Characterizations of Gelatin from the Skin of American Bullfrog ( Rana catesbeiana ) as Affected by Extraction Temperature

: We investigated the effect of extraction temperature on the gel properties of gelatin from the skin of the American bullfrog ( Rana catesbeiana ) and the mechanisms. The textural and rheological properties of bullfrog gelatin extracted at 45 ◦ C (G45), 55 ◦ C (G55), and 65 ◦ C (G65) were measured. The molecular weight distributions, microstructures, and amino acid compositions of the bullfrog gelatins were also determined. G45, G55, and G65 had gel strengths of 272.1, 225.6, and 205.8 g and hardness values of 28.1, 24.0, and 22.5 N, respectively. The gelling temperatures ranged from 19.3 to 23.9 ◦ C, and the melting temperatures ranged from 28.9 to 31.5 ◦ C. All the results were compared with those of commercial porcine gelatin. We propose that the higher gel strength of G45 with a higher band intensity of α 2-chains compared with G55 and G65 was more likely to form ordered and strong cross-links. The gelatin extracted at a lower temperature (G45) had a ﬁner gel structure, suggesting that it would be more difﬁcult to disrupt by applied force. Gelatin extracted at a lower temperature demonstrated better properties with α 2-chains and a ﬁne gel structure. These results provide basic information on the extraction of American bullfrog skin gelatin for industrial applications.


Introduction
Gelatin is a fibrous protein hydrolyzed from a collagen that is mainly derived from the skin, connective tissues, and bones of animals [1]. Gelatin has been widely used as a stabilization, gelation, and emulsion agent in food and non-food industries [2,3]. The world usage of gelatin is about 200,000 metric tons yearly [4], which is predominantly manufactured from the skin and bones of pigs and cows. However, the outbreaks of foot-and-mouth ailments and bovine spongiform encephalopathy (BSE) have caused panic among customers [5].
Recently, chicken skin [6], chicken deboner residue [7], duck feet [8], camel skins [9], goat skin [10], cod fish skin [11,12], salmon fish skin [13], and shark byproducts [14] as additional sources have been tapped for gelatin extraction in attempts to increase safety. American bullfrogs (Rana catesbeiana) may be a new and safe source of gelatin with no threat of BSE. In China, the bullfrog annual production currently exceeds 100,000 tons, which is processed into products with no skin [15]. Gelatin can be extracted from different species of frogs in the genus Rana, such as Rana tigerina [2], Rana nigromaculata [16], and Rana esculanta [17].
The American bullfrog (Rana catesbeiana) is an important economic amphibian and one of the largest frogs in the genus Rana [15]. As bullfrog meat has developed in international gastronomy in various styles [18], the hatcheries of American bullfrogs have increased all around the world [19], generating a great amount of skin as by-products. Extracting gelatin from American bullfrog skin can reduce waste in the bullfrog industry and increase the

Extraction of Gelatin from Bullfrog Skin
Bullfrog skin gelatin was extracted using the procedure described by Hafsteinsson et al. [23] with some modifications. The bullfrog skins were first cleaned to eliminate all residues of muscle then cut into small pieces (2 × 3 cm). The skin was soaked in 0.1 mol·L −1 NaOH solution 1:10 (w/v) below 10 • C for 3 h, followed by washing with water until the wash water was almost neutral. The skin was soaked in 0.05 mol·L −1 CH 3 COOH solution 1:10 (w/v) under gentle stirring below 10 • C for 3 h. This was followed by washing with water until the wash water was almost neutral. Extractions with water 1:6 (w/v) were stirred for 6 h at 45, 55, and 65 • C The gelatin samples were filtered by cheesecloth and then centrifuged at 1589× g for 10 min. The supernatant was freeze-dried.

Yield Determination
The yield of gelatin was calculated based on the following equation:

Proximate Analysis
The moisture content was calculated by the weight lost during drying. The freezedried gelatin samples were first weighed and then placed in an oven at 105 • C After 2 h of drying, the gelatin was re-weighed, then put back in the oven at 105 • C for 1 h, and then re-weighed again. The above operation was repeated until the difference between the two masses was less than 2 mg. The protein content of the gelatin samples was determined using the Kjeldahl method. The nitrogen conversion factor used for the gelatin was 5.55.

Determination of Gel Strength
The method of Fernandez-Daz et al. [24] was used to determine the gel strength. Gelatin solutions (6.67%, w/v) were prepared at 60 • C in distilled water and kept at 10 • C for 16-18 h before analysis. The gel strength of the samples was determined by a Brookfield CT3 Texture Analyzer (Leatherhead Food Research Association Texture Analyzer Brookfield, Sustainability 2021, 13, 4390 3 of 11 USA) with a 12.7 mm diameter probe. The speed of the plunger was 0.5 mm/s. The maximum force was recorded when the penetration distance reached 4 mm.

Rheological Behavior
The rheological behaviors of the gelatin solutions (6.67%, w/v) were analyzed using a rheometer (AR1500ex, TA Instruments, New Castle, DE, USA) using a 40 mm parallel plate. Temperature sweeps were performed from 50 to 10 • C and 10 to 50 • C with cooling/heating rates of 1.0 • C/min. The frequency and strain amplitude were set at 1 Hz and 0.1%. The cross-over point of the storage modulus (G ) and loss modulus (G") was considered as the gelling or melting temperature of each gelatin gel. The angular frequency sweep of the gelatin solutions with a range of 0.1-100 rad/s were measured with a stress value of 1 Pa. All the analyses were performed in triplicate.

Texture Profile Analysis (TPA)
The texture profiles of gelatin gels (6.67%, w/v) were analyzed using the previously published method of Huang et al. [25]. A TMS-Pro Texture Analyzer (Food Technology Corporation, Sterling, VA, USA) with a 50-mm diameter aluminum cylindrical probe (P/50) was used to measure the hardness, springiness, cohesiveness, and chewiness. Each sample was poured into a mold and kept at 10 • C for 16-18 h. The gelatin samples (d: 3 cm × h: 2.5 cm) were compressed to 50% of the original height for two cycles at a speed of 60 mm/min.

Electrophoretic Analysis
The gelatin samples were measured as described by Laemmli et al. [26]. A gelatin solution (1 mg protein/mL water) was mixed in a 1:4 (v/v) ratio with loading buffer (6% 1 M Tris-HCl, 50% glycerol, 10% [w/v] SDS, 1% [w/v] bromophenol blue, and 0.5% 2-mercaptoethanol, pH 6.8). The mixture was heated in a boiling water bath for 5 min. The stacking and resolving gel from the PAGE Gel Fast Preparation Kit (Shanghai epizyme Biotech Co., Ltd. Shanghai, China) was subjected to electrophoresis.
The electrophoresis was run at a constant voltage of 80 V/gel for the stacking gel, then run at 120 V/gel for 60 min until the resolving gel reached the bottom of the gel. This was followed by staining with Coomassie brilliant blue R-250 (0.25% w/v). A rainbow protein maker (5-245 kDa) was used to estimate the molecular weight distributions. The gels were scanned with an Imager 600 (Amersham, UK) gel-imaging system.

Microstructure Analysis of Gelatin
The microstructure of the gelatin (6.67%, w/v) was elucidated using S-3000N cryoscanning electron microscopy (cryo-SEM, Hitachi Co., Tokyo, Japan). The samples were deposited in the slots of a stub with rivets and then frozen by plunging them into slush nitrogen. After being fractured, the free water of the gels was sublimated at −85 • C for 30 min, and the gel was sputter-coated with gold (Model PP3000T, Quorum Technologies, East Grinstead, UK).

Amino Acid Composition Analysis
The amino acid composition was measured according to GB 5009.124-2016 [27].

Statistical Analysis
The experiments were performed in triplicate (except for the amino acid composition data). All data underwent analysis of variance, and significant differences (p < 0.05) between the means were determined using Tukey's test using SPSS 17.0, SPSS Inc. (Chicago, IL, USA).

Yield and Proximate Composition
The gelatin yields extracted at the three temperatures from bullfrog skin (45, 55, and 65 • C) are shown in Table 1. The yields of G45, G55, and G65 were 9.6%, 11.7%, and 12.3% (on a wet weight basis), respectively. Generally, an increase in extraction temperature was associated with a higher yield. The results were consistent with those of Nagarajan et al. [22] and Kittiphattanabawon et al. [28], who reported that gelatin yield increased as the extraction temperature increased. Increasing the extract temperature provides more energy for the disruption of the stabilizing collagen structures by breaking hydrogen bonds and peptide bonds [29]. As the extraction temperature increases, gelatin undergoes more helix-to-coil transitions and becomes easier to extract into the water, leading to a higher yield [30]. The moisture and protein of gelatin extracted at different temperatures (45, 55, and 65 • C from the bullfrog skin were 6.1-7.3% and 87.2-87.8%, respectively. The moisture contents of all bullfrog skin gelatin samples were below the prescribed limit (15%), which was lower than commercial porcine gelatin (PG) (p > 0.05). There were no significant differences in the moisture or protein contents among G45, G55, and G65. All bullfrog skin gelatin samples showed low levels of moisture content and high levels of proteins. The bullfrog skin moisture as a major component was 67.6%. Zhang et al. [16] showed a similar result that the moisture content of bullfrog (Rana nigromaculata) skin was 74.0%.

Gel Strength of Gelatin
The gel strength of the gelatins from bullfrog skin at different extraction temperatures are shown in Figure 1. G45 showed the highest gel strength (b: 272.1 g) compared with G55 (c: 225.6 g) and G65 (d: 205.8 g) (p < 0.05), which were lower than PG (a: 474.3 g). Therefore, the extraction temperatures directly affected the gel strength of the bullfrog gelatin. This agrees with the studies by Liu et al. [31] and Sinthusamran et al. [21], who reported that high extraction temperatures significantly decreased the gel strength of gelatin.
Gelatins with different molecular weight distributions and amino acid compositions have different gel strength. Higher extraction temperatures might cause more hydrolysis, the more hydrolysis likely leads to shorter chains. The shorter chains cannot align properly, and the junction zone cannot form to a higher degree [20]. The amino acid composition has been reported to be one of the most important factors affecting the gel strength of gelatin [22].  Figure 2 shows the temperature development of G′ upon cooling and heating. G′ was higher than of G″ (not shown) at lower temperatures indicated that gelatin exhibited solid behavior, demonstrating gelatin molecules in a triple-helix arrangement. As shown in Figure 2, the G′ of all gelatin samples increased sharply as the extraction temperature decreased, the cross-over point of G′ and G″ (not shown) indicated that the formation of the gel was considered to be the gelling temperature of the gelatin gels.

Temperature Sweep
The gelation process for gelatin is the transition from single strands to a triple helix via hydrogen bonding, ionic interaction, hydrophobic association, van der Waals forces, and self-assembly [32]. The maximum values of the G′ values of G45, G55, and G65 were 2814, 2470, and 2266 Pa, respectively, showing that higher temperature extraction reduced the crosslinking between gelatin molecules. This result agrees with the gel strength (Figure 1).
As shown in Table 1, the gelling and melting temperatures of G45, G55, and G65 were 19.3-23.4 °C and 28.9-32.3 °C, respectively, and there was no marked difference among all bullfrog skin gelatins and PG (p > 0.05). The gelling and melting temperatures in this study were higher than those of gelatins from the skins of camel (15.2-11.1 and 18.4-21.6 °C, respectively) [9], croaker fish (17.4 °C and 23.8 °C, respectively) [33] and similar with goat skin gelatin (21.2-25.2 °C and 30.7-34.1 °C, respectively) [20]. The difference in the gelling and melting properties of bullfrog gelatin compared with other gelatins can be attributed to the difference in extraction conditions, amino acid compositions, and protein chain length [29].   Figure 2 shows the temperature development of G upon cooling and heating. G was higher than of G" (not shown) at lower temperatures indicated that gelatin exhibited solid behavior, demonstrating gelatin molecules in a triple-helix arrangement. As shown in Figure 2, the G of all gelatin samples increased sharply as the extraction temperature decreased, the cross-over point of G and G" (not shown) indicated that the formation of the gel was considered to be the gelling temperature of the gelatin gels.  Figure 2 shows the temperature development of G′ upon cooling and heating. G′ was higher than of G″ (not shown) at lower temperatures indicated that gelatin exhibited solid behavior, demonstrating gelatin molecules in a triple-helix arrangement. As shown in Figure 2, the G′ of all gelatin samples increased sharply as the extraction temperature decreased, the cross-over point of G′ and G″ (not shown) indicated that the formation of the gel was considered to be the gelling temperature of the gelatin gels.

Temperature Sweep
The gelation process for gelatin is the transition from single strands to a triple helix via hydrogen bonding, ionic interaction, hydrophobic association, van der Waals forces, and self-assembly [32]. The maximum values of the G′ values of G45, G55, and G65 were 2814, 2470, and 2266 Pa, respectively, showing that higher temperature extraction reduced the crosslinking between gelatin molecules. This result agrees with the gel strength (Figure 1).
As shown in  [20]. The difference in the gelling and melting properties of bullfrog gelatin compared with other gelatins can be attributed to the difference in extraction conditions, amino acid compositions, and protein chain length [29].  The gelation process for gelatin is the transition from single strands to a triple helix via hydrogen bonding, ionic interaction, hydrophobic association, van der Waals forces, and self-assembly [32]. The maximum values of the G values of G45, G55, and G65 were 2814, 2470, and 2266 Pa, respectively, showing that higher temperature extraction reduced the crosslinking between gelatin molecules. This result agrees with the gel strength ( Figure 1).
As shown in Table 1, the gelling and melting temperatures of G45, G55, and G65 were 19.3-23.4 • C and 28.9-32.3 • C, respectively, and there was no marked difference among all bullfrog skin gelatins and PG (p > 0.05). The gelling and melting temperatures in this study were higher than those of gelatins from the skins of camel (15.2-11.1 and 18.4-21.6 • C, respectively) [9], croaker fish (17.4 • C and 23.8 • C, respectively) [33] and similar with goat skin gelatin (21.2-25.2 • C and 30.7-34.1 • C, respectively) [20]. The difference in the gelling and melting properties of bullfrog gelatin compared with other gelatins can be attributed to the difference in extraction conditions, amino acid compositions, and protein chain length [29].

Frequency Sweep
Measuring the angular frequency of the modulus can be used to evaluate the strength of the gel network [34]. The cross-linking behavior of bullfrog skin gelatin was characterized by performing a dynamic rheological test on a 6.67% (w/v) gelatin sample at a constant temperature (10 • C) (Figure 3). The G values were higher than the G" values (not shown) during the angular frequency range studied, confirming that gelatins were capable of forming a network and possessed a solid-like gel structure at 10 • C [25]. G45 showed numerically higher G values compared with G55 and G65 (Figure 3). This observation agrees with the results of a study by Abedinia et al. [8]. As explained above, the stronger inter-molecular interactions in G45 compared with those in G55 and G65 may result in higher G values [34].

Frequency Sweep
Measuring the angular frequency of the modulus can be used to evaluate the strength of the gel network [34]. The cross-linking behavior of bullfrog skin gelatin was characterized by performing a dynamic rheological test on a 6.67% (w/v) gelatin sample at a constant temperature (10 °C) (Figure 3). The G′ values were higher than the G″ values (not shown) during the angular frequency range studied, confirming that gelatins were capable of forming a network and possessed a solid-like gel structure at 10 °C [25]. G45 showed numerically higher G′ values compared with G55 and G65 (Figure 3). This observation agrees with the results of a study by Abedinia et al. [8]. As explained above, the stronger inter-molecular interactions in G45 compared with those in G55 and G65 may result in higher G′ values [34].

Texture Profile Analysis
Texture profile analysis (TPA) is closely related to the sensory evaluation of gels [25]. The TPA results of the gelatins in this study are presented in Table 2. The hardness of G45, G55, and G65 were 28.1, 24.0, and 22.5 N, respectively. The higher extraction temperature could result in the lower hardness of gelatin gels. The hardness of all bullfrog skin gelatin was lower than porcine skin gelatin. This result is consistent with the gel strength findings (Figure 1). There were no significant differences in the springiness, cohesiveness, or chewiness among all gelatin samples (p > 0.05). A study reported that the textural properties of gelatin gels can be influenced by the amino acid compositions and molecular weight distributions [25].

Texture Profile Analysis
Texture profile analysis (TPA) is closely related to the sensory evaluation of gels [25]. The TPA results of the gelatins in this study are presented in Table 2. The hardness of G45, G55, and G65 were 28.1, 24.0, and 22.5 N, respectively. The higher extraction temperature could result in the lower hardness of gelatin gels. The hardness of all bullfrog skin gelatin was lower than porcine skin gelatin. This result is consistent with the gel strength findings (Figure 1). There were no significant differences in the springiness, cohesiveness, or chewiness among all gelatin samples (p > 0.05). A study reported that the textural properties of gelatin gels can be influenced by the amino acid compositions and molecular weight distributions [25].

SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
The protein patterns of the gelatin samples are shown in Figure 4. All the gelatin samples contained αand β-chains with molecular weights of approximately 100 and 200 kDa, as the major components. This indicates that the αand β-chains of the mother collagen were retained with rare degradation [29]. Among all the gelatin samples, G65 possessed the lowest αand β-chain band intensity (as observed visually), while G45 showed a higher band intensity of α2-chains over G55 or G65. Gomez-Guillen et al. [35] reported that gelatins with higher α-chain contents possessed better functional properties.

SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
The protein patterns of the gelatin samples are shown in Figure 4. All the gelatin samples contained α-and β-chains with molecular weights of approximately 100 and 200 kDa, as the major components. This indicates that the α-and β-chains of the mother collagen were retained with rare degradation [29]. Among all the gelatin samples, G65 possessed the lowest α-and β-chain band intensity (as observed visually), while G45 showed a higher band intensity of α2-chains over G55 or G65. Gomez-Guillen et al. [35] reported that gelatins with higher α-chain contents possessed better functional properties.
We noted that the bands at around 70 kDa were more intense in G65, suggesting that more drastic degradation occurred during the extraction process, which is consistent with the findings of Pang et al. [36]. It is likely that more degradation occurred in G65 as a result of the higher extraction temperature. These results suggest that the intensities of the αand β-chains bands of bullfrog skin gelatin were influenced by the extraction temperature. The result was partly in accordance with Tan et al. [37], who found a high extraction temperature (75 °C) resulted in a decrease in the major protein components (a-and β-chains) of black tilapia (Oreochromis mossambicus) gelatin.

Microstructures of Gelatin Gels
The gel strength of gelatin was affected by the generally conformation and association of the proteins in the gel matrix [38]. Figure 5 shows the microstructures of the gelatin gels. The structures of all gelatins were sponge or coral-like. Among all the bullfrog gelatins, G45 showed the finest gel network with very small voids. Gelatin extracted at lower temperatures with fine gel structures is consistent with a higher gel strength ( Figure 1).
As observed, the gel network of G65 was found to be coarse and heterogenous. This result was partially in agreement with the findings of Sinthusamran et al. [29] who described gelatin extracted at a lower temperature for less time as having a finer gel structure. The microstructure of the gel is known to be closely related to its physical properties [36], and a heterogenous network may be more easily disrupted by applied force [28]. We noted that the bands at around 70 kDa were more intense in G65, suggesting that more drastic degradation occurred during the extraction process, which is consistent with the findings of Pang et al. [36]. It is likely that more degradation occurred in G65 as a result of the higher extraction temperature. These results suggest that the intensities of the αand β-chains bands of bullfrog skin gelatin were influenced by the extraction temperature. The result was partly in accordance with Tan et al. [37], who found a high extraction temperature (75 • C) resulted in a decrease in the major protein components (aand β-chains) of black tilapia (Oreochromis mossambicus) gelatin.

Microstructures of Gelatin Gels
The gel strength of gelatin was affected by the generally conformation and association of the proteins in the gel matrix [38]. Figure 5 shows the microstructures of the gelatin gels. The structures of all gelatins were sponge or coral-like. Among all the bullfrog gelatins, G45 showed the finest gel network with very small voids. Gelatin extracted at lower temperatures with fine gel structures is consistent with a higher gel strength ( Figure 1).
As observed, the gel network of G65 was found to be coarse and heterogenous. This result was partially in agreement with the findings of Sinthusamran et al. [29] who described gelatin extracted at a lower temperature for less time as having a finer gel structure. The microstructure of the gel is known to be closely related to its physical properties [36], and a heterogenous network may be more easily disrupted by applied force [28].
The proline content of G45 (13.6%) was lower than in PG (14.6%) and was higher than shark extracts (9.0%) [14]. This result was consistent with G45 showing a lower gel strength compared with PG. Proline can form hydrogen bonds between hydroxyl groups in hydroxyproline and water molecules, and these bonds contribute to the strength of the gelatin gel [29]. The hydrophobic amino acid content of G45 (18.3%) was lower than that of PG (20.1%), and these amino acids can form hydrophobic associations, which also contribute to the gel strength [40]. The differences in the amino acid content might be due to the different sources and manufacturing processes of the gelatin [41].