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Extraction and physicochemical characterization of broiler (Gallus gallus domesticus) skin gelatin compared to commercial bovine gelatin
Extraction and physicochemical characterization of broiler (Gallus gallus domesticus) skin gelatin compared to commercial bovine gelatin E. Aykın-Din¸cer, A. Ko¸c, and M. Erba¸s1 Department of Food Engineering, Engineering Faculty, Akdeniz University, Antalya, 07058, Turkey ABSTRACT Gelatin was extracted from broiler (Gallus gallus domesticus) skins and analyzed to compare its physicochemical properties with those of commercial bovine gelatin. The average yield of broiler skin gelatin was 6.5% on a wet weight basis. Broiler skin gelatin had more α1 -and α2 -chains than β -chain and contained high molecular weight (γ -chain) polymers. Glycine was the dominant amino acid in broiler skin gelatin (20.26%), followed by proline (Pro) (15.12%) then hydroxyproline (Hyp) (11.36%). Compared to commercial bovine
gelatin, broiler skin gelatin had less total imino acids (Pro and Hyp) but a higher (33.65 vs. 31.38◦ C) melting temperature (P < 0.01). The differences in physical properties between the broiler and commercial bovine gelatins appeared to be associated with differences in their amino acid composition and molecular weight distribution. The sensory evaluation results revealed that broiler skin gelatin could be a potential alternative to commercial bovine gelatin, useful in various food products.
Key words: broiler, commercial bovine gelatin, gel strength, gelatin, SDS-PAGE 2017 Poultry Science 96:4124–4131 http://dx.doi.org/10.3382/ps/pex237
INTRODUCTION Raising broilers (Gallus gallus domesticus) in the livestock sector has many advantages, including a very short raising period, potentially intense production per unit area, and a very high rate of feed transformation into low-cost white meat and with lower cholesterol and fat contents combined with more easily digested amino acids compared to meat (Jung et al., 2011; Jayasena et al., 2013). Global production of broiler meat has made great progress since 1990, increasing from approximately 402,000 (Terin et al., 2010) tons to 2,000,000 tons in 2015 (Anonymous, 2016). Efficient utilization of chicken by-products is important for the profitability of the poultry industry. By-products, such as internal organs, feet, head, skin, bones, and feathers (Lasekan et al., 2013) are important protein resources that can be used to produce value-added products such as feed or fertilizer, protein hydrolysates, protein isolates, and gelatin (Du et al., 2013; Khiari et al., 2013; Mart´ınezAlvarez et al., 2015). Gelatin is a denatured protein that is produced by controlled hydrolysis of collagen, extracted from the skin, bones, and connective tissues of animals, such as pigs and cows (Niu et al., 2013; Hanani et al., 2014; Ktari et al., 2014; Lassoued et al., 2014). Al least 28 types of collagen have been identified and described (Sherman et al., 2015), but 80 to 90% of the collagen in C 2017 Poultry Science Association Inc. Received December 5, 2016. Accepted August 9, 2017. 1 Corresponding author: [email protected]
the body consists of Types I, II, and III (Lodish et al., 1995). Type I is located in the skin, bones, and tendons. Type II is located in cartilage tissue, and Type III appears in young skins, while other collagen types present in relatively low amounts are distinctive to organs (G´ omez-Guill´en et al., 2011). The primary structure of Type I collagen is a triple helix comprising 3 α-chains that consist mostly of continuous repetitions of glycine (Gly)-X-Y amino acid sequences, where X and Y are generally proline (Pro) and hydroxyproline (Hyp), respectively (G´ omez-Guill´en et al., 2011; Duconseille et al., 2015). The Gly-Pro-Hyp motif, which fits neatly into the primary structure, is responsible for the secondary structure of collagen. Gly and Pro-Hyp, the main amino acids in collagen, are present at approximately 33 and 22%, respectively (Okuyama et al., 2012). The global gelatin production is approximately 326,000 tons per annum (Shyni et al., 2014). The most important resources of gelatin production are pig skin (46%), cattle skin (29%), and pig and cattle bones (23%) (Duconseille et al., 2015). In Europe, gelatin is produced primarily from pigs (60%) and the remaining 40% from other resources, such as the skin and bones of cattle and other animals. Recent studies have focused on fish- and poultry-based gelatin production for consumers with special preferences and sensibilities (Lee et al., 2012; Du et al., 2013; Nikoo et al., 2014; Rafieian et al., 2015). Also, in food, the use of gelatin originating from skin and bones derived from fish and poultry decreases the risk of bovine spongiform encephalopathy infection (Hanani et al., 2014).
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This study aimed to produce high-quality gelatin from broiler skins and to compare its physicochemical properties with those of commercial bovine gelatin.
Yield of Extracted Gelatin The gelatin extraction yield obtained from broiler skin samples was determined by gravimetric method and calculated using the following formula:
MATERIALS AND METHODS Materials As a raw material for gelatin production, dressed broiler skins were obtained from a local poultry farm, and any meat residues were removed before the skins were washed thoroughly with cold water. Afterwards, skin samples were cut into small pieces (≈2 × 2 cm) with a knife and freeze dried (Operon fdu & fdb type, Gyeonggi-do, South Korea). Next, the fat was removed by Soxhlet method. Commercial bovine skin gelatin was purchased from ˙ a well-known producer (Natura, Izmir, Turkey) and stored according to the manufacturer’s recommendations. All chemicals were analytical grade.
Gelatin Extraction Gelatin was extracted from skin samples using the method of Badii and Howell (2006) with slight modifications. Briefly, NaOH (250 mL, 0.15%) was added to 15 g of the fat-removed, freeze-dried skin samples and blended at 25◦ C for 30 min in a shaking water bath (WiseBath WSB-30, Seoul, South Korea) and then centrifuged at 4,500 × g for 10 min (Eppendorf Centrifuge 5430, Hamburg, Germany). Afterwards, the supernatant, containing non-collagen proteins and pigments, was removed. After repeating this process 3 times, the remaining alkali pellet was rinsed with distilled water, blended with 250 mL H2 SO4 (0.15%) at 25◦ C for 30 min in a shaking water bath, and then was centrifuged at 4,500 × g for 10 min to denature the collagen. This process was repeated 3 times, then the remaining acid pellet was rinsed with distilled water. Finally, the obtained pellet was blended with 250 mL of citric acid (0.7%) at 25◦ C for 30 min and then centrifuged at 4,500 × g for 10 minutes. This process also was repeated 3 times. Any residual salts remaining in the pellets after the extraction stages were removed by washing with distilled water before centrifugation at 4,500 × g for 10 min. The extract in the distilled water was left overnight at 50◦ C without blending and then filtered using a Buchner funnel with Whatman filter paper (no. 4). The filtrate was processed through ion exchangers to remove any salts. The resultant solution was adjusted to pH 6 with 0.1 M NaOH, then its volume lowered to 1/10 of its original volume by rotary evaporation (Laborota 4000, Heidolph Instruments GmbH & Co. KG, Schwabach, Germany) under vacuum at 50◦ C. Finally, the concentrated solution was freeze-dried and ground into a powder.
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% Yield =
Weight of freeze dried gelatin (g) Wet weight of fresh skin (g)
× 100
Determination of Proximate Composition The moisture content was determined gravimetrically after oven drying the samples at 105◦ C until they reached a constant weight. The ash content was determined gravimetrically by burning the samples at 550◦ C until they acquired a gray-white ash color. The fat content of the skin samples was determined by Soxhelet extraction method. The protein content of the samples was determined by Kjeldahl method with 5.4 as the protein conversion factor for skin (AOAC, 2000). The conversion factor of 5.4 was used because collagen, the main protein in the skin, contains a nitrogen ratio of 18.7% (Eastoe and Eastoe, 1952).
Determination of pH Distilled water (10 mL) was added to 1 g of sample and the solution then heated at 45◦ C for 5 min to dissolve the gelatin powder. The solutions were cooled to room temperature and the pH values determined during magnetic stirring using a pH-meter (Hanna HI 2210, Woonsocket, RI) (Shyni et al., 2014) calibrated against pH 4 and pH 7 buffers.
Determination of Molecular Weight Distribution SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (Owl, Model P8D8, Thermo Fisher Scientific, Rockford, IL, USA) was performed using a gradient gel (5% w/v loading gel and 10% w/v separating gel, 0.8 mm). The samples (5 mg) were dissolved in 5% SDS, heated at 85◦ C for one h, and then centrifuged at 8,500 × g for 5 min at room temperature. The supernatants were treated with SDS-PAGE loading buffer (200 mM TrisHCl, pH 6.8, 8% SDS, 0.4% bromophenol blue, 40% glycerol, and 100 mM DTT) at 3:1 ratio and incubated at 70◦ C for 10 minutes. After loading the sample mixtures and protein marker (20 μL) (PageRuler Unstained Protein Ladder, Thermo Fisher Scientific, Waltham, MA, USA) (Sambrook and Russell, 1989) onto the gel, they were electrophoresed using 1× TGS buffer (0.025 M Tris base, 0.192 M Glycine, 0.1% SDS, pH 8.3) at a constant voltage of 150 V. The protein bands were visualized by Coomassie Blue (G-250)
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staining and scanned using the Odyssey Infrared Imaging system (LI-COR Biosciences, Lincoln, NE).
Determination of Amino Acid Composition The homogenized sample (0.4 g) and HCl (5 mL, 6 N) were weighed into screw-cap tubes. Then, phenol (250 μL, 2 mM) was added to prevent oxidation. For the optimization of cysteine, methionine, and tyrosine recovery, 0.1 g Na2 SO3 was also added. The tubes were then closed under nitrogen gas and hydrolyzed at 110◦ C for 24 hours. Afterwards, the pH was adjusted to near neutral (6.7−7.3) and the tube filled to 50 mL with ultra-water according to the total amino acid amount. After the filtration through a membrane filter (Sigma, Stenheim, Germany) with a nominal pore size of 0.45 μm, the sample was analyzed by HPLC (Agilent HPLC 1100, Agilent Technologies, Inc., Santa Clara, CA) using a Zorbax Eclipse-AAA column (4.6 × 250 mm, 5 μm). The eluents used were buffer A (40 mM Na2 H2 PO4 , pH: 7.8) and buffer B (methanol: acetonitrile: ultra pure water [45:45:10]). The eluted amino acids are subjected to post-column derivatization with ortho-phthalaldehyde (OPA) regent delivered using a peristaltic pump at the rate of 2 mL/min at 40◦ C. Detection was done using a FL detector with absorption wavelength of 340 nm and emission wavelength of 450 nm. Additionally, the content of hydroxyproline in the same sample was analyzed using ACE 5C18 column (4.6 × 250 mm, 5 μm) with a UV detector in 265 nm wavelength at the rate of 1 mL/min at 25◦ C. The mobile phase that was used in hydroxyproline analysis contains 650 mL sodium acetate buffer prepared with 3% acetic acid and 350 mL acetonitrile. Amino acid standards (Sigma 21 L-Amino Acids plus kit) were run to calculate the concentration of amino acids in the sample. Norvaline (Sigma-Aldrich Inc., Oakville, ON, Canada) was added as an internal standard. The amino acid content was expressed as a percentage of the total amino acids in the sample.
Determination of Viscosity The viscosity (cP) of the gelatin solutions (6.67%) was determined at 30 ± 0.5◦ C using a Brookfield digital viscometer (Brookfield Engineering, Middleboro, MA) equipped with a no. 1 spindle at 60 rpm (Shakila et al., 2012).
Determination of Melting Point The melting point was determined by the Wainewright (1977) method. A 5 mL aliquot of gelatin solution (10% w/v) was placed into tubes and screw-capped, then cooled rapidly in an ice water bath. Five drops of chloroform and red food dye (3:1) were placed on the surface of the gels. The tubes were then placed in a 10◦ C water bath, and the temperature of the bath was gradually increased. The temperature at which the red drops started to move freely into the gel was recorded as the melting point.
Determination of Color Values Gelatin solution (6.67% w/v) was heated at 60◦ C for one h and then cooled to room temperature. The color was then measured at 6 different locations on the sample using a CR-400 chromameter (Konica Minolta, Tokyo, Japan). The L∗ , a∗ , and b∗ values obtained refer to the black-white, red-green, and yellow-blue color values, respectively, in accordance with the CIE Lab color system. The equipment was calibrated using its own calibration plate prior to analysis (Aykın et al., 2016).
Determination of Gel Clarity The gelatin solution (6.67%) was heated at 60◦ C for one h, and the clarity of the obtained gel samples was determined spectrophotometrically by transmittance (%T) measurement at 620 nm (Avena-Bustillos et al., 2006).
Determination of Gel Strength
Determination of Odor
Gelatin (7.5 g) and distilled water (105 mL) were blended in a bloom bottle to give 6.67% (w/v) gelatin solution, which was then rested at room temperature for one h to allow the gelatin to absorb the water. Afterwards, the bloom bottles were held in a 65◦ C water bath for 25 min to dissolve the gelatin, then rested at room temperature for 15 min before being placed in a 10◦ C water bath for 18 hours. The gel strength (bloom value) was determined by texture analysis (TA.XTPlus, Stable Microsystems, Godalming, UK). When the plunger (Delrin probe, P/0.5R caliber) had penetrated into the gel to a depth of 4 mm, the maximum strength (g) was determined, and the results were expressed as bloom (g) (Shyni et al., 2014).
A 6.67% gelatin solution was prepared by placing 0.5 g gelatin samples and 7 mL distilled water into test tubes. The tubes were capped and then placed into a 50◦ C water bath to dissolve. Afterwards, the test tubes were covered with aluminum foil so the panelists could not see the color of the samples. The odor specifications were evaluated by a 15-person educated panelist group, consisting of undergraduate and graduate students of the Food Engineering Department at Akdeniz University (Turkey) using a 6-point scale (0: no odor, 1: very light, can be sensed when carefully evaluated, 2: mild, easily detectable, 3: strong but not offensive, 4: strong and offensive, 5: very strong and offensive) (Muyonga et al., 2004).
CHARACTERIZATION OF BROILER SKIN GELATIN Table 1. Proximate composition of broiler skin and commercial bovine gelatins. Content
Broiler skin gelatin
Protein (%) Moisture (%) Ash (%) Fat (%) pH
84.29 12.57 2.13 0.45 5.82
± ± ± ± ±
Commercial bovine gelatin
1.60a 0.16a 0.07a 0.06a 0.08a
88.45 10.10 1.22 0.45 5.73
± ± ± ± ±
1.07a 0.21b 0.02b 0.05a 0.01a
a,b Different letters in the same row of each effect indicate significant (P < 0.05) differences between the means.
Statistical Analysis Physical and chemical properties of the broiler skin and commercial bovine gelatins were studied as 2 replications, and the analyses were duplicated. The data were analyzed by analysis of variance (ANOVA) using the SAS statistical software package (v.7.00, SAS Institute Inc., Cary, NC). Duncan’s multiple range test was used to determine significant differences at the 5% level. The results were expressed as the mean ± standard deviation.
RESULTS AND DISCUSSION Yield of Broiler Skin Gelatin The broiler skin gelatin yield was 6.5% on a wet weight basis. Sarbon et al. (2013) reported a yield of extracted chicken skin gelatin of 2.16% wet weight basis, while the yield of duck feet gelatin, extracted using different techniques, varied from 0.75 to 3.31% (Park et al., 2013). In contrast, the yields obtained in this study were lower than those found by Lassoued et al. (2014) for thornback ray skin (18.32 to 30.16%.); Shyni et al. (2014) for shark skin (19.7%), tuna skin (11.3%), and rohu skin (17.2%); and Ktari et al. (2014) for zebra blenny skin (14.8%) gelatins. The gelatin yields have been reported to vary among different raw materials, mainly due to differences in the extraction time, pretreatment process, washing step, collagen content, and the skin compositions (Du et al., 2013; Lassoued et al., 2014; Sinthusamran et al., 2014). The yield of gelatin from the chicken skin may be increased by using more intense extraction conditions, such as a higher temperature, more extreme acid-alkali treatment, and/or enzymes.
Proximate Composition of Broiler Skin Gelatin The proximate composition of broiler skin and commercial bovine gelatins is given in Table 1. The protein, moisture, ash, and fat contents of broiler skin gelatin were determined as 84.29, 12.57, 2.13, and 0.45%, respectively. The low fat content indicated that there was an efficient removal of fat from the skin. The broiler skin gelatin exhibited higher (P < 0.05) moisture and ash contents than the commercial bovine gelatin. How-
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ever, the moisture content of broiler skin gelatin was lower than the limit (15%) prescribed for edible gelatin (GME, 2008). The relatively low ash content suggested that the broiler skin gelatin was of good quality, considering that the maximum limit recommended for edible gelatin is 2.5% (Jones, 1977). The difference in the ash content of broiler gelatin compared with commercial bovine gelatin may have been due to the formation of inorganic compounds during the extraction procedure using acid and alkali solutions.
pH Values of Gelatin Samples The extracted broiler skin and commercial bovine gelatins had a pH of 5.82 and 5.73, respectively, as shown in Table 1, indicating their category as Type B. The conversion of collagen to gelatin leads to changes in the molecular structure of several amino acids. Type B gelatin has a pH ∼5 because the alkaline process deaminates glutamine into glutamic acid and asparagine into aspartic acid (Duconseille et al., 2015; Hattrem et al., 2015). It has been reported that Type B gelatin has minimum viscosity and maximum gel strength at pH 5 (Cole, 2000), signifying the importance of pH during the rheological properties of gelatin. In previous research, the pH was 4.83 in chicken deboner residue gelatin (Rafieian et al., 2015), 4.29 in tuna skin gelatin (Shyni et al., 2014), 4.60 in pink perch skin gelatin (Koli et al., 2012), 5.12 in striped catfish gelatin (Jamilah et al., 2011), and 6.21 to 6.84 in octopus skin gelatins (Jridi et al., 2015). The differences among pH values of gelatin samples may be due to the different acid and alkali pretreatments used during the extraction procedure.
Molecular Weight Distribution of Gelatin Samples The broiler and commercial bovine gelatins had different molecular weight distributions (Figure 1). The α1 - and α2 -chains with a molecular weight ∼120 kDa were present in both gelatin samples. While the β -chain with a molecular weight ∼200 kDa was absent in commercial bovine gelatin, it was present in broiler skin gelatin. The α-chain band was less intense in the commercial bovine gelatin than broiler skin gelatin samples. It was reported that gelatins with higher content of α-chain possess better functional properties (G´ omezGuill´en et al., 2002). Also, in contrast to the commercial bovine gelatin, the broiler skin gelatin included peptide fragments with a molecular weight less than 100 kDa, which could be attributed to the excessive cleavage of collagen by the heat treatment used during gelatin preparation. It was reported that the presence of these fragments is responsible for the low viscosity, low melting point, and decreased bloom strength of gelatin (Nagarajan et al., 2012). However, polymers with very high molecular weight (∼240 kDa) (γ -chain), plausibly
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AYKIN-DINC ¸ ER ET AL. Table 2. Amino acid composition of broiler skin and commercial bovine gelatins. Amino acids Aspartic acid (Asx) Glutamic acid (Glx) Serine (Ser) Histidine (His) Glycine (Gly) Threonine (Thr) Arginine (Arg) Alanine (Ala) Tyrosine (Tyr) Valine (Val) Methionine (Met) Phenylalanine (Phe) Isoleucine (Ile) Leucine (Leu) Lysine (Lys) Hydroxyproline (Hyp) Proline (Pro)
Broiler skin gelatin (%) 5.91 9.59 2.67 0.74 20.26 2.70 7.57 8.32 0.82 2.07 1.12 2.76 1.48 3.25 3.21 11.36 15.12
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.42a 0.54a 0.18a 0.02a 0.59a 0.40a 0.40a 0.37a 0.12a 0.13a 0.08a 0.19a 0.08a 0.20a 0.28a 0.11a 0.49b
Commercial bovine gelatin (%) 4.87 8.93 3.11 0.72 20.60 1.84 7.46 8.18 0.60 1.90 0.47 1.96 1.38 2.89 2.76 9.80 21.23
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.47a 0.66a 0.18a 0.01a 0.70a 0.09a 0.13a 0.21a 0.03a 0.10a 0.02b 0.13b 0.08a 0.13a 0.23a 0.61a 0.81a
a,b Different letters in the same row of each effect indicate significant (P < 0.05) differences between the means. Asx = Asp + Asn; Glx = Glu + Gln.
Figure 1. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) image of gelatin. M: molecular weight protein markers. B1: Broiler skin gelatin (5 mg) were dissolved in 5 mL of 5% SDS. B2: Broiler skin gelatin (5 mg) were dissolved in 10 mL of 5% SDS. C1: Commercial bovine gelatin (5 mg) were dissolved in 5 mL of 5% SDS. C2: Commercial bovine gelatin (5 mg) were dissolved in 10 mL of 5% SDS.
residual heat-stable cross-linked proteins, were present in both gelatins. These results suggested that broiler skin gelatin with a high content of α- and β -chains showed high molecular stability.
Amino Acid Composition of Gelatin Samples As shown in Table 2, the amino acid composition of broiler skin and commercial bovine gelatins exhibited a low content of methionine, histidine, and tyrosine, and no tryptophan or cysteine. This is in agreement with several studies, which reported that tryptophan and cysteine are typically absent in Type I collagen (Ktari et al., 2014; Lassoued et al., 2014; Jridi et al., 2015). Gly was the dominant amino acid in broiler skin gelatin (20.26%), followed by Pro (15.12%) then Hyp (11.36%). Gly, Pro, Hyp, and alanine were the main amino acids in both gelatins, which is a characteristic of all gelatins. The aspartic and glutamic acid contents were higher in comparison to those previously reported (Sarbon et al., 2013; Ktari et al., 2014), which may be associated with the acid pretreatment, as some of the glutamine and asparagine may have transformed to their acidic forms during the gelatin extraction. The amino acid compositions of the gelatin samples from broiler skin were not significantly (P > 0.05) different from that of bovine gelatin, except for methionine, phenylalanine, and Pro (P < 0.05). Broiler skin gelatin had higher methionine and phenylalanine
Table 3. Rheological properties of broiler skin and commercial bovine gelatins. Rheological properties Gel strength (g) Melting temperature (◦ C) Viscosity (cP)
Broiler skin gelatin
Commercial bovine gelatin
166.65 ± 1.63b 33.65 ± 0.07a 1.35 ± 0.10b
238.25 ± 2.47a 31.38 ± 0.11b 3.12 ± 0.12a
a,b Different letters in the same row of each effect indicate significant (P < 0.05) differences between the means.
contents than commercial bovine gelatin, as was similarly observed for thornback ray skin gelatin (Lassoued et al., 2014) and tuna skin gelatin (G´ omez-Estaca et al., 2009). Broiler skin gelatin had less total imino acid content (Pro and Hyp) than commercial bovine gelatin. The higher imino acid content suggested that the broiler skin gelatin may exhibit better rheological properties by structural stabilization of the triple helix of collagen, developing a strong gel structure (Ktari et al., 2014), as the hydroxyl groups of Hyp form hydrogen bonds with available water molecules. Lassoued et al. (2014) reported that bovine gelatin with a higher imino acid content had a higher gel strength than thornback ray skin gelatin.
Gel Strength of Gelatin Samples Gel strength is one of the most important physical properties of gelatin. As shown in Table 3, the gel strength of broiler skin gelatin (166.65 g) was significantly (P < 0.01) lower than that of commercial bovine gelatin (238.25 g). This may be due to the lower imino acid content (Pro and Hyp) of broiler skin, which could result in a less organized triple-helix structure because both imino acids are responsible for stabilizing the secondary protein structure. In previous research, the gel strength was 355 g for chicken skin gelatin (Sarbon
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et al., 2013), 260 g for bovine gelatin and 140 g for thornback ray skin gelatin (Lassoued et al., 2014), 124 g for rohu skin gelatin (Shyni et al., 2014), and 62 to 284 g for gelatins extracted from octopus skin using various pepsin concentrations (Jridi et al., 2015). The gel strength of commercial bovine gelatins is reported to range from 100 to 300 g (G´ omez-Guill´en et al., 2011). The differences in gel strength among samples could be explained by differences in the ages of the animals, the extraction process used, the presence of other hydrocolloids, and the intrinsic properties of collagen, which vary among species.
Table 4. Color and transmittance values of broiler skin and commercial bovine gelatins. Parameter L∗ a∗ b∗ Transmittance (%)
Broiler skin gelatin 27.62 − 2.98 1.33 44.28
± ± ± ±
Commercial bovine gelatin
0.04a 0.04b 0.04b 0.14b
23.64 − 1.91 3.91 86.01
± ± ± ±
0.06b 0.03a 0.04a 0.58a
a,b Different letters in the same row of each effect indicate significant (P < 0.05) differences between the means.
hence it has a higher gel strength, viscosity, and melting temperature (Sarbon et al., 2013).
Viscosity of Gelatin Samples The viscosity of broiler skin gelatin (1.35 cP) was significantly (P < 0.01) lower than commercial bovine gelatin (Table 3) and previously reported values for chicken deboner residue and commercial bovine gelatins (5.55 and 2.90 cP, respectively) (Rafieian et al., 2015), which could be attributed to the low molecular weight fragments present in broiler gelatin (Figure 1). Viscosity is correlated to the molecular weight and molecular size distribution of the extracted proteins (Sperling, 2005). It was reported that gelatins containing a smaller proportion of large molecules, such as β -chains, than small molecules, exhibit low viscosity and result in weak gels (Silva et al., 2014). A low viscosity also has been observed for gelatin at pH 6 to 8. Also, gelatin prepared at a high (≥0.05 M citric acid or ≥0.07 M HCl) or weak (0.01 to 0.03 M acetic acid or 0.01 M HCl) acid concentration, had less β -chains and low viscosity (Niu et al., 2013).
Melting Temperature of Gelatin Samples The melting temperature of broiler skin gelatin was significantly (P < 0.01) higher than that of commercial bovine gelatin (Table 3), which can be correlated with its higher imino acid content, but was similar to that reported for chicken gelatin (33.57◦ C) (Sarbon et al., 2013). It seems, therefore, that broiler skin gelatin could be used to replace the other gelatins in some food products in which gelatin is used. In comparison with other species, fish gelatin (3.4 to 25.8◦ C) (Koli et al., 2012) and acid bovine skin gelatin (29.7◦ C) have a relatively low melting temperature, whereas that of acid pork skin gelatin is comparatively high (32.3◦ C) (Gudmundsson, 2002). It is known that fish gelatins, which have a low content of imino acids (Gudmundsson, 2002), usually have a lower melting temperature than mammalian gelatins (Karim and Bhat, 2009). Thus, variation in melting temperature among different gelatin samples is probably due to the differences in imino acid contents (Pro and Hyp). The gelatin with high imino acid content may exhibit better rheological properties by structural stabilization of the triple helix of collagen, and
Color and Gel Clarity Values of Gelatin Samples The L∗ , a∗ , and b∗ color values of the broiler and commercial bovine gelatin samples were significantly (P < 0.01) different (Table 4). The L∗ value, or lightness value, of broiler gelatin was slightly higher than that of commercial bovine gelatin. Both gelatin samples showed negative a∗ values, and it was significantly lower for broiler gelatin. The b∗ value of broiler gelatin was slightly lower than that of commercial bovine gelatin, indicating that broiler gelatin was less yellow than commercial bovine gelatin. These results showed that factors, such as raw material and extraction conditions, influenced the color of extracted gelatin. While commercial bovine gelatin showed good transmittance (%), broiler skin gelatin had a significantly poor value (Table 4). This might be due to inorganic, proteinaceous, and mucosubstance contaminants, either introduced or not removed, during its extraction. Filtration efficiency during extraction directly affects the degree of clarity of gelatin solutions, and, accordingly, non-settling and unfilterable particulate matter in gelatin solution may be the cause of turbidity (Muyonga et al., 2004). The color and gel clarity are important aesthetic properties of gelatin, depending on its intended use.
Odor of Gelatin Samples The odor of the broiler skin gelatin (2.07 ± 0.28) was not significantly (P > 0.05) different from that of commercial bovine gelatin (1.70 ± 0.05). Both gelatins were found to have a mild odor because the hedonic score was ∼2. However, the odor of broiler skin gelatin was more apparent than that of commercial bovine gelatin. It can be concluded that the odor of broiler skin gelatin did not affect consumers and was similar to the odor of gelatin products available on the market. Choi and Regenstein (2000) reported that activated carbon treatment at the final stage of extraction can further decrease the odor and improve the acceptability of gelatin.
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CONCLUSIONS Broiler skin as an animal by-product obtained from the poultry industry could be a promising source of gelatin. The yield of broiler skin gelatin was 6.5% on a wet weight basis, and more intensive extraction conditions should be investigated in future studies, as this may further increase the yield. The gelatin extracted from broiler skin showed a higher melting temperature than that of commercial bovine gelatin. The difference in the rheological properties of broiler gelatin compared with commercial bovine gelatin may be due to the intrinsic properties associated with differences in their amino acid composition. The main structural difference between broiler skin and commercial bovine gelatins was the imino acid content, which was higher in the commercial bovine gelatin. Considering its sensory evaluation, broiler skin gelatin could potentially be an alternative to commercial bovine gelatin, added in various food products.
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CHARACTERIZATION OF BROILER SKIN GELATIN Niu, L., X. Zhou, C. Yuan, Y. Bai, K. Lai, F. Yang, and Y. Huang. 2013. Characterization of tilapia (Oreochromis niloticus) skin gelatin extracted with alkaline and different acid pretreatments. Food Hydrocolloid. 33:336–341. Okuyama, K., K. Miyama, K. Mizuno, and H. P. B¨ achinger. 2012. Crystal structure of (Gly-Pro-Hyp) 9: Implications for the collagen molecular model. Biopolymers 97:607–616. Park, J.-H., J.-H. Choe, H.-W. Kim, K.-E. Hwang, D.-H. Song, E.J. Yeo, H.-Y. Kim, Y.-S. Choi, S.-H. Lee, and C.-J. Kim. 2013. Effects of various extraction methods on quality characteristics of duck feet gelatin. Korean J. Food Sci. Anim. Resour. 33: 162–169. Rafieian, F., J. Keramat, and M. Shahedi. 2015. Physicochemical properties of gelatin extracted from chicken deboner residue. LWT-Food Sci. Technol. 64:1370–1375. Sambrook, J., and D. W. Russell. 1989. SDS-polyacrylamide gel electrophoresis of proteins. Pages 47–59 in Molecular Cloning: A Laboratory Manual 3. J. Sambrook, E. F. Fritsch, and T. Maniatis, eds. Cold Spring Harbor Laboratory, New York. Sarbon, N. M., F. Badii, and N. K. Howell. 2013. Preparation and characterisation of chicken skin gelatin as an alternative to mammalian gelatin. Food Hydrocolloid. 30:143–151. Shakila, R. J., E. Jeevithan, A. Varatharajakumar, G. Jeyasekaran, and D. Sukumar. 2012. Functional characterization of gelatin ex-
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tracted from bones of red snapper and grouper in comparison with mammalian gelatin. LWT-Food Sci. Technol. 48:30–36. Sherman, V. R., W. Yang, and M. A. Meyers. 2015. The materials science of collagen. J. Mech. Behav. Biomed. Mater. 52:22–50. Shyni, K., G. Hema, G. Ninan, S. Mathew, C. Joshy, and P. Lakshmanan. 2014. Isolation and characterization of gelatin from the skins of skipjack tuna (Katsuwonus pelamis), dog shark (Scoliodon sorrakowah), and rohu (Labeo rohita). Food Hydrocolloid. 39:68–76. Silva, R. S., S. F. Bandeira, and L. A. Pinto. 2014. Characteristics and chemical composition of skins gelatin from cobia (Rachycentron canadum). LWT-Food Sci. Technol. 57:580–585. Sinthusamran, S., S. Benjakul, and H. Kishimura. 2014. Characteristics and gel properties of gelatin from skin of seabass (Lates calcarifer) as influenced by extraction conditions. Food Chem. 152:276–284. Sperling, L. H. 2005. Introduction to physical polymer science. John Wiley & Sons. New York. Terin, M., I. Yildirim, and K. C ¸ ift¸ci. 2010. Chicken meat production and poultry meat consumption in turkey and its progress. Proceedings of the 2nd Mediterrean Summit of WPSA:215–220. Wainewright, F. 1977. Physical tests for gelatin and gelatin products. Pages 507–531 in The Science and Technology of Gelatin. A. G. Ward, and A. Courts, eds. Academic Press, London, UK.
gelatin, broiler skin gelatin had less total imino acids (Pro and Hyp) but a higher (33.65 vs. 31.38◦ C) melting temperature (P < 0.01). The differences in physical properties between the broiler and commercial bovine gelatins appeared to be associated with differences in their amino acid composition and molecular weight distribution. The sensory evaluation results revealed that broiler skin gelatin could be a potential alternative to commercial bovine gelatin, useful in various food products.
Key words: broiler, commercial bovine gelatin, gel strength, gelatin, SDS-PAGE 2017 Poultry Science 96:4124–4131 http://dx.doi.org/10.3382/ps/pex237
INTRODUCTION Raising broilers (Gallus gallus domesticus) in the livestock sector has many advantages, including a very short raising period, potentially intense production per unit area, and a very high rate of feed transformation into low-cost white meat and with lower cholesterol and fat contents combined with more easily digested amino acids compared to meat (Jung et al., 2011; Jayasena et al., 2013). Global production of broiler meat has made great progress since 1990, increasing from approximately 402,000 (Terin et al., 2010) tons to 2,000,000 tons in 2015 (Anonymous, 2016). Efficient utilization of chicken by-products is important for the profitability of the poultry industry. By-products, such as internal organs, feet, head, skin, bones, and feathers (Lasekan et al., 2013) are important protein resources that can be used to produce value-added products such as feed or fertilizer, protein hydrolysates, protein isolates, and gelatin (Du et al., 2013; Khiari et al., 2013; Mart´ınezAlvarez et al., 2015). Gelatin is a denatured protein that is produced by controlled hydrolysis of collagen, extracted from the skin, bones, and connective tissues of animals, such as pigs and cows (Niu et al., 2013; Hanani et al., 2014; Ktari et al., 2014; Lassoued et al., 2014). Al least 28 types of collagen have been identified and described (Sherman et al., 2015), but 80 to 90% of the collagen in C 2017 Poultry Science Association Inc. Received December 5, 2016. Accepted August 9, 2017. 1 Corresponding author: [email protected]
the body consists of Types I, II, and III (Lodish et al., 1995). Type I is located in the skin, bones, and tendons. Type II is located in cartilage tissue, and Type III appears in young skins, while other collagen types present in relatively low amounts are distinctive to organs (G´ omez-Guill´en et al., 2011). The primary structure of Type I collagen is a triple helix comprising 3 α-chains that consist mostly of continuous repetitions of glycine (Gly)-X-Y amino acid sequences, where X and Y are generally proline (Pro) and hydroxyproline (Hyp), respectively (G´ omez-Guill´en et al., 2011; Duconseille et al., 2015). The Gly-Pro-Hyp motif, which fits neatly into the primary structure, is responsible for the secondary structure of collagen. Gly and Pro-Hyp, the main amino acids in collagen, are present at approximately 33 and 22%, respectively (Okuyama et al., 2012). The global gelatin production is approximately 326,000 tons per annum (Shyni et al., 2014). The most important resources of gelatin production are pig skin (46%), cattle skin (29%), and pig and cattle bones (23%) (Duconseille et al., 2015). In Europe, gelatin is produced primarily from pigs (60%) and the remaining 40% from other resources, such as the skin and bones of cattle and other animals. Recent studies have focused on fish- and poultry-based gelatin production for consumers with special preferences and sensibilities (Lee et al., 2012; Du et al., 2013; Nikoo et al., 2014; Rafieian et al., 2015). Also, in food, the use of gelatin originating from skin and bones derived from fish and poultry decreases the risk of bovine spongiform encephalopathy infection (Hanani et al., 2014).
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CHARACTERIZATION OF BROILER SKIN GELATIN
This study aimed to produce high-quality gelatin from broiler skins and to compare its physicochemical properties with those of commercial bovine gelatin.
Yield of Extracted Gelatin The gelatin extraction yield obtained from broiler skin samples was determined by gravimetric method and calculated using the following formula:
MATERIALS AND METHODS Materials As a raw material for gelatin production, dressed broiler skins were obtained from a local poultry farm, and any meat residues were removed before the skins were washed thoroughly with cold water. Afterwards, skin samples were cut into small pieces (≈2 × 2 cm) with a knife and freeze dried (Operon fdu & fdb type, Gyeonggi-do, South Korea). Next, the fat was removed by Soxhlet method. Commercial bovine skin gelatin was purchased from ˙ a well-known producer (Natura, Izmir, Turkey) and stored according to the manufacturer’s recommendations. All chemicals were analytical grade.
Gelatin Extraction Gelatin was extracted from skin samples using the method of Badii and Howell (2006) with slight modifications. Briefly, NaOH (250 mL, 0.15%) was added to 15 g of the fat-removed, freeze-dried skin samples and blended at 25◦ C for 30 min in a shaking water bath (WiseBath WSB-30, Seoul, South Korea) and then centrifuged at 4,500 × g for 10 min (Eppendorf Centrifuge 5430, Hamburg, Germany). Afterwards, the supernatant, containing non-collagen proteins and pigments, was removed. After repeating this process 3 times, the remaining alkali pellet was rinsed with distilled water, blended with 250 mL H2 SO4 (0.15%) at 25◦ C for 30 min in a shaking water bath, and then was centrifuged at 4,500 × g for 10 min to denature the collagen. This process was repeated 3 times, then the remaining acid pellet was rinsed with distilled water. Finally, the obtained pellet was blended with 250 mL of citric acid (0.7%) at 25◦ C for 30 min and then centrifuged at 4,500 × g for 10 minutes. This process also was repeated 3 times. Any residual salts remaining in the pellets after the extraction stages were removed by washing with distilled water before centrifugation at 4,500 × g for 10 min. The extract in the distilled water was left overnight at 50◦ C without blending and then filtered using a Buchner funnel with Whatman filter paper (no. 4). The filtrate was processed through ion exchangers to remove any salts. The resultant solution was adjusted to pH 6 with 0.1 M NaOH, then its volume lowered to 1/10 of its original volume by rotary evaporation (Laborota 4000, Heidolph Instruments GmbH & Co. KG, Schwabach, Germany) under vacuum at 50◦ C. Finally, the concentrated solution was freeze-dried and ground into a powder.
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% Yield =
Weight of freeze dried gelatin (g) Wet weight of fresh skin (g)
× 100
Determination of Proximate Composition The moisture content was determined gravimetrically after oven drying the samples at 105◦ C until they reached a constant weight. The ash content was determined gravimetrically by burning the samples at 550◦ C until they acquired a gray-white ash color. The fat content of the skin samples was determined by Soxhelet extraction method. The protein content of the samples was determined by Kjeldahl method with 5.4 as the protein conversion factor for skin (AOAC, 2000). The conversion factor of 5.4 was used because collagen, the main protein in the skin, contains a nitrogen ratio of 18.7% (Eastoe and Eastoe, 1952).
Determination of pH Distilled water (10 mL) was added to 1 g of sample and the solution then heated at 45◦ C for 5 min to dissolve the gelatin powder. The solutions were cooled to room temperature and the pH values determined during magnetic stirring using a pH-meter (Hanna HI 2210, Woonsocket, RI) (Shyni et al., 2014) calibrated against pH 4 and pH 7 buffers.
Determination of Molecular Weight Distribution SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (Owl, Model P8D8, Thermo Fisher Scientific, Rockford, IL, USA) was performed using a gradient gel (5% w/v loading gel and 10% w/v separating gel, 0.8 mm). The samples (5 mg) were dissolved in 5% SDS, heated at 85◦ C for one h, and then centrifuged at 8,500 × g for 5 min at room temperature. The supernatants were treated with SDS-PAGE loading buffer (200 mM TrisHCl, pH 6.8, 8% SDS, 0.4% bromophenol blue, 40% glycerol, and 100 mM DTT) at 3:1 ratio and incubated at 70◦ C for 10 minutes. After loading the sample mixtures and protein marker (20 μL) (PageRuler Unstained Protein Ladder, Thermo Fisher Scientific, Waltham, MA, USA) (Sambrook and Russell, 1989) onto the gel, they were electrophoresed using 1× TGS buffer (0.025 M Tris base, 0.192 M Glycine, 0.1% SDS, pH 8.3) at a constant voltage of 150 V. The protein bands were visualized by Coomassie Blue (G-250)
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staining and scanned using the Odyssey Infrared Imaging system (LI-COR Biosciences, Lincoln, NE).
Determination of Amino Acid Composition The homogenized sample (0.4 g) and HCl (5 mL, 6 N) were weighed into screw-cap tubes. Then, phenol (250 μL, 2 mM) was added to prevent oxidation. For the optimization of cysteine, methionine, and tyrosine recovery, 0.1 g Na2 SO3 was also added. The tubes were then closed under nitrogen gas and hydrolyzed at 110◦ C for 24 hours. Afterwards, the pH was adjusted to near neutral (6.7−7.3) and the tube filled to 50 mL with ultra-water according to the total amino acid amount. After the filtration through a membrane filter (Sigma, Stenheim, Germany) with a nominal pore size of 0.45 μm, the sample was analyzed by HPLC (Agilent HPLC 1100, Agilent Technologies, Inc., Santa Clara, CA) using a Zorbax Eclipse-AAA column (4.6 × 250 mm, 5 μm). The eluents used were buffer A (40 mM Na2 H2 PO4 , pH: 7.8) and buffer B (methanol: acetonitrile: ultra pure water [45:45:10]). The eluted amino acids are subjected to post-column derivatization with ortho-phthalaldehyde (OPA) regent delivered using a peristaltic pump at the rate of 2 mL/min at 40◦ C. Detection was done using a FL detector with absorption wavelength of 340 nm and emission wavelength of 450 nm. Additionally, the content of hydroxyproline in the same sample was analyzed using ACE 5C18 column (4.6 × 250 mm, 5 μm) with a UV detector in 265 nm wavelength at the rate of 1 mL/min at 25◦ C. The mobile phase that was used in hydroxyproline analysis contains 650 mL sodium acetate buffer prepared with 3% acetic acid and 350 mL acetonitrile. Amino acid standards (Sigma 21 L-Amino Acids plus kit) were run to calculate the concentration of amino acids in the sample. Norvaline (Sigma-Aldrich Inc., Oakville, ON, Canada) was added as an internal standard. The amino acid content was expressed as a percentage of the total amino acids in the sample.
Determination of Viscosity The viscosity (cP) of the gelatin solutions (6.67%) was determined at 30 ± 0.5◦ C using a Brookfield digital viscometer (Brookfield Engineering, Middleboro, MA) equipped with a no. 1 spindle at 60 rpm (Shakila et al., 2012).
Determination of Melting Point The melting point was determined by the Wainewright (1977) method. A 5 mL aliquot of gelatin solution (10% w/v) was placed into tubes and screw-capped, then cooled rapidly in an ice water bath. Five drops of chloroform and red food dye (3:1) were placed on the surface of the gels. The tubes were then placed in a 10◦ C water bath, and the temperature of the bath was gradually increased. The temperature at which the red drops started to move freely into the gel was recorded as the melting point.
Determination of Color Values Gelatin solution (6.67% w/v) was heated at 60◦ C for one h and then cooled to room temperature. The color was then measured at 6 different locations on the sample using a CR-400 chromameter (Konica Minolta, Tokyo, Japan). The L∗ , a∗ , and b∗ values obtained refer to the black-white, red-green, and yellow-blue color values, respectively, in accordance with the CIE Lab color system. The equipment was calibrated using its own calibration plate prior to analysis (Aykın et al., 2016).
Determination of Gel Clarity The gelatin solution (6.67%) was heated at 60◦ C for one h, and the clarity of the obtained gel samples was determined spectrophotometrically by transmittance (%T) measurement at 620 nm (Avena-Bustillos et al., 2006).
Determination of Gel Strength
Determination of Odor
Gelatin (7.5 g) and distilled water (105 mL) were blended in a bloom bottle to give 6.67% (w/v) gelatin solution, which was then rested at room temperature for one h to allow the gelatin to absorb the water. Afterwards, the bloom bottles were held in a 65◦ C water bath for 25 min to dissolve the gelatin, then rested at room temperature for 15 min before being placed in a 10◦ C water bath for 18 hours. The gel strength (bloom value) was determined by texture analysis (TA.XTPlus, Stable Microsystems, Godalming, UK). When the plunger (Delrin probe, P/0.5R caliber) had penetrated into the gel to a depth of 4 mm, the maximum strength (g) was determined, and the results were expressed as bloom (g) (Shyni et al., 2014).
A 6.67% gelatin solution was prepared by placing 0.5 g gelatin samples and 7 mL distilled water into test tubes. The tubes were capped and then placed into a 50◦ C water bath to dissolve. Afterwards, the test tubes were covered with aluminum foil so the panelists could not see the color of the samples. The odor specifications were evaluated by a 15-person educated panelist group, consisting of undergraduate and graduate students of the Food Engineering Department at Akdeniz University (Turkey) using a 6-point scale (0: no odor, 1: very light, can be sensed when carefully evaluated, 2: mild, easily detectable, 3: strong but not offensive, 4: strong and offensive, 5: very strong and offensive) (Muyonga et al., 2004).
CHARACTERIZATION OF BROILER SKIN GELATIN Table 1. Proximate composition of broiler skin and commercial bovine gelatins. Content
Broiler skin gelatin
Protein (%) Moisture (%) Ash (%) Fat (%) pH
84.29 12.57 2.13 0.45 5.82
± ± ± ± ±
Commercial bovine gelatin
1.60a 0.16a 0.07a 0.06a 0.08a
88.45 10.10 1.22 0.45 5.73
± ± ± ± ±
1.07a 0.21b 0.02b 0.05a 0.01a
a,b Different letters in the same row of each effect indicate significant (P < 0.05) differences between the means.
Statistical Analysis Physical and chemical properties of the broiler skin and commercial bovine gelatins were studied as 2 replications, and the analyses were duplicated. The data were analyzed by analysis of variance (ANOVA) using the SAS statistical software package (v.7.00, SAS Institute Inc., Cary, NC). Duncan’s multiple range test was used to determine significant differences at the 5% level. The results were expressed as the mean ± standard deviation.
RESULTS AND DISCUSSION Yield of Broiler Skin Gelatin The broiler skin gelatin yield was 6.5% on a wet weight basis. Sarbon et al. (2013) reported a yield of extracted chicken skin gelatin of 2.16% wet weight basis, while the yield of duck feet gelatin, extracted using different techniques, varied from 0.75 to 3.31% (Park et al., 2013). In contrast, the yields obtained in this study were lower than those found by Lassoued et al. (2014) for thornback ray skin (18.32 to 30.16%.); Shyni et al. (2014) for shark skin (19.7%), tuna skin (11.3%), and rohu skin (17.2%); and Ktari et al. (2014) for zebra blenny skin (14.8%) gelatins. The gelatin yields have been reported to vary among different raw materials, mainly due to differences in the extraction time, pretreatment process, washing step, collagen content, and the skin compositions (Du et al., 2013; Lassoued et al., 2014; Sinthusamran et al., 2014). The yield of gelatin from the chicken skin may be increased by using more intense extraction conditions, such as a higher temperature, more extreme acid-alkali treatment, and/or enzymes.
Proximate Composition of Broiler Skin Gelatin The proximate composition of broiler skin and commercial bovine gelatins is given in Table 1. The protein, moisture, ash, and fat contents of broiler skin gelatin were determined as 84.29, 12.57, 2.13, and 0.45%, respectively. The low fat content indicated that there was an efficient removal of fat from the skin. The broiler skin gelatin exhibited higher (P < 0.05) moisture and ash contents than the commercial bovine gelatin. How-
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ever, the moisture content of broiler skin gelatin was lower than the limit (15%) prescribed for edible gelatin (GME, 2008). The relatively low ash content suggested that the broiler skin gelatin was of good quality, considering that the maximum limit recommended for edible gelatin is 2.5% (Jones, 1977). The difference in the ash content of broiler gelatin compared with commercial bovine gelatin may have been due to the formation of inorganic compounds during the extraction procedure using acid and alkali solutions.
pH Values of Gelatin Samples The extracted broiler skin and commercial bovine gelatins had a pH of 5.82 and 5.73, respectively, as shown in Table 1, indicating their category as Type B. The conversion of collagen to gelatin leads to changes in the molecular structure of several amino acids. Type B gelatin has a pH ∼5 because the alkaline process deaminates glutamine into glutamic acid and asparagine into aspartic acid (Duconseille et al., 2015; Hattrem et al., 2015). It has been reported that Type B gelatin has minimum viscosity and maximum gel strength at pH 5 (Cole, 2000), signifying the importance of pH during the rheological properties of gelatin. In previous research, the pH was 4.83 in chicken deboner residue gelatin (Rafieian et al., 2015), 4.29 in tuna skin gelatin (Shyni et al., 2014), 4.60 in pink perch skin gelatin (Koli et al., 2012), 5.12 in striped catfish gelatin (Jamilah et al., 2011), and 6.21 to 6.84 in octopus skin gelatins (Jridi et al., 2015). The differences among pH values of gelatin samples may be due to the different acid and alkali pretreatments used during the extraction procedure.
Molecular Weight Distribution of Gelatin Samples The broiler and commercial bovine gelatins had different molecular weight distributions (Figure 1). The α1 - and α2 -chains with a molecular weight ∼120 kDa were present in both gelatin samples. While the β -chain with a molecular weight ∼200 kDa was absent in commercial bovine gelatin, it was present in broiler skin gelatin. The α-chain band was less intense in the commercial bovine gelatin than broiler skin gelatin samples. It was reported that gelatins with higher content of α-chain possess better functional properties (G´ omezGuill´en et al., 2002). Also, in contrast to the commercial bovine gelatin, the broiler skin gelatin included peptide fragments with a molecular weight less than 100 kDa, which could be attributed to the excessive cleavage of collagen by the heat treatment used during gelatin preparation. It was reported that the presence of these fragments is responsible for the low viscosity, low melting point, and decreased bloom strength of gelatin (Nagarajan et al., 2012). However, polymers with very high molecular weight (∼240 kDa) (γ -chain), plausibly
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AYKIN-DINC ¸ ER ET AL. Table 2. Amino acid composition of broiler skin and commercial bovine gelatins. Amino acids Aspartic acid (Asx) Glutamic acid (Glx) Serine (Ser) Histidine (His) Glycine (Gly) Threonine (Thr) Arginine (Arg) Alanine (Ala) Tyrosine (Tyr) Valine (Val) Methionine (Met) Phenylalanine (Phe) Isoleucine (Ile) Leucine (Leu) Lysine (Lys) Hydroxyproline (Hyp) Proline (Pro)
Broiler skin gelatin (%) 5.91 9.59 2.67 0.74 20.26 2.70 7.57 8.32 0.82 2.07 1.12 2.76 1.48 3.25 3.21 11.36 15.12
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.42a 0.54a 0.18a 0.02a 0.59a 0.40a 0.40a 0.37a 0.12a 0.13a 0.08a 0.19a 0.08a 0.20a 0.28a 0.11a 0.49b
Commercial bovine gelatin (%) 4.87 8.93 3.11 0.72 20.60 1.84 7.46 8.18 0.60 1.90 0.47 1.96 1.38 2.89 2.76 9.80 21.23
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.47a 0.66a 0.18a 0.01a 0.70a 0.09a 0.13a 0.21a 0.03a 0.10a 0.02b 0.13b 0.08a 0.13a 0.23a 0.61a 0.81a
a,b Different letters in the same row of each effect indicate significant (P < 0.05) differences between the means. Asx = Asp + Asn; Glx = Glu + Gln.
Figure 1. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) image of gelatin. M: molecular weight protein markers. B1: Broiler skin gelatin (5 mg) were dissolved in 5 mL of 5% SDS. B2: Broiler skin gelatin (5 mg) were dissolved in 10 mL of 5% SDS. C1: Commercial bovine gelatin (5 mg) were dissolved in 5 mL of 5% SDS. C2: Commercial bovine gelatin (5 mg) were dissolved in 10 mL of 5% SDS.
residual heat-stable cross-linked proteins, were present in both gelatins. These results suggested that broiler skin gelatin with a high content of α- and β -chains showed high molecular stability.
Amino Acid Composition of Gelatin Samples As shown in Table 2, the amino acid composition of broiler skin and commercial bovine gelatins exhibited a low content of methionine, histidine, and tyrosine, and no tryptophan or cysteine. This is in agreement with several studies, which reported that tryptophan and cysteine are typically absent in Type I collagen (Ktari et al., 2014; Lassoued et al., 2014; Jridi et al., 2015). Gly was the dominant amino acid in broiler skin gelatin (20.26%), followed by Pro (15.12%) then Hyp (11.36%). Gly, Pro, Hyp, and alanine were the main amino acids in both gelatins, which is a characteristic of all gelatins. The aspartic and glutamic acid contents were higher in comparison to those previously reported (Sarbon et al., 2013; Ktari et al., 2014), which may be associated with the acid pretreatment, as some of the glutamine and asparagine may have transformed to their acidic forms during the gelatin extraction. The amino acid compositions of the gelatin samples from broiler skin were not significantly (P > 0.05) different from that of bovine gelatin, except for methionine, phenylalanine, and Pro (P < 0.05). Broiler skin gelatin had higher methionine and phenylalanine
Table 3. Rheological properties of broiler skin and commercial bovine gelatins. Rheological properties Gel strength (g) Melting temperature (◦ C) Viscosity (cP)
Broiler skin gelatin
Commercial bovine gelatin
166.65 ± 1.63b 33.65 ± 0.07a 1.35 ± 0.10b
238.25 ± 2.47a 31.38 ± 0.11b 3.12 ± 0.12a
a,b Different letters in the same row of each effect indicate significant (P < 0.05) differences between the means.
contents than commercial bovine gelatin, as was similarly observed for thornback ray skin gelatin (Lassoued et al., 2014) and tuna skin gelatin (G´ omez-Estaca et al., 2009). Broiler skin gelatin had less total imino acid content (Pro and Hyp) than commercial bovine gelatin. The higher imino acid content suggested that the broiler skin gelatin may exhibit better rheological properties by structural stabilization of the triple helix of collagen, developing a strong gel structure (Ktari et al., 2014), as the hydroxyl groups of Hyp form hydrogen bonds with available water molecules. Lassoued et al. (2014) reported that bovine gelatin with a higher imino acid content had a higher gel strength than thornback ray skin gelatin.
Gel Strength of Gelatin Samples Gel strength is one of the most important physical properties of gelatin. As shown in Table 3, the gel strength of broiler skin gelatin (166.65 g) was significantly (P < 0.01) lower than that of commercial bovine gelatin (238.25 g). This may be due to the lower imino acid content (Pro and Hyp) of broiler skin, which could result in a less organized triple-helix structure because both imino acids are responsible for stabilizing the secondary protein structure. In previous research, the gel strength was 355 g for chicken skin gelatin (Sarbon
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et al., 2013), 260 g for bovine gelatin and 140 g for thornback ray skin gelatin (Lassoued et al., 2014), 124 g for rohu skin gelatin (Shyni et al., 2014), and 62 to 284 g for gelatins extracted from octopus skin using various pepsin concentrations (Jridi et al., 2015). The gel strength of commercial bovine gelatins is reported to range from 100 to 300 g (G´ omez-Guill´en et al., 2011). The differences in gel strength among samples could be explained by differences in the ages of the animals, the extraction process used, the presence of other hydrocolloids, and the intrinsic properties of collagen, which vary among species.
Table 4. Color and transmittance values of broiler skin and commercial bovine gelatins. Parameter L∗ a∗ b∗ Transmittance (%)
Broiler skin gelatin 27.62 − 2.98 1.33 44.28
± ± ± ±
Commercial bovine gelatin
0.04a 0.04b 0.04b 0.14b
23.64 − 1.91 3.91 86.01
± ± ± ±
0.06b 0.03a 0.04a 0.58a
a,b Different letters in the same row of each effect indicate significant (P < 0.05) differences between the means.
hence it has a higher gel strength, viscosity, and melting temperature (Sarbon et al., 2013).
Viscosity of Gelatin Samples The viscosity of broiler skin gelatin (1.35 cP) was significantly (P < 0.01) lower than commercial bovine gelatin (Table 3) and previously reported values for chicken deboner residue and commercial bovine gelatins (5.55 and 2.90 cP, respectively) (Rafieian et al., 2015), which could be attributed to the low molecular weight fragments present in broiler gelatin (Figure 1). Viscosity is correlated to the molecular weight and molecular size distribution of the extracted proteins (Sperling, 2005). It was reported that gelatins containing a smaller proportion of large molecules, such as β -chains, than small molecules, exhibit low viscosity and result in weak gels (Silva et al., 2014). A low viscosity also has been observed for gelatin at pH 6 to 8. Also, gelatin prepared at a high (≥0.05 M citric acid or ≥0.07 M HCl) or weak (0.01 to 0.03 M acetic acid or 0.01 M HCl) acid concentration, had less β -chains and low viscosity (Niu et al., 2013).
Melting Temperature of Gelatin Samples The melting temperature of broiler skin gelatin was significantly (P < 0.01) higher than that of commercial bovine gelatin (Table 3), which can be correlated with its higher imino acid content, but was similar to that reported for chicken gelatin (33.57◦ C) (Sarbon et al., 2013). It seems, therefore, that broiler skin gelatin could be used to replace the other gelatins in some food products in which gelatin is used. In comparison with other species, fish gelatin (3.4 to 25.8◦ C) (Koli et al., 2012) and acid bovine skin gelatin (29.7◦ C) have a relatively low melting temperature, whereas that of acid pork skin gelatin is comparatively high (32.3◦ C) (Gudmundsson, 2002). It is known that fish gelatins, which have a low content of imino acids (Gudmundsson, 2002), usually have a lower melting temperature than mammalian gelatins (Karim and Bhat, 2009). Thus, variation in melting temperature among different gelatin samples is probably due to the differences in imino acid contents (Pro and Hyp). The gelatin with high imino acid content may exhibit better rheological properties by structural stabilization of the triple helix of collagen, and
Color and Gel Clarity Values of Gelatin Samples The L∗ , a∗ , and b∗ color values of the broiler and commercial bovine gelatin samples were significantly (P < 0.01) different (Table 4). The L∗ value, or lightness value, of broiler gelatin was slightly higher than that of commercial bovine gelatin. Both gelatin samples showed negative a∗ values, and it was significantly lower for broiler gelatin. The b∗ value of broiler gelatin was slightly lower than that of commercial bovine gelatin, indicating that broiler gelatin was less yellow than commercial bovine gelatin. These results showed that factors, such as raw material and extraction conditions, influenced the color of extracted gelatin. While commercial bovine gelatin showed good transmittance (%), broiler skin gelatin had a significantly poor value (Table 4). This might be due to inorganic, proteinaceous, and mucosubstance contaminants, either introduced or not removed, during its extraction. Filtration efficiency during extraction directly affects the degree of clarity of gelatin solutions, and, accordingly, non-settling and unfilterable particulate matter in gelatin solution may be the cause of turbidity (Muyonga et al., 2004). The color and gel clarity are important aesthetic properties of gelatin, depending on its intended use.
Odor of Gelatin Samples The odor of the broiler skin gelatin (2.07 ± 0.28) was not significantly (P > 0.05) different from that of commercial bovine gelatin (1.70 ± 0.05). Both gelatins were found to have a mild odor because the hedonic score was ∼2. However, the odor of broiler skin gelatin was more apparent than that of commercial bovine gelatin. It can be concluded that the odor of broiler skin gelatin did not affect consumers and was similar to the odor of gelatin products available on the market. Choi and Regenstein (2000) reported that activated carbon treatment at the final stage of extraction can further decrease the odor and improve the acceptability of gelatin.
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CONCLUSIONS Broiler skin as an animal by-product obtained from the poultry industry could be a promising source of gelatin. The yield of broiler skin gelatin was 6.5% on a wet weight basis, and more intensive extraction conditions should be investigated in future studies, as this may further increase the yield. The gelatin extracted from broiler skin showed a higher melting temperature than that of commercial bovine gelatin. The difference in the rheological properties of broiler gelatin compared with commercial bovine gelatin may be due to the intrinsic properties associated with differences in their amino acid composition. The main structural difference between broiler skin and commercial bovine gelatins was the imino acid content, which was higher in the commercial bovine gelatin. Considering its sensory evaluation, broiler skin gelatin could potentially be an alternative to commercial bovine gelatin, added in various food products.
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