 15 g of trichloroacetic acid into 82.9 mL of distilled water. An oil sample of 0.2 mL was incorporated into the 3.8 mL TBA reagent in a test tube with slight shaking and then placed in a boiling water bath for 20 min. The mixture was centrifuged at 3000× g for 15 min after cooling down in an ice water bath and then measured at 532 nm with a UV spectrophotometer. A standard curve was prepared using 1,1,3,3-tetraethoxypropane (malonaldehydebis(dimethyl acetal)) in the TBA reagent at concentrations ranging from 0 to 0.1 mL/2mL. The results of TBARs concentrations were expressed as the malonaldehyde equivalent in mL per g of oil.

K 270 Extinction Coefficient
The oil samples were diluted in acetone and then, after homogenization, the optical density was measured at 270 nm using a spectrophotometer (SAFAS UVmc, Monaco city, Monaco) in order to assess the development of secondary oxidation products [19]. Pure acetone was used as a reference here. Indeed, the specific extinction coefficient was used to characterize the degree and the stage of oxidation of oil. During the oxidation, the hydroperoxides, the primary oxidation products, are converted into various secondary products, including volatile and non-volatile off-flavor products, namely, esterified aldehydes, which absorb in the UV range of about 270 nm [19].

Free Fatty Acid Content (FFA)
The free fatty acid content (FFA), used as an index of hydrolysis, was determined according to the method of Takeungwongtrakul et al. [20]. An oil sample (0.1 g) was added with 5 mL of isooctane and swirled vigorously to dissolve the sample. The mixture was then added with 1 mL of a 5% (w/v) cupric acetate-pyridine reagent, freshly prepared by dissolving 5 g of the reagent grade cupric acetate in 100 mL of water, filtering, and then adjusting the pH to 6.0-6.2 using pyridine. The mixture was shaken vigorously for 90 s using a vortex and allowed to stand for 20 s. The upper layer was subjected to absorbance measurement at 715 nm. A standard curve was prepared using oleic acid in isooctane at concentrations ranging from 0 to 50 µmol/5 mL. Free fatty acid (FFA) content was then expressed as g of oleic acid per 100 g of oil (g oleic acid /100 g oil ).

Intrinsic Viscosity of FO
The intrinsic viscosity of flaxseed oil, before and after oxidation, was assisted using an NMR apparatus (Bruker Minispec mq20, Palaiseau, France). This technique allows the measurement of the oil relaxation time (T 2 ). The analysis was carried out using a gain of around 65 dB, with 16 scans, a recycle delay of 20 s, and a frequency of 20 MHz. Then, T 2 was directly related to the local viscosity according the Boltzman and Stokes-Einstein laws.

Color
A CIE colorimeter (Chroma Meter CR-400; Konica Minolta, Roissy, France) was used in order to assess the color changes of flaxseed oil and gelatin pouches before and after application. A white standard color plate (L 0 = 97.5, a 0 = −0.1 and b 0 = 2.3) was used as a background for the color measurements. The color of the gelatin pouches and that of the flaxseed oil were expressed as L (lightness/brightness), a (redness/greenness), and b (yellowness/blueness) values.

Light Transmission
Gelatin pouches samples (1 × 3 cm) were placed in the test cell of a UV-visible spectrophotometer (SAFAS UVmc, Monaco city, Monaco). An empty test cell was used as a reference. The UV-vis absorption spectra was recorded in the wavelength range of 200 to 800 nm.

Differential Scanning Calorimetry (DSC)
The thermal properties of the gelatin pouches were studied using a differential scanning calorimeter (DSC Q20, TA Instruments, Guyancourt, France). Pouches (5 mg) were placed into aluminum pans, sealed, and subjected to a double heating-cooling cycle from −50 • C to 150 • C at a rate of 10 • C/min. An empty aluminum pan was used as a reference. Nitrogen was used as the purge gas at a flow rate of 25 mL/min. The glass transition temperature (Tg) for each sample was then determined from the mid-point of the second heating cycle using the TA Universal Analysis 2000 software (version 4.5 A, TA instruments, Guyancourt, France).

Statistical Analysis
Statistical analyses were performed with SPSS version 17.0, professional edition, using ANOVA analysis at a p-value < 0.05. Duncan's multiple range test (p-value < 0.05) was used to detect differences among the mean values of all the analyzed parameters.

Peroxide Value (PV)
The oxidation of flaxseed oil was assessed firstly by the measurement of the peroxide value. This index is used for the determination of the concentration of hydroperoxides formed at the initial stage of lipid oxidation, which are unstable and easily spontaneously decomposed into secondary oxidation products [21,22]. The results in Figure 2 display the variation of the peroxide value (PV) as a function of storage time. The peroxide index of the oil stored in the gelatin-based pouches increased slightly on the 4th day and then was maintained as almost stable until the 15th day. During the early stages of storage, the lipid oxidation was propagated by consuming oxygen present in the headspace of the pouch [23]. Among all the formulations, FO packaged in non-heated gelatin-glucose (FG-Glc 25 • C) pouches exhibited the best oxidation stability, as it showed the lower peroxide values during the storage period of 15 days. Indeed, FG-Glc 25 • C pouches contain glucose and were exposed to a moderate heat treatment (50 • C, 15 days) during the storage test, which induced the development of Maillard reaction and the possibility of the release of antioxidants (MR products). Therefore, this fact may explain the higher stability of FO packaged in FG-Glc 25 • C pouches as compared to the other pouches formulations. At the 21st day, a significant decrease in the PV to 0.48, 0.83, 0.87, 0.72 mMoles cumene hydroperoxide equivalent/g of oil was noted for FO packaged in FG 25 • C, FG 120 • C, FG-Glc 25 • C and FG-Glc 120 • C, respectively. This decrease in the PV suggest the decomposition of peroxides to secondary oxidation products. However, for the positive and negative controls, peroxide values increased significantly as a function of storage time and reached 6.75 and 9.40 mMoles cumene hydroperoxide equivalent/g of oil at the 21st day, respectively. This increase in PV evidences the continuous progression of the oil oxidation process until the 21st day. Indeed, lipid radicals react with oxygen to form peroxyl radicals, which act as the chain carriers of the rapid progressing reaction by attacking new lipid molecules [21]. These results are in accordance with those of Nilsuwan et al. [14], who reported that chicken skin oil packaged in the pouches based on fish gelatin and those containing epigallocatechin gallate showed a lower peroxide value after 30 days of storage at 25-28 • C, in comparison with that packaged in low-density polyethylene pouches (which were used as a control). The development of secondary oxidation products in FO was monitored by the measurement of the thiobarbituric acid reactive substances ( Figure 3). Indeed, TBARs have been used to measure the concentration of relatively polar secondary lipid oxidation products, especially aldehydes, including malondialdehyde (MDA) [24]. The results reveal that the oil stored in the pouches, especially in  The development of secondary oxidation products in FO was monitored by the measurement of the thiobarbituric acid reactive substances ( Figure 3). Indeed, TBARs have been used to measure the concentration of relatively polar secondary lipid oxidation products, especially aldehydes, including malondialdehyde (MDA) [24]. The results reveal that the oil stored in the pouches, especially in gelatin-glucose ones, display less TBARs content, decreasing with oxidation time from 10.28 and 13.69 mL equivalent MDA/g of oil at the 4th day to 5.56 and 7.27 mL equivalent MDA/g of oil at the 21st day, respectively, for the FG-Glc 25 • C and FG-Glc 120 • C pouches. The protective effect seen in the gelatin-glucose pouches for flaxseed oil oxidation could be explained by their antioxidant potential, which is in agreement with our previous study which revealed the in vitro lipid peroxidation inhibition ability of Maillard reaction-treated gelatin-glucose films during linoleic acid storage at 45 • C [15]. However, higher TBARs values have been obtained for the controls, with a tendency to continuously increase until the 21st day to reach 25

K270 Extinction Coefficient
Furthermore, the absorbance at 270 nm has been determined for FO packaged or not in gelatin pouches in order to confirm the development of secondary oxidation products. Indeed, the greater the extinction at 270 nm, the greater the richness of secondary oxidation products in the oil, which reflects its poor preservation ability [25]. As can be seen in Figure 4

K 270 Extinction Coefficient
Furthermore, the absorbance at 270 nm has been determined for FO packaged or not in gelatin pouches in order to confirm the development of secondary oxidation products. Indeed, the greater the extinction at 270 nm, the greater the richness of secondary oxidation products in the oil, which reflects its poor preservation ability [25]. As can be seen in Figure 4, both the positive and negative controls displayed higher absorbance values compared to that of oil packaged in gelatin pouches. In addition, absorbance values increased significantly as a function of storage time. This increase in the K 270 nm values reveals the continuous propagation of the lipid oxidation reaction to the development of secondary products in the control FO, as demonstrated previously by the TBARs test.
pouches in order to confirm the development of secondary oxidation products. Indeed, the greater the extinction at 270 nm, the greater the richness of secondary oxidation products in the oil, which reflects its poor preservation ability [25]. As can be seen in Figure 4, both the positive and negative controls displayed higher absorbance values compared to that of oil packaged in gelatin pouches. In addition, absorbance values increased significantly as a function of storage time. This increase in the K270 nm values reveals the continuous propagation of the lipid oxidation reaction to the development of secondary products in the control FO, as demonstrated previously by the TBARs test.

Free Fatty Acid (FFA) Content
The variation of free fatty acid content of FO packaged in gelatin pouches as a function of storage time was investigated and results are presented in Figure 5. FO packaged in gelatin pouches revealed a lower free fatty acid content compared to the control oils, especially at the third week of storage. Among the different formulations, FO packaged in unheated FG and FG-Glc pouches displayed a lower FFA content (p < 0.05) and the values found on the 21st day were 0.260 and 0.310 g oleic acid/100 g of oil, respectively. Indeed, the release of free fatty acids reveals the presence of hydrolysis, which seems to be favored by heat-treated films. However, on the 21st day, both the positive and negative controls displayed the highest hydrolysis rate of polyunsaturated fatty acids and the obtained values were 0.85 and 1.26 g oleic acid/100 g of oil, respectively. Indeed, the increase in free fatty acid content in uncovered flaxseed oils (positive and negative controls) at the end of the storage period (21st day) can be explained by the oxidation process, which is favored by temperature (50 • C) and the presence of oxygen. Furthermore, as the lipid oxidation process was favored and reached a maximum in the 21st day (as proven by the peroxide index and TBARs results), the hydrolysis rate of polyunsaturated fatty acids will be bigger, and the free fatty acid content will be more important. The hydrolysis of glycerol-fatty acid esters occurs normally in lipids and is catalyzed by lipases and phospholipases [26]. Generally, lipids can undergo hydrolysis in the presence of moisture and heat [24]. According to Takeungwongtrakul et al. [20], released free fatty acids are likely prone to oxidation, leading to lipid oxidation, as indicated by the increases in TBARs values, particularly at the end of storage.

Intrinsic Viscosity
A NMR technique was used in order to determine the changes in flaxseed oil intrinsic viscosity before and after the storage period. The results in Table 1 reveal that flaxseed oil (d0) displayed a relatively lower T 2 value (159.2 ± 1 ms) compared to that stored during 21 days at 50 • C in the different pouch formulations. After 21 days of storage, the T 2 values of the FO packaged in gelatin pouches increased to 167.5, 168.5, 165.5, and 167.5 ms for the FG 25 • C, FG 120 • C, FG-Glc 25 • C, and FG-Glc 120 • C pouches, respectively. For the positive and negative control oils, the T 2 values were 163.5 and 160 ms, which are lower than those of packaged FO. Indeed, the T 2 parameter corresponds to the relaxation time. Generally, if T 2 decreases, the sample becomes less viscous and maybe related to the oil degradation [27]. According to the obtained results, we can conclude that the FO intrinsic viscosity decreased, especially for the uncovered oil samples, due to the oxidation process. The T 2 , or relaxation time measured by NRM, represents the time required for the transverse magnetization to fall to approximately 37% (1/e) of its initial value. It is directly related to the local viscosity according the Boltzman and Stokes-Einstein laws. Thus, if the oil degradation or oxidation modify (as often) the viscosity of the oil, the T 2 value will be modified. displayed a lower FFA content (p < 0.05) and the values found on the 21st day were 0.260 and 0.310 g oleic acid/100 g of oil, respectively. Indeed, the release of free fatty acids reveals the presence of hydrolysis, which seems to be favored by heat-treated films. However, on the 21st day, both the positive and negative controls displayed the highest hydrolysis rate of polyunsaturated fatty acids and the obtained values were 0.85 and 1.26 g oleic acid/100 g of oil, respectively. Indeed, the increase in free fatty acid content in uncovered flaxseed oils (positive and negative controls) at the end of the storage period (21st day) can be explained by the oxidation process, which is favored by temperature (50 °C) and the presence of oxygen. Furthermore, as the lipid oxidation process was favored and reached a maximum in the 21st day (as proven by the peroxide index and TBARs results), the hydrolysis rate of polyunsaturated fatty acids will be bigger, and the free fatty acid content will be more important. The hydrolysis of glycerol-fatty acid esters occurs normally in lipids and is catalyzed by lipases and phospholipases [26]. Generally, lipids can undergo hydrolysis in the presence of moisture and heat [24]. According to Takeungwongtrakul et al. [20], released free fatty acids are likely prone to oxidation, leading to lipid oxidation, as indicated by the increases in TBARs values, particularly at the end of storage.

Intrinsic Viscosity
A NMR technique was used in order to determine the changes in flaxseed oil intrinsic viscosity before and after the storage period. The results in Table 1 reveal that flaxseed oil (d0) displayed a relatively lower T2 value (159.2 ± 1 ms) compared to that stored during 21 days at 50 °C in the different pouch formulations. After 21 days of storage, the T2 values of the FO packaged in gelatin pouches increased to 167.5, 168.5, 165.5, and 167.5 ms for the FG 25 °C, FG 120 °C, FG-Glc 25 °C, and FG-Glc 120 °C pouches, respectively. For the positive and negative control oils, the T2 values were 163.5 and 160 ms, which are lower than those of packaged FO. Indeed, the T2 parameter corresponds to the relaxation time. Generally, if T2 decreases, the sample becomes less viscous and maybe related

Color
The changes in FO color were assessed before (day 0) and after (day 21) the storage period, as illustrated in Table 2. FO color parameters were maintained as almost stable after being packaged in gelatin pouches during 21 days under heating conditions (50 • C). However, both the positive and negative controls displayed a notable increase in their a-values and a decrease in their b-values after 21 days of storage at 50 • C. These results prove the variation of the uncovered flaxseed oil color parameters and correlate well with the visual observations, revealing that the oxidation process causes the lightening in the control FO. These findings are in agreement with the observations of Choo et al. [28], who explained that the decrease in color trends (the loss of yellowness) of heated flaxseed oils may be due to the reduction of natural carotenoids that are naturally present in flaxseed oil, resulting in their oxidation (bleaching of the carotenoids) or decomposition during heating.

Physicochemical Properties of Gelatin Pouches Related to the Application with Flaxseed Oil
Firstly, the oxygen transfer rate of the gelatin films was measured at 0% relative humidity and the obtained values were 0.3 ± 0.1, 0.5 ± 0.1, 0.33 ± 0.1 and 0.4 ± 0.1 cm 3 /m 2 ·d for FG 25 • C, FG 120 • C, FG-Glc 25 • C, and FG-Glc1 20 • C, respectively. These results reveal the very low oxygen permeability of gelatin films, with no significant effect from heat treatment nor the Maillard reaction. Therefore, the tested gelatin films can be effectively used as efficient barriers against oxygen transfer. Moreover, the oxygen values are very low compared to that of many edible films, where the values range from to 5 to 2000 cm 3 /m 2 ·d, and, compared to common plastic films, the most efficient (PET/PA/PET three layers) values is about 0.1, or, for polyolefine films, this ranges from 1000 to 150,000 cm 3 /m 2 ·d [29][30][31].
Secondly, the optical properties of the gelatin pouches were investigated by the measurement of their color before (day 0) and after (day 21, after being in contact with oil) the storage period. The data in Table 2 display almost the same color parameters (L, a and b) for the gelatin pouches before and after 21 days of storage at 50 • C, except for the FG-Glc 25 • C pouches. These pouches showed a slight decrease in their L and a values and a noticeable increase in their b values on the 21st day as compared to the initial day. This variation in color parameters affirms that the storage period (21 days at 50 • C) induces a moderate Maillard reaction development in unheated gelatin-glucose-based pouches. Indeed, it is well known that the Maillard reaction occurs rather slowly at 35 • C but accelerates at temperatures above 55 • C, and most studies dealing with the Maillard reaction have usually been carried out between 55 • C to 120 • C. Regarding the incubation time, it ranges from as low as 30 min to as high as 24 h, and even up to several days [32].
These findings were confirmed by the results of UV-visible spectroscopy ( Figure 6). As can be seen, only a slight increase in the absorbance spectra (in the range of 250-300 nm) has been revealed for the FG 25 • C and FG 120 • C pouches on the 21st day due to the heating process (50 • C). However, for the FG-Glc 25 • C pouches, a notable increase of light absorbance was obtained in the range of 250-400 nm. Indeed, this absorbance is specific of Maillard reaction products (MRPs), as previously demonstrated by Etxabide et al. [33]. The development of MRPs in the FG-Glc 25 • C pouches suggests that these compounds can successfully retard primary lipid oxidation based on their ability to break radical chains, breakdown hydrogen peroxide, and scavenge oxygen reactive species [34]. For the FG-Glc 120 • C pouches, the same UV-visible spectra were obtained before and after 21 days of storage at 50 • C. In addition, the spectra revealed that the FG-Glc 120 • C pouches exhibit a high protection against UV light in the range of 200-400 nm due to presence of brown MRPs. This protection against UV light suggests the potential preventive effect of FG-Glc 120 • C pouches on the retardation of product oxidation induced by UV light.
These findings were confirmed by the results of UV-visible spectroscopy ( Figure 6). As can be seen, only a slight increase in the absorbance spectra (in the range of 250-300 nm) has been revealed for the FG 25 °C and FG 120 °C pouches on the 21st day due to the heating process (50 °C). However, for the FG-Glc 25 °C pouches, a notable increase of light absorbance was obtained in the range of 250-400 nm. Indeed, this absorbance is specific of Maillard reaction products (MRPs), as previously demonstrated by Etxabide et al. [33]. The development of MRPs in the FG-Glc 25 °C pouches suggests that these compounds can successfully retard primary lipid oxidation based on their ability to break radical chains, breakdown hydrogen peroxide, and scavenge oxygen reactive species [34]. For the FG-Glc 120 °C pouches, the same UV-visible spectra were obtained before and after 21 days of storage at 50 °C. In addition, the spectra revealed that the FG-Glc 120 °C pouches exhibit a high protection against UV light in the range of 200-400 nm due to presence of brown MRPs. This protection against UV light suggests the potential preventive effect of FG-Glc 120 °C pouches on the retardation of product oxidation induced by UV light. On the other hand, the thermal properties of gelatin pouches before and after the storage period were studied using the differential scanning calorimetry and the glass transition temperature (Tg) was determined from the second heating cycle. As shown in Table 3 and Figure 7, before application, the Tg value of the unheated glucose-free gelatin pouches was 53.8 °C and increased to 57.8 °C after the films were heated at 120 °C during 24 h. Regarding the gelatin-glucose pouches, their Tg values were 48.4 °C and 59.8 °C for the unheated and 120 °C-heated ones, respectively. Further, the thermal properties of the gelatin pouches were investigated for the inner (the pouch part that is in contact with the oil) and outer part of the pouches after the storage period (21 days at 50 °C). As can be seen, a slight decrease in the Tg values has been noted for the outer part of the gelatin pouches. However, a notable increase in the Tg values was obtained for gelatin pouch inner parts, probably due to the contact between the flaxseed oil and the pouches. In this context, Taktak et al. [35] noted that the Tg values of European eel oil emulsified gelatin/protein isolate blend films are higher than those of non-emulsified films. Indeed, it has been reported that Tg increases with the amount of chain stiffness and bonding, along with the crosslinking between chains and the degree of crystallinity [36]. Table 3. Glass transition temperature (Tg) of the FG and FG-Glc pouches at the initial time (d0) and 21 days after application (d21). The inner part consists of the film sample in contact with the oil, whereas the outer part is not in contact with the oil. The average relative error of the data is about 10%.

Pouches
Tg (  On the other hand, the thermal properties of gelatin pouches before and after the storage period were studied using the differential scanning calorimetry and the glass transition temperature (Tg) was determined from the second heating cycle. As shown in Table 3 and Figure 7, before application, the Tg value of the unheated glucose-free gelatin pouches was 53.8 • C and increased to 57.8 • C after the films were heated at 120 • C during 24 h. Regarding the gelatin-glucose pouches, their Tg values were 48.4 • C and 59.8 • C for the unheated and 120 • C-heated ones, respectively. Further, the thermal properties of the gelatin pouches were investigated for the inner (the pouch part that is in contact with the oil) and outer part of the pouches after the storage period (21 days at 50 • C). As can be seen, a slight decrease in the Tg values has been noted for the outer part of the gelatin pouches. However, a notable increase in the Tg values was obtained for gelatin pouch inner parts, probably due to the contact between the flaxseed oil and the pouches. In this context, Taktak et al. [35] noted that the Tg values of European eel oil emulsified gelatin/protein isolate blend films are higher than those of non-emulsified films. Indeed, it has been reported that Tg increases with the amount of chain stiffness and bonding, along with the crosslinking between chains and the degree of crystallinity [36]. Table 3. Glass transition temperature (Tg) of the FG and FG-Glc pouches at the initial time (d 0 ) and 21 days after application (d 21 ). The inner part consists of the film sample in contact with the oil, whereas the outer part is not in contact with the oil. The average relative error of the data is about 10%.

Conclusions
A novel formulation of gelatin and gelatin-glucose based pouches has successfully been prepared and investigated here for packaging flaxseed oil and its protection against oxidation under accelerated conditions (50 °C, 21 days). The results found for the peroxide index, TBARs, and FFA assays, monitoring the development of primary and secondary oxidation products, indicate that the different gelatin-based pouch formulations protect flaxseed oil against oxidation, as compared to

Conclusions
A novel formulation of gelatin and gelatin-glucose based pouches has successfully been prepared and investigated here for packaging flaxseed oil and its protection against oxidation under accelerated conditions (50 • C, 21 days). The results found for the peroxide index, TBARs, and FFA assays, monitoring the development of primary and secondary oxidation products, indicate that the different gelatin-based pouch formulations protect flaxseed oil against oxidation, as compared to uncovered oil. Particularly, the non-heated gelatin-glucose pouches showed higher stability for the flaxseed oil, approved by the lower peroxide index, secondary metabolites and free fatty acids values, suggesting therefore a moderate and gradual development of the MR at 50 • C. Indeed, the protective effect of MR-treated gelatin pouches is due to both their UV and oxygen barrier properties and also due to the bioactive compounds generated by the MR. These interesting findings suggest the potential use of the produced bioactive gelatin pouches as an alternative to synthetic packaging for the protection of sensitive foods against oxidation.
Author Contributions: The individual contribution of H.K. is methodology, experiments, data analyses, validation and original draft preparation and paper writing, M.J. contribution was methodology, data analyses and curation, validation, supervision, professors F.D. and M.N. contributions were mainly focused on supervision, validation of the paper review and editing, project administration and funding acquisition. All authors have read and agreed to the published version of the manuscript.
Funding: Some equipment were financed by the Regional Council of Bourgogne Franche Comté and the Fonds Européen de Développement Régional (FEDER). The cotutelle PhD of Ms Kchaou has been supported by the Utique PHC program (project SeaCoatPack) N • 39290YK of Campus France, funded by the Ministries of Education and Research of both France and Tunisia and the French Embassy in Tunisia.