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Effect of Gamma Radiation on Novel Gelatin Extracted from Camel Skin for Pharmaceutical Application

PJZ_55_2_819-830

Effect of Gamma Radiation on Novel Gelatin Extracted from Camel Skin for Pharmaceutical Application

Salma Bessalah1, Samira Jebahi2, Asim Faraz3*, Amel Raoufi2, Cem Tırınk4, Walid Dridi2, Mahmoud Daouad2, Hassib Keskes5, Touhami Khorchani1, Khaled Farah2, Abdul Waheed3 and Mohamed Hammadi1

1Livestock and Wildlife Laboratory, Arid Lands Institute (I.R.A), University of Gabès, Médenine, Tunisia.

2Laboratory on Energy and Matter for Nuclear Sciences Development (LR16CNSTN02), National Centre for Nuclear Sciences and Technologies, Sidi Thabet Technopole, 2020 Sidi Thabet, Tunisia.

3Department of Livestock and Poultry Production, Bahauddin Zakariya University Multan, Multan, Pakistan.

4Department of Animal Science, Biometry and Genetic Unit, Faculty of Agriculture, Igdir University, Igdir, Turkey.

5Experimental Surgery of the Musculoskeletal System, Faculty of Medicine of Sfax, Tunisia.

ABSTRACT

Gelatin is a natural polymer that can be derived from collagen through hydrolysis. It most extensively used in diverse fields, mainly in pharmaceuticals and therapeutic. Herein Gelatin was newly extracted from camel (Camelus dromedarius) skin and irradiated with gamma rays from 60Co source. Gelatin was optimized and characterization and functional properties were determined. The structural changes occurring after γ-irradiation at doses from 5 to 30 kGy were reported by physico-chemical techniques such as electron paramagnetic resonance (EPR), Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) for the first time. Results revealed that gelatin yield increased significantly (P<0.05) as the extraction temperature and time increased (19.71%) at 70°C for 12 h. EPR spectroscopy demonstrated that 10 kGy of radiation-induced the decomposition of side groups. New peak positions (Paramagnetic centers) were located at around 3800 G. Moreover, an enhancement of EPR peak (3300 and 3400 G) (g-factor = 2.0) was detected. In addition, free radicals trapped in the crystalline regions were moved toward the boundary regions and the amorphous phases disappeared. XRD patterns showed a new peak at 2θ= 65.8° and a diminution of the relative intensity for the peak of 2θ= 20.54° after 30 kGy. FTIR measurements revealed structural deformations from 5 kGy through chain scission of C–H chains as well as the deformation of the C=O carboxyl groups with increasing γ-radiation doses. The camel skin was thus proved to be a source of gelatin. Irradiation induced a structural deformation with desirable functionalities that make it a potent source of gelatin that could be used in food and biomedical applications.


Article Information

Received 18 February 2022

Revised 25 March 2022

Accepted 08 April 2022

Available online 20 June 2022

(early access)

Published 02 January 2023

Authors’ Contribution

SB and SJ conceived and designed research. SB, SJ, AR, WD and MD conducted experiments. HK, TK, KF, MH contributed analytical tools. SB, AF, AW, SJ and CT analyzed data and wrote the manuscript. All authors read and approved the manuscript.

Key words

Camel skin gelatin, γ-irradiation, Electron paramagnetic resonance, X-Ray diffraction

DOI: https://dx.doi.org/10.17582/journal.pjz/20220218020207

* Corresponding author: drasimfaraz@bzu.edu.pk

0030-9923/2023/0002-819 $ 9.00/0

Copyright 2023 by the authors. Licensee Zoological Society of Pakistan.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).



INTRODUCTION

Gelatin is a water-soluble protein derived from collagen, an abundant connective tissue in bones, skin and animal hides. Most commercial gelatin sources are from mammalians (bovine and mostly porcine). Today, despite its wide applications, the use of this type of gelatin tends to be limited due to the occurrence of several socio-cultural and safety issues (Kittiphattanabawon et al., 2010; Sae-Leaw et al., 2016). Skin gelatin from different aquatic animals has also been extracted and characterized (Arnesen and Gildberg, 2007; Abdelmalek et al., 2016; Jridi et al., 2015). However, the use of fish gelatin as supplement or food ingredient is hindered by several factors such as fishy odor, allergic reactions, thermal stability and weak rheological properties (Sae-Leaw and Benjakul, 2015; Binsi et al., 2017). Today, there is need to explore alternative sources of gelatin which will have no cultural or health concerns. Among these, camel skin could be an excellent candidate. Still, the extraction procedure is yet to be optimized. Several studies reported that the extraction process parameters (temperature, time, and pH) could influence the yield and the length of the polypeptide chains. As a consequence, the biological properties of the resulting gelatin can be greatly affected (Regenstein and Zhou, 2007; Nagarajan et al., 2012). Therefore, optimizing the extraction conditions is required to avoid over-hydrolysis for generating the desired product with excellent functionalities. Nevertheless, data is unavailable about the impact of extraction temperature and time on the properties of gelatin from camel skin. Also, extraction methods under strong basic and strong acidic conditions have caused gelatin to leach during the washing steps. To the best of our knowledge, extraction of camel skin gelatin using a combination of strong basic (NaOH) and weak acid (citric acid) solutions has not been reported previously. Being a renewable and biodegradable polymer, camel skin can be considered as a sustainable alternative in food industry in terms of developing materials, enhancing resource efficiency and reducing environmental problems associated with packaging waste. Added to that, the range of novel applications of gelatin in biomedical and pharmaceutical fields can be developed. Gelatin materials for tissue engineering applications must be submitted to sterilization (Hara et al., 2010; Ouyang and Bai, 2015). Ionizing radiation such as γ-rays provides physical cross-linking reactions (Dorati et al., 2012). It allows avoiding the supplementation of potentially toxic compounds in these polymer networks, which might eventually result in cytotoxic effects. Irradiation causes irreversible molecular alterations due to the breakage of the covalent bonds of the polypeptide chains of gelatin. Therefore, it is important to evaluate radiation effects on gelatin at different doses (5–30 kGy), which are normally used in the sterilization of food and medical products (Diehl, 2002). The application of solid gelatin in food and pharmaceuticals has extensively been investigated (Pan et al., 2018). However, the effect of the γ -radiation on the variation of functional and bioactive properties of solid camel skin gelatin has not been explored. As irradiation causes cleavage of various chains and bonds in gelatin molecules, the present study, attempts to investigate radiation effects on gelatin and valorize the camel skin gelatin as an ingredient in food and pharmaceutical products.

MATERIALS AND METHODS

Collection and preparation of camel skin

The skin of camel (Camelus dromedarius) was obtained from a slaughtering house in Gabes (Tunisia). Fresh skin was put in polyethylene bags and taken to the laboratory. The skin was washed with cool tap water, cut into small pieces, placed in polyethylene bags and stored at -20°C until use. The storage time was less than 2 months.

Pre-treatment of camel skin

Camel skin was soaked in 0.5 M NaOH with ratio of skin/solution at 1:5 (w/v) for 3 days to remove non-collagenous proteins; the solution was changed every day. The alkaline-treated skin was washed with tap water until neutral pH. The samples were then soaked in 0.1 M citric acid with ratio of 1:5 (w/v) for 1 h. The samples were again washed with tap water until neutral pH.

Extraction of gelatin

The swollen skin was mixed with distilled water at 1:5 (w/v) at different temperatures (50, 60 and 70°C) for various times (3, 6, 9 and 12 h). The mixtures were then filtered using filter paper to remove insoluble materials. The supernatant was freeze-dried and subjected to analyses.

Determination of yield

The yield of gelatin was calculated based on the wet weight of fresh skin as follows:

Yield (%) = [weight of dry gelatin (g)/ weight of initial skin (g)] x 100 …. (1)

Determination of color

Colors of the gelatin samples (66.7 g L−1) were measured by Minolta CM-2006 d spectrophotometer (Konica Minolta Holdings, Inc, Osaka, Japan). L*, a* and b* values, indicating lightness/brightness, redness/greenness and yellowness/blueness, respectively, were recorded. The total difference in color (∆E*) was calculated as described previously (Pan et al., 2018).

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

The protein pattern of extracted gelatins was analyzed using SDS-PAGE, following a method adopted previously (Laemmli, 1970). Gelatin samples were dissolved in SDS 5%, heated at 60°C for 20 min and centrifuged at 12000 rpm. The supernatant was mixed in a 1:2 (v/v) ratio with loading buffer (0.5 M Tris-HCl, pH 6.8, glycerol, 10% (w/v) SDS, 0.5% (w/v) bromophenol blue and 5% 2-mercaptoethanol). The mixed solution was heated in water bath (95 °C) for 5 min and loaded into 4% stacking gel and 7.5% resolving gel.

Determination of functional properties

Emulsifying properties

Emulsion activity index (EAI) and emulsion stability index (ESI) of gelatin samples were determined, following the conditions adopted previously (Pearce and Kinsella 1978). Soybean oil (2 ml) and gelatin solution (1, 2 and 3%, 6 ml (w/v)) were homogenized at a speed of 20,000 rpm for 1 min. Emulsions were diluted 100- fold with 0.1% (w/v) SDS. The absorbance at 500 nm of the resulting dispersion was measured at 0 min and 10 min using a spectrophotometer. All determinations are means of at least three measurements.

EAI and ESI were calculated by the following formula:

EAI (m2/g) = (2 x 2.3 x A x DF)/ løC … (2)

ESI= A0x ∆t/∆A … (3)

Where A = A500, DF = dilution factor, l = path length of cuvette (m), ø = oil volume fraction (0.25), C= protein concentration (g/m3), ∆A = A0 - A10 and ∆t = 10 min.

Foaming properties

Foam expansion (FE) and foam stability (FS) of gelatin solutions were determined (Shahidi et al., 1995). Gelatin solutions (1, 2 and 3%, w/v) were homogenized using a homogenizer (model system polytron PT 1200 E, KI-11030031 PT-DA 07/2EC-E107) at 13,000 rpm for 1 min and then transferred into 100 ml cylinders. The sample was allowed to stand for 0, 15, 30 and 60 min. FE and FS were then calculated using the following equations:

FE (%) = (VT/V0) x 100 … (4)

FS (%) = (Vt/VT) x 100 … (5)

Where VT is total volume after whipping; V0 is the original volume before whipping and Vt is total volume after leaving at room temperature for different times (15, 30 and 60 min). All determinations are means of at least three measurements.

Viscosity

The denaturation temperature (Td) was measured following the methods previously described (Zhao et al., 2018). Briefly, dried gelatin was dissolved in distilled water to obtain a concentration of 0.6% (w/v). Gelatin samples were heated at different temperatures (from 4 °C to 40°C) with a heating rate of 4°C. The relative viscosity of the prepared solution was contrasted with that obtained at 4°C. The temperature at which the relative viscosity was 50% was defined as the Td of the sample.

Setting time for gelatin gel

Setting time for gelatin was determined at 4 °C and room temperature (25–26 °C) as described previously (Muyonga et al., 2004). Gelatin solution (6.67%, w/v) (2 ml) was transferred to a thin wall (12 mm - 75 mm) test tubes (PYREX®, Corning, NY, USA) and preheated at 60°C for 10 min, followed by incubation in an ice bath (4°C) and at room temperature (25–26 °C). An aluminum needle with a diameter of 0.1 cm was inserted into the gelatin sample every 10 s until the time it could not come off. This time is considered as the setting time. The measurement was carried out in three determinations.

Physico-chemical characterization

Gamma irradiation

The irradiations of gelatin were performed at the Tunisian Cobalt-60 gamma irradiation facility with energies of 1.173 and 1.332 MeV at a dose rate of 36 Gy/min. The dose rate was determined using Fricke dosimeter chemical standard dosimeter. The traceability to Aerial, the Secondary Standard Dosimetry Laboratory (SSDL), was established using the Alanine/EPR dosimetry system. Gelatin was placed in a polystyrene phantom to ensure electronic equilibrium and was irradiated at room temperature (293–298 K) with a dose range from 5 kGy to 30 kGy.

Electron paramagnetic resonance spectroscopy (EPR)

EPR spectra of the gelatin samples were recorded at room temperature on a Bruker ER-200D spectrometer operating at 9.8 GHz X-Band frequencies with modulation amplitude of 0,2 mT, modulation frequency of 100 khz, sweep width of 210 mT and microwave power of 63 mW.

X-ray diffraction (XRD)

The XRD analysis of gelatin was conducted using Brucker D8 advance with Cu-Kα radiation of wavelength λ = 1.541 Å in 2θ values in the range of 15–90°. The results obtained by X-ray measurement were analyzed with the X’Pert High Score Plus program.

Fourier transform infrared (FTIR)

FTIR spectroscopy was used to study the structural and chemical properties of un-irradiated and irradiated gelatin. The measurement was recorded by Vertex 70 infrared spectrometer from 400 to 4000 cm-1 at a spectral resolution of 2 cm-1 and 32 scans (Jebahi et al., 2012).

Statistical analyses

The obtained data set was performed to determine the effects of determination the effect of gamma radiation on novel gelatin extracted from camel skin with the analysis of variance (ANOVA) test. For this purpose, Levene homogeneity test and Kolmogorov-Smirnov normality test were applied to test the homogeneity of variances and normality assumptions, which are necessary conditions for performing ANOVA. For this purpose, Levene homogeneity test and Kolmogorov-Smirnov normality test were applied to test the homogeneity of variances and normality assumptions, which are necessary conditions for performing ANOVA. As a result of these analyzes, it was determined statistically that the data set was distributed homogeneously and provided the assumption of normality (p>0.05). In the light of this information, as a result of the ANOVA test, it was determined that there was a statistical difference between the groups (p<0.05). In the light of this information, as a result of the ANOVA test, it was determined that there was a statistical difference between the groups (p<0.05).

All authors hereby declare that all experiments have been examined and approved by the appropriate ethics committee.

RESULTS

Gelatin yield

Yield of gelatin from camel skin extracted under various temperatures (50°C, 60°C and 70°C) and times (3h, 6h, 9h and 12 h) are shown in Figure 1. Extraction yields with different conditions ranged from 3.7% to 19.26% on a dry weight basis. Increased yield was observed with increasing extraction temperatures and times and the highest yield (19.26%) was recorded for gelatin extracted at 70 °C for 12h (P < 0.05).

 

SDS–PAGE

The SDS-PAGE pattern of camel skin gelatin for different extraction temperatures (50°C, 60°C and 70°C) and times (3h, 6h, 9h and 12h) are presented in Figure 2. All samples showed typical electrophoresis profiles of gelatin that consisted of α-chain (α1 and α2) and β-chain as the major components with the small amount of γ-chain. In this study, no severe degradation in protein patterns at higher temperature was observed.

 

Color

Color of gelatin from camel skin with different extraction conditions, expressed in terms of L*(lightness), a*(redness) and b* (yellowness), are shown in Table I. The lightness of gelatin gel decreased as extraction temperature and time increased (P < 0.05). However, increases in a* and ΔE* values were found when the extraction time increased (P<0.05). The increases in a* and ΔE* values were in accordance with the increased yield.

 

Table I. Color of gelatins from the skin of Camelus dromedarius at different temperatures for various times.

Extraction conditions

Color value

L*

a*

b*

∆ E

50°C

3h

55.12±0.40j

-0.53±0.33k

-0.06±0.21k

39.94±0.11a

6h

66.05±1.35e

-0.32±1.22g

4.49±1.44g

29.26±1.18h

9h

57±1.55i

-0.27±0.25e

3.85±0.11e

38.19±0.20c

12h

68.19±1.62b

-0.18±1.36c

3.39±1.27c

27.01±1.25k

60°C

3h

68.72±1.22a

-0.28±1.33f

4.03±1.19f

26.55±1.59l

6h

66.99±0.48d

-0.27±0.46e

3.55±0.28e

28.21±0.36i

9h

62.14±1.56h

-0.16±1.10b

7.27±1.55b

33.58±1.12d

12h

56.9±1.41i

-0.02±1.22a

5.81±1.17a

38.5±1.14b

70°C

3h

67.34±0.23c

-0.39±0.30h

5.57±0.17h

28.15±0.27j

6h

65.5±1.34f

-0.44±1.51i

5.09±1.51i

29.89±1.28g

9h

65.44±1.44f

-0.22±1.40d

5.22±1.31d

29.97±1.22f

12h

63.69±1.20g

-0.49±1.45j

6.99±1.16j

32±1.33e

 

Values are presented as mean ± SD (n = 3). Different superscripts within the same column.

 

Emulsifying properties

Emulsifying properties are used to enhance the protein performances. Emulsifying activity index (EAI) and emulsion stability index (ESI) of camel skin gelatin extracted under different conditions and at different concentrations are shown in Table II. EAI of all gelatin samples decreased with the increase of gelatin (1%, 2% and 3%) (P<0.05). The EAI of camel skin gelatin was significantly (P < 0.05) affected by the extraction temperature and the lowest EAI values were observed at 70°C. In general, ESI values significantly decreased with increasing temperature (P < 0.05). The best results were obtained for samples extracted at 50°C.

Foaming properties

Foam expansion (FE) and foam stability (FS) at 15, 30 and 60 min after whipping were determined to evaluate the foam capacity and foam stability of camel skin gelatin. FE and FS of camel skin gelatin at various concentrations (1, 2, and 3 g/100 mL) extracted under various conditions are shown in Table II. Foam expansion values increased with increasing gelatin concentration (P<0.05). In addition, FE increases with increasing extraction temperature. However, slight decreases in FE were observed in gelatin extracted at 70°C for 12 h. Results show also that the highest FS value was found when using 60°C and 70°C at different concentrations.

Setting time for gel formation of gelatin extracted with different conditions

Setting time required for gel formation at 4 °C and room temperature (25 °C) were depicted in Figure 3. The setting time at 4 °C of camel skin gelatin increased when the extraction temperature and time increased (P < 0.05). Gelling time at 4°C ranged from 1.43 min to 2.15 min without significant difference. At 25°C, camel skin gelatin extracted under different temperatures (50°C, 60°C and 70°C) and for different times (3h, 6h, 9h and 12h) were able to set within 16 min.

 

Viscosity and denaturation temperature

The relative viscosity of all gelatin solutions is shown in Figure 4. The denaturation values of all gelatin samples, with different extraction conditions, were ranged from 32°C to 40°C.

 

Table II. Emulsion activity index (EAI) and emulsion stability index (ESI) of camel skin gelatin at different concentrations. Values are given as mean ± SD from triplicate determinations. Different superscripts in the same column indicate significant differences (p < 0.05). Different uppercase letters in the same column within the same gelatin sample indicate significant differences (P < 0.05). Different lowercase letters in the same column within the same concentration and at the same extraction time indicate significant differences (P < 0.05).

Extraction conditions

Concentration (g/100 ml)

Emulsion activity index (EAI)

(m2/g)

Emulsion stability index (ESI) (min)

50°C-3h

1

15,62±0.5aA

176,67±0.4aA

2

6,26±0.45aB

97,14±0.31aB

3

4,3±0.37abC

58,42±0.22aC

50°C-6h

1

13,26±0.3aA

90,00±4.99aA

2

7,36±0.22aB

88,89±0.2aA

3

6,07±0.32aC

80,00±5.1aB

50°C-9h

1

12,15±0.01aA

44,00±3.2aB

2

7,16±0.13aB

90,97±3.4bA

3

4,97±0.56aC

50,63±2.1aB

50°C-12h

1

12,89±0.3bA

21,88±1.1cC

2

7,81±0.36aB

50,78±0.41aA

3

5,52±0.25aC

27,27±0.36bB

60°C-3h

1

11,96±0.12bA

26,96±1.5bB

2

5,71±0.03aB

23,64±2.1bC

3

4,79±0.15aC

41,18±0.5bA

60°C-6h

1

12,34±0.46aA

29,13±0.9bB

2

6,72±0.26aB

27,04±0.5bC

3

5,46±0.11aC

42,38±0.31bA

60°C-9h

1

11,28±0.51abA

23,72±0.3cC

2

5,46±0.05bB

61,13±1.5cA

3

5,01±0.12aB

38,86±0.41cB

60°C-12h

1

12,16±0.11cA

38,15±0.2aA

2

5,52±0.21cB

31,58±0.61bB

3

4,67±0.15bC

33,04±1.1aB

70°C-3h

1

7,92±0.31cA

9,25±0.7cC

2

4,14±0.07bB

13,24±0.45cB

3

3,93±0.21bB

18,82±0.11cA

70°C-6h

1

11,23±0.33bA

20,33±0.33cC

2

4,97±0.15bB

24,55±0.1cA

3

4,17±0.16bC

21,25±0.2cB

70°C-9h

1

10,43±0.21bA

33,69±0.7bB

2

6,23±0.54abB

169,00±7.1aA

3

4,81±0.44aC

43,16±0.1bB

70°C-12h

1

15,10±0.07aA

27,92±2.2bA

2

6,17±.11bB

28,53±0.2cA

3

4,77±0.1bC

28,53±0.15bA

 

 

FTIR analysis

The chemical shift of amide A, I and II for non-irradiated and γ-irradiated gelatin is shown in Figure 5. The absorption for hydrogen bonded N–H associated to amid A amide at 3.362 cm-1 was shifted to higher wave number 3.430 cm-1 (very acute) and to 3.332 cm-1 (very broad) for 5 and 20 kGy dose, respectively. The results also showed that the absorption band at 1.656 cm-1 of C=O stretching (amide I) for non-irradiated gelatin has also shifted to 1646 cm-1 (broad) for 5 kGy dose and then to higher wave number at 1.650 cm-1 (very cute) for 20 kGy dose. Furthermore, the absorption for N–H bending (amide II) coupled with C–N stretching showed no similar trend to that of amide I throughout the studied irradiation ranges. The increase of radiation doses induces the shift of amide A, I and II wave number gradually due to the degradation of protein molecules. However, the shift of absorption of amide A, I and II peaks after 20 kGy dose with increased intensity is associated with crosslinking and due to the formation of hydrogen bond between the amino acid residues of the chains. The frequency of the nitrile (C≡N) stretching vibration was appeared after irradiation in the range 2.100–2.400 cm−1and increased at 15 kGy.

EPR analysis

Figure 6 illustrated the EPR spectra of un-irradiated and γ-irradiated gelatin. It is clearly seen that in the case of slightly irradiation (10 kGy), two new peaks were signaled due to the existence of free radicals in gelatin system at around of 3300 G and 3400 G in the vicinity of g=2. Moreover, the amplitude of the EPR spectrum of the first paramagnetic center was one and a half times greater than the non-irradiated one. Doses from 5 to 30 kGy were showed the disappearance of the new peaks. More interesting spectrum EPR demonstrated the augmentation of the first and the second paramagnetic center resulted in the increase in free radical concentration after irradiation with Co gamma rays the spectrum changed notably as a function of doses. In fact, the increase of gamma radiation induces increase of paramagnetic center intensities.

 

XRD pattern

Diffraction pattern of non-irradiated gelatins were obtained and compared with that of irradiated ones (Fig. 7). XRD pattern of gelatin powder showed amorphous morphology with a characteristic broad hump in the range of 15-30 • 2θ. Moreover, a sharp peak with low intensity were located at 2θ = ~7˚ (not shown). At the dose of 5 kGy, a modification of crystallinity was detected. But the most important modification can be occurred at high doses (25 and 30 kGy), a new sharp peak 2θ = 25.54° was appeared. As showed in Table III, peaks at 2θ= 29.027 characteristic of amorphous phase signaled an important relative intensity 83.27% and minimum of area and FMWM (677) (Table IV). However, after irradiation, the relative intensities were decreased because the crystallinity was increased, and the amorphous regions phases were disappeared.

 

Table III. Foaming properties of camel skin gelatin at different concentrations. Values are given as mean ± SD from triplicate determinations. Different uppercase letters in the same column within the same gelatin sample indicate significant differences (P < 0.05). Different lowercase letters in the same column within the same concentration and for the same extraction time indicate significant differences (P < 0.05).

Extraction conditions

Concentration (g/100 ml)

Foam expansion

(FE) (%)

Foam stability (FS) (%)

t=15 min

t= 30 min

t= 60 min

50-3h

1

40±0.4bB

36±0.1cC

36±0.15cC

36±0.12cC

2

60±1.1cA

60±0.5cA

56±0.3cA

56±0.22bA

3

60±3.5cA

52±2.5cB

52±1.7cB

52±1.15cB

50-6h

1

60±0.5cC

50±2.5bC

50±1bC

44±3.2bC

2

70±1.5cB

62±0.5bB

60±0.3cB

60±0.35cB

3

96±1.2aA

96±2.15aA

96±0.2aA

94±0.5aA

50-9h

1

60±0.5aC

56±0.5cC

52±1.3cC

52±2.8bC

2

90±1.1aB

84±4bB

80±1.5bB

70±5.1aB

3

96±3.2bA

96±1.5aA

90±2.4aA

80±2.5bA

50-12h

1

96±4bA

96±2.5aA

80±1.7bA

80±1aA

2

96±5.5bA

96±4.5aA

80±5.2bA

80±4.7bA

3

98±7.2aA

80±5.5bB

80±3.5bA

76±2.2bA

60-3h

1

100±0.5aA

100±0.3aAB

98±1.1aAB

94±2.5aA

2

110±0.5aA

104±0.2aA

102±0.5aA

98±0.8aA

3

100±4.5bA

96±3bB

96±2.5bB

95±1.2bA

60-6h

1

100±0.5bB

96±0.3aB

96±0.4aB

94±0.2aB

2

110±0.7bA

100±0.22aA

100±0.35aA

100±0.1aA

3

90±0.5aC

90±0.3aC

80±0.4bC

80±0.5bC

60 °C 9h

1

100±3.7aA

78±1.5bA

70±0.5bB

52±2.5bB

2

90±3.5aAB

80±2.8bA

60±3.5cC

50±3bB

3

80±8.5bB

78±1.5bA

78±2bA

70±2.5cA

60°C 12h

1

120±6.5aA

80±2.5bC

80±2.5bB

70±1.2bB

2

110±4aAB

100±1.5aA

100±1aA

90±0.5aA

3

96±7.5aB

90±0.2abB

60±1.2cC

52±0.5cC

70°C 3h

1

100±0.5aB

98±0.7bB

96±0.3bB

80±1.5bB

2

100±0.5bB

98±1bB

96±0.5bB

60±2.5bC

3

120±1.5aA

120±0.8aA

114±1.8aA

104±2.5aA

70-6h

1

110±0.5aAB

100±1aA

98±1aA

90±1.5aA

2

120±2.5aA

100±1.5aA

98±0.5bA

92±1bA

3

100±7aB

96±3.5aA

90±4.5aA

78±2bB

70-9h

1

100±1aB

96±2aA

94±1.5aA

90±2aA

2

104±3aB

96±2.5aA

90±1.5aA

52±1.2bB

3

120±4.5aA

96±1.8aA

90±2.2aA

90±1.5aA

70°C 12h

1

100±0.5bA

98±1.5aA

90±2aB

60±2.5cB

2

100±2abA

98±2aA

92±2.5aB

90±1aA

3

104±1.5aA

98±0.5aA

98±0.5aA

90±0.5aA

 

 

Table IV. Relative intensity observed at peak 2θ= 29,027.

Treatment dose (Kgy)

0

5

10

15

20

25

30

Relative intensity (%)

83.27

62.19

41.60

59.95

64.04

52.25

69.73

 

Table V. Determination of peak position, area and FWHM of XRD data.

Dose (kGy)

Peak position

Area

FWHM

0

29,027

1040,386

1,350

5

28,986

677,794

0,879

10

29,518

869,157

0,388

15

29,272

915,892

0,675

20

29,231

1148,621

1,391

25

29,292

1574,817

0,573

30

29,292

1341,835

1,534

 

DISCUSSION

In this study, gelatin was extracted from camel skin wastes, as an alternative source. Gelatin yield is influenced by the extraction condition. Herein gelatin yield is in agreement with previous works Kaewruang et al. (2013) reported that an increase in water extraction temperature from 45 to 75 °C caused an increase in yield of gelatin

 

from the skin of unicorn leatherjacket. This result was higher than yields of cuttlefish gelatin Balti et al. (2011) and chicken skin gelatin Sarbon et al. (2013) reported as 7.84% and 2.16%, respectively. In this study, no severe degradation in protein patterns at higher temperature was observed, suggesting a high thermal stability of gelatin of camel skin. With increasing extraction temperature and time, covalent bonds stabilizing the triple helix of native mother collagen can be destroyed a consequence, more free α chains and β chains were released from the skin complex leading to higher yield. The color parameter showed a congruence with pervious work that reported the highest L* for gelatin extracted for short time (3h) (Mad-Ali et al., 2016). The decrease of the L value may be due to the occurrence of a non-enzymatic browning reaction, especially Maillard reaction, during extraction at higher temperature for longer time. Concerning emulsifying properties, several authors have reported the decrease of emulsifying capacity with increasing protein concentration. Moreover, foams with higher protein concentrations were denser and more stable, resulting in an increase in the thickness at the interface (Zayas, 1997). Camel skin gelatin extracted at 60 and 70°C had a better foaming ability compared with gelatin extracted at 50°C, though the difference was not statistically significant (p>0.05). In general, viscosity of each sample was decreased continuously with increasing the temperature from 4 to 40°C. Td defined as the temperature at which the relative viscosity was 50%. At this temperature, the gelatin molecules dissociates into random coils (Zhang et al., 2007). Lower Td is one of the most drawbacks limiting the use of gelatin molecule for food and pharmaceutical applications. Furthermore, these values were higher than those for the fish skins (Zhao et al., 2018; Wang et al., 2019). As a result, the triple helix structure was converted into one, two or three random chain gelatin molecules. Thereby, relative viscosity was directly correlated with the thermal stability of the gelatin gel. Considering the importance of thermal stability of gelatin in both food and biomedical applications, camel skin gelatin could be an excellent alternative to other gelatin sources. After extraction and irradiation, FTIR spectroscopy has been used to monitor the structural change in the functional polymer groups. Un-irradiated gelatin contained Amide I, II, III, A and B. These peaks were present with the irradiated ones but at modified intensities. Gelatin lost its triple helix structure when the amide I was affected (Derkach et al., 2019). The stretching vibrations of C=O and C-N groups frequency is found in the range between 1600 and 1700 cm-1. At 5 kGy irradiation, the absorption band at 1,656 cm-1 of C=O stretching (I) has shifted at 1.644 cm-1 (broad) and then at 30 kGy, wave number moved to 1.657 cm-1 (broader). This value is close to un-irradiated gelatin which proves that gelatin undergoes denaturation then crosslinking at higher doses. For amide III, the recorded peak indicates a blend of N-H deformation and C-N stretching vibrations. Bands were present in the 1320-1295 cm-1 (largely affected with 5 kGy but returned to the normal structure at 30 kGy suggesting the same interpretation with that of amide I. Both amide A (3323 cm-1) and amide B band (3057 cm-1) were not affected by the action of radiation. Also, the alkyl C–H stretching bands of the amino acid residues at 2969, 2927 and 2859 cm –1 remained unaffected. However, the changes occurred only at 20 kGy and then the normal aspect was retrieved at 30 kGy. Moreover, the frequency of the nitrile (C≡N) stretching containing unnatural amino vibration in the range 2.100–2.400 cm-1 was accentually apparent at 20 kGy (Ma et al., 2015). With low dose ionizing radiation (5 kGy), irradiated camel gelatin acquired a highly disordered molecular structure that affected the triple helix and α helix structure. This denaturation was caused by hydrogen bond rupture that is responsible for the stabilization protein conformation. A high dose of gamma radiation (25 kGy) makes cross linking the major phenomenon that dominates chain scission mechanisms. A typical XRD pattern was obtained from pure and un-irradiated powder of gelatin. This spectrum is archetypal of an incompletely crystalline biomaterial with a large peak positioned at 2θ = ~19°. A second small peak placed in the region of 2θ = ~7˚ was related to gelatin like triple helix structure (Badii et al., 2014). The intensity of the peak was related to the content of triple helix. The latter molecule confers strict amino acid order that necessitates the recurrence of a (Gly-X-Y) (n) with a high quantity of amino acid in the gelatin (Ramshaw et al., 1998). However, it is clear that sharper peaks (increased crystal size) were obtained from the γ-irradiated gelatin sample at 2θ= ~10°, 15° and 20°. XRD showed a significant alteration in the arrangement of molecules in the crystal lattice. On the other hand, EPR spectroscopy permits to determine the paramagnetic centers. In fact, gelatin spectra recorded at least two components at 3300 and 3300 G. Signal intensities of irradiated and unirradiated samples of the camel gelatin powder were shown. This first band was allocated to the comparatively stable radical at the end of the CH2–CH2~chain • α showing interaction with two protons. The species found in the paramagnetic field were determined as anions radical with an unpaired electron positioned on the carboxyl carbon (Silva et al., 2008). It is seen that the hyperfine constants are slighter than anion radicals of other amino acids (Aydin, 2010). At around 3300 G (g-factor = 2.0), the peak is related to glycine residue, appeared~•CH as a minor product. The other compound of homolytic scission is ~ (O) C• radical that demonstrated an important singlet (Aydin, 2010). The methylene group is capable to offer hydrogen atoms to both radicals •~ which leads to the ~CH formation. At higher radiation doses (25 and 30 kGy), the free radicals were increased. In fact, spectrum EPR demonstrated the augmentation of the free radical concentration after irradiation with Co gamma rays and the spectrum changed notably as a function of doses. Macromolecules generate a three-dimensional hydrogen network actively contributing in the transfer of protons to make possible the conversion/recombination of radicals (Przybytniak et al., 2019). These results provide important data for studying the feasibility of gelatin for biological and medical applications in the future. In fact, these results prove that gamma irradiation is an effective sterilization technique that should be taken into account because its application can alter the chemical structure of the pharmaceutical, skin allograft or food product.

CONCLUSION

Large quantities of gelatin are found in camel skin waste. It underwent chain scission when irradiated and sterilized. FTIR spectroscopy revealed that cross linking phenomenon started to dominate over degradation/denaturation process with the increase of irradiation. It is due to the formation of hydrogen bonds between the amino acid residues of the chains. The effect of radiation over gelatin caused modification in the arrangement of molecules in the crystal lattice was signaled with XRD analysis. A new sharp peak 2θ = 25.54° appeared at high doses (25 and 30 kGy). The spectrum EPR demonstrated the augmentation of the free radical concentration after irradiation with Co gamma rays and the spectrum changed notably as a function of doses. The radicals found in camel skin gelatin might initiate (i) damages in backbone substituent (• CH2CH2~) which affect the structure of the peptide, (ii) strand breaks at glycine residues (~CH2•~).and (iii) release of hydrogen atom from glycine (~CH•~).

Statement of conflict of interest

The authors have declared no conflict of interest.

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