The study hypothesizes that different temperatures and times will affect the physicochemical characteristics of gelatine extracted from the skin and bones of snakehead fish.

Wild Snakehead (Channa striata) fish of between 600 to 700 g with a length of approximately 42.07 ±0.75 cm were obtained from Bili Bili Dam, Gowa South Sulawesi, Indonesia (Figure 1).

The analytical grade chemicals Ca(OH)₂,Citric acid, HCl, KBr (Merck, Germany) were used in the experiments. The commercial gelatin used was bone bovine gelatin, 150 g gel strength (Green Valley, Global Capsule Ltd. Dhaka 1000-Bangladesh).

The instrument used in this study were oven (Memmert UM 400, Germany), dry blender (Miyako 152PF, PT. Kencana, Indonesia), weighted (Mettler Toledo, J81603-C/FACT, Germany), water-bath (Memmert W350, Germany), TAXT2 Texture Analyzer (Stable Micro System, UK), ultra-performance liquid chromatography (UPLC, ACQUITY UPLC-H Class, Waters, USA), vortex (IKA Labortechnik VF2, Germany), homogenizer (IKA T25 digital-ULTRA TURRAX, Germany), spectrophotometer (Thermo Fisher Scientific 4001/4, Genesys VF2, Germany), centrifuged (High speed refrigerated micro centrifuge, Tomy Mx-305) and Fourier Transform Infrared Spectrophotometry (FTIR) (IR Prestige-21, FTIR-8400, Shimadzu, Japan).

The experimental data were tested using one-way analysis of variance (ANOVA) based on a completely randomized factorial design, namely yield, gel strength, proximate composition, amino acid composition, emulsifying properties, water holding and fat binding capacity of the skin and bone gelatin. The validity of diversity was checked using the Duncan test. The data was running using SPSS software (IBM Corporation, US).

The yields were an important factor that was always considered in the conversion of collagen to gelatin because it was related to the effectiveness of the process applied and the economic benefits. The yield produced in the process of transforming collagen from the skin and bone of snakehead fish as raw material into gelatin at different extraction temperatures and times showed significant differences (p <0.05), as shown in Table 1.

The yield of snakehead gelatin was influenced by the type of raw material, temperature, and extraction time. According to Karim and Bhat (2009); Kołodziejska et al. (2008), that the conversion of collagen to gelatin can be influenced by processing parameters, such as the nature of the raw material and the method of extraction, including the pre-treatment conditions, temperature, and time. The higher the extraction temperature, the yield of SSG and SBG significantly increased. Likewise, the longer the extraction time, the yield of the two types of gelatin also significantly increased. Gelatin yield was influenced by differences in the type of raw material, temperature, and extraction time.

However, extraction at a temperature of 50 °C with different extraction times had not obtained an increase in the SBG yield (p >0.05). Sims and Bailey (1992) explained that skin collagen from immature animals contained an intermediate crosslink of dehydroxylysinonorleucine and when it reached adulthood it was converted into trivalent cross-linked of histidinohydroxylysinonorleucine which was more stable, while collagen in bones contained hydroxylysinocetonorleucine which was then converted to pyridinoline. According to Bailey, Paul and Knott (1998), pyridinoline was more stable to heating. This possible phenomenon was one of the factors that cause the SBG yield to be lower than SSG due to the slower conversion process of collagen in bones compared to skin.

There were significant differences due to the interaction of raw materials and time; and the interaction of raw materials and temperature, as well as the interaction between temperature and time; and the interaction of raw materials, temperature, and time on gelatin yield (p <0.05). Table 1 showed that SSG was significantly higher than SBG at the same temperature and extraction time. During the extraction process, temperature affects the solubility rate of collagen, where high temperatures caused the collagen bond structure to open and result in the breaking of the bonds from the collagen protein molecule. Extraction time was the most significant factor affecting gelatin yield (Ahmad and Benjakul, 2011; Hanjabam et al., 2015). The higher temperatures used the gelatin yield tended to increase. During the extraction process in the hydrogen bonds, the heating process in a water bath which stabilized the triple helix of the parent collagen is damaged, causing a transition from helix to coil resulting in the conversion of collagen into gelatin solution (Benjakul, Kittiphattanabawon and Regenstein, 2012).

In general, the yield of SBG was relatively lower than SSG, either due to a single factor of raw material, temperature or time, or the interaction between treatments. The skin was composed of organic material that can easily change its character due to chemical treatment rather than bone. This was because the bone structure consists of organic and inorganic composites (Szpak, 2011). The demineralization process of bone to ossein at the beginning of the pre-treatment process causes the release of several minerals, as suggested by Muyonga, Cole and Duodu (2004a) and Muyonga, Cole and Duodu (2004b). The demineralization process causes the release of mineral components that fill the spaces between collagen molecules in the bone matrix, allowing easier conversion of collagen to gelatin. The yields of SSG and SBG were 18.25 ±0.26% and 8.19 ±0.17%, respectively, higher than the results obtained by Muyonga, Cole and Duodu (2004b) on the skin and bones of Lates niloticus fish as much as 12.3 ±2.1% to 16.0 ±0.3% and 1.3 ±1.0 to 2.4 ±0.7%, respectively.

The main parameter of gelatin was gel strength, which was used as a method for measuring the impact of production factors and optimizing the quality of the final product. According to Karim and Bhat (2009), raw material characteristics, temperature, and extraction time play an important role in gelatin gel strength. There was a difference in gel strength between the skin and snakehead fish bones extracted at different temperatures and times (p <0.05). The effect of the gel strength gelatin of skin and bone snakehead fish on the temperature treatment and extraction time were presented in Table 1.

The interaction of treatment combinations between raw materials and temperature; raw materials and time; and between raw materials, temperature and time had different effects on the gel strength of snakehead fish gelatin (p <0.05). The existence of this interaction showed that there was a different gel strength on SSG and SBG. The longer the extraction time, the gel strength of SSG tends to decrease but vice versa in SBG was tending to increase.

The same properties were found in the gel strength of Aluterus monoceros skin gelatin (Ahmad and Benjakul, 2011) which tended to decrease with increasing extraction time. There was an increase in gel strength when the SSG was heated between 50 °C and 60 °C but decreased when the extraction temperature was increased by 10 degrees, while the gel strength of SBG tended to increase as the extraction temperature increased. It seems that the snakehead bone takes a relatively long extraction time to match the gel strength of snakehead skin. Compared to snakehead skin, the relatively long extraction process on the bones was thought to be related to the different chemical compositions of the two materials.

The skin consists of organic material and very little inorganic material, but bones contain organic material with a high proportion of inorganic material (Olszta et al., 2007; Rosmawati et al., 2018a; Szpak, 2011). Demineralization of bone before pretreatment and extraction was an effective method for releasing inorganic materials (Muyonga, Cole and Duodu, 2004a; Muyonga, Cole and Duodu, 2004b; Ranasinghe et al., 2020).

Based on Table 1 above, the shown that SSG and SBG have relatively the same gel strength when each extracted at different temperature and time combinations, SS4 (60 °C, 12 h) vs SB6 (60 °C, 24 h). Skin and bone have structural differences and cross-linkages (Sims and Bailey, 1992) so that different gel strengths can occur under the same extraction conditions. The extraction process allows the chain to break, the shorter the chain, the lower the gel strength. The temperature and extraction time contribute to the breaking of this chain. This is inseparable from the influence of the structure, sequence, and configuration of amino acids (Kittiphattanabawon et al., 2012). The best gel strength was found in SSG extracted at 60 °C for 12 hours, while SBG at the same temperature with 24 hours extraction time was 278.37 ±22.23 g and 239.53 ±20.68 g, respectively. The gel strength of SSG and SBG was higher than BCG, namely 150 g (according to product label).

The chemical composition of SSG and SBG compared with commercial bovine gelatin (BCG) was shown in Table 2. Analysis of variance showed the type of gelatin had a significant effect (p <0.05) on moisture, protein, fat, and ash content.

The water content of the three types of gelatin showed significant differences (p <0.05), where the water content of SBG was lower than SSG and BCG. The difference in gelatin water content was influenced by differences in raw materials and extraction processes used. The maximum level of gelatin water content based on the Indonesian National Standard (SNI, 1995) was 16%, while the British Standard Institution (BSI, 1975) regulates maximum water content between 13 to 14%. Based on this, the snakehead gelatin has fulfilled the standard regulations imposed.

The protein content of SSG was lower than SBG and BCG (p <0.05). This difference may be related to the water content which tends to be low on SBG. Protein levels according to Pranoto, Marseno and Rahmawati (2011) can be used as a benchmark for evaluating the purity of gelatin. Protein levels in gelatin do not have a standard, but the levels can vary depending on the source of the raw material used. Different methods of extraction and pretreatment will affect the protein content obtained. The protein content of SBG was higher than SSG, in contrast to the results of research by (Muyonga, Cole and Duodu, 2004a) on the skin and bone (Lates niloticus) which produced a higher protein range in the skin (87.4 ±5.6 to 88.7 ±2.4) than bone (82.0 ±2.1 to 82.9 ±4.3) as was the case with Wulandari, Suptijah and Tarman (2015) and Ratnasari et al. (2013) in snakehead skin, were 96.21% and 87.27 ±0.78, respectively. Although See et al. (2010) were also only able to produce snakehead skin gelatin with lower protein levels, namely 75.63 ±1.05. Different contents of protein can be influenced by factors such as raw materials, pretreatment processes, and extraction methods (Gomez-Guillen et al., 2011; Jamilah and Harvinder, 2002; Nagarajan et al., 2012).

The other important chemical compositions of gelatin were fat and ash. The highest SBG fat content was 1.05 ±0.05%. The relatively high-fat content in SBG compared to SSG can be attributed to the fat content of raw materials, where the fat of the snakehead bone was higher than the skin (Rosmawati et al., 2018a). There was no definite standard for the minimum level of gelatin fat, but may be high levels of fat that can affect the quality of gelatin, including the shelf life. The fat content of commercial bovine skin gelatin was 0.23% (Pranoto, Marseno and Rahmawati, 2011). This value was lower than the fat content of SSG but relatively higher than the fat content of BCG.

The gelatin ash content of the research results showed a fairly low value than Indonesian National Standard (1995), which was less than 3.25%, whereas according to GMIA (2012) standard for type A gelatin which is about 0.3 to 2%. Ash content of SSG was lower than SBG and BCG, respectively (p <0.05). The demineralization process before the extraction effectively has reduced the ash content of SBG.

The variation in chemical composition between gelatin occurs mainly due to differences in methods during the process of changing collagen to gelatin (Gomez-Guillen et al., 2011), protein content in raw materials, including collagen levels, amount of dissolved components in the skin, initial treatment and washing process and extraction method used (Abedinia et al., 2017; Jamilah and Harvinder, 2002). Meanwhile, according to Irwandi et al. (2009), the chemical composition of gelatin produced can be related to the environment and type of fish feed.

The amino acid composition of skin gelatin (SSG) and snakehead bone (SBG) compared to commercial gelatin (BCG) was presented in Figure 2. Glycine and proline were two types of the main amino acid (Jamilah and Harvinder, 2002) and were the highest in all types of gelatin were observed.

The characteristic feature of collagen and its derivatives was that the amino acid content of glycine and proline was higher than other amino acids (Nagarajan et al., 2012), and glycine is the main amino acid (Irwandi et al., 2009). However, specifically, the presence of proline and hydroxyproline were the main determinants of gelatin quality related to the gel strength, unfortunately, hydroxyproline gelatin was not analyzed, and so it was rather difficult to predict gel strength really due to the presence of proline alone. It can be seen in Figure 2 that the highest proline levels of BCG then SSG and SBG, whereas the strength of the BCG gel was the lowest compared to SSG and SBG. The content of amino acids between fish and mammals was different, but its presence determines the stability of the gelatin conformation during the gel formation process (Haug, Draget and Smidsrød, 2004). As Gautieri et al. (2009) emphasized that glycine was the most important amino acid in stabilizing collagen protein tertiary molecular structure whereas Pranoto, Marseno and Rahmawati (2011) suggested that the percentage of glycine can affect the binding properties of gelatin by water and lead to high values of gel strength and viscosity. The higher the temperature and the longer the extraction time affects the gel strength, this is assumed because the amino acids undergo cleavage in the peptide chain, and at the same time degradation occurs during the acid hydrolysis process (Darragh et al., 1996).

Glutamate acid and alanine show levels that were not very different, and it seems that the levels of these two amino acids were higher in succession in the SSG than SBG and BCG. Arginine was also found to be quite abundant, respectively SBG, SSG, and BCG. The high levels of three amino acids may be linked to the source of the raw material of gelatin which was rich in these amino acids (Rosmawati et al. 2018b). The other amino acids such as serine, aspartate acid, phenylalanine, threonine, leucine, isoleucine, methionine, tyrosine, valine, and histidine show an average of fewer than 5 residues/100 residues. Cysteine and tryptophan were not found in gelatin (Haug and Draget, 2009), apart from the fact that both amino acids were present in sufficiently small amounts in their raw materials, these amino acids were thought to be susceptible to damage during the hydrolysis process. Amino acids were organic materials, that were bipolar by the presence of amine groups (-NH2) and carboxyl (-COOH-), and side-chain groups (-R) that were specific and determine the level of reactivity of amino acids. Differences in side-chains cause differences in the physicochemical properties of proteins (Aftabuddin and Kundu, 2007). Amino acids were grouped according to the type of side chain based on their solubility in water, hydrophilic, and hydrophobic amino acid (Barrett and Elmore, 1998). Amino acids of gelatin with side chains were hydrophobic are alanine, leucine, valine, isoleucine, methionine, and phenylalanine, and hydrophilic side chains namely glycine, glutamate acid, lysine, serine, threonine, tyrosine, histidine, proline, arginine, and aspartate. The total percentage of amino acid groups based on their solubility in water was presented in Figure 3.

The hydrophilic amino acid of BCG was slightly higher than in SSG and SBG, and vice versa in the hydrophobic amino acids SSG and SBG were relatively slightly higher than the hydrophilic amino acids.

Aftabuddin and Kundu (2007) stated that the average level of the hydrophobic node tends to be greater than the hydrophilic node. Consequently, the ability of these amino acids to bind to fat greater than their ability to attract water. Different from BCG which has a hydrophilic amino acid that was relatively larger than its hydrophobic amino acids, it will have an impact on the ability to attract water that was greater than its ability to bind fat.

Between SSG and SBG tends to show differences that were not so striking in the amino acid levels. But this relatively small difference still influences the functional characteristics of the two types of gelatin, as well as those reported in the gelatin of the skin and bones of Lates niloticus (Muyonga, Cole and Duodu, 2004a; Muyonga, Cole and Duodu, 2004b). Extraction methods and different types of raw materials can result in differences in amino acid composition between the three types of gelatin observed.

Gelatin is one type of hydrocolloid that has been widely used as an emulsifier in the industry, including food, pharmaceuticals, medicines, and other technical applications because it has surface-active properties (Jellouli et al., 2011). This was a function of the ability of a surfactant to be hydrophilic and lipophilic at once, to reduce the surface tension caused by mixing oil and water. Gelatin was a type of natural surfactant, as a function of amino acids. The emulsion activity index and stability index of SSG, SBG, and BCG emulsion were presented in Table 3.

The index of emulsion activity of the three types of gelatin differed from one another (p <0.05).

The index of emulsion activity (IAE) of each type of gelatin was relatively different. The index of emulsion activity of SSG was higher than BCG and SBG. The value of IAE reported by Jellouli et al. (2011) in the gelatin of gray triggerfish (Balistes capriscus) skin was lower (21.44 ±0.09), and as was reported by (Jridi et al., 2013) in gelatin cuttlefish skin (Sepia officinalis) were 25.97 ±1.05, 25.42 ±0.95, 27.64 ±1.26 and 27.65 ±1.35 respectively. The difference of IAE of gelatin can be caused by differences in sources of raw material (Jellouli et al., 2011; Jridi et al., 2013; Zhang et al., 2012) and extraction processes applied (Ahmad and Benjakul, 2011; Kaewruang et al., 2013), which has implications for the intrinsic character of gelatin (Khiari et al., 2013). The important intrinsic factor that influences the activity of gelatin emulsion was the contribution of amino acids making up the gelatin protein. The amino acid of gelatin with an active side chain gives the gelatin the ability to be hydrophilic (polar charge) and hydrophobic (non-polar charge). The peptide chain of hydrophobic amino acids contributes to the emulsifying properties of gelatin, because of the presence of surface-active properties, where the greater the content of hydrophobic amino acids, the more emulsion activity tends to be higher (Karim and Bhat, 2009). Ahmad and Benjakul (2011) further explained that surface activity in the interface of water and oil in gelatin gave the ability to facilitate the formation and stabilization of droplets during and after the emulsion process. The emulsion activity index was related to the area between surfaces, where the higher the emulsion activity index value, the smaller the size of the fat globule. The emulsion activity index shows the ability of proteins to stabilize oil and water interfaces.

In Table 3, there was a significant effect (p <0.05) of the gelatin type on the index of emulsion stability (ISE). The index of emulsion stability of SBG was smaller than BCG and SSG. Among the three types of gelatin, SSG appeared to show a better level of emulsion stability than BCG and SBG. However, the ISE of SBG was better than BCG, and in the same emulsion activity, SBG can achieve its stability in a shorter period. The same properties were reported by Jellouli et al. (2011) in gray triggerfish (Balistes capriscus) which had better ISE than bovine gelatin.

The emulsifying properties of gelatin were described as emulsion capacity or emulsion activity, which showed the ability of gelatin to aids the formation and stabilization of newly created emulsions and their ability to give emulsion strength for the resistance of pressure. The index of emulsion activity was an interface measurement area. It was stabilized over a unit of protein weight (m².g^-1), and it was related to the ability of proteins to coat an interface (Pearce and Kinsella, 1978). The emulsion activity index was influenced by the intrinsic factor of gelatin protein. Adding gelatin to an oil-water emulsion solution affects the oil-water interface. Emulsifying properties were produced due to the presence of hydrophobic regions in the peptide chain (Karim and Bhat, 2009). The hydrophobic nature of the side chains of gelatin amino acid peptides reduces surface tension so that emulsion stability can be achieved. However, this depends on how large the ability of hydrophobicity of gelatin. The higher level of hydrophobic amino acid, the greater the IAE, and the shorter the ISE to achieve its stability. According to Ahmad and Benjakul (2011); Jellouli et al. (2011), the molecular weight can also influence the dispersion ability between oil and water, where the level of stability of emulsion from gelatin at high molecular weights results in better levels of stability than gelatin from lower molecular weights.

Water holding and fat binding capacity were functional properties that were closely related to the texture of food products as interactions between components such as water, oil, and other components (Ismail et al., 2010; Rawdkuen, Thitipramote and Benjakul, 2013). The results of the analysis in Table 4 showed that the type of gelatin has a significant effect (p <0.05) on the water holding capacity (WHC).

Snakehead bone gelatin (SBG) has the lowest WHC and was highest in BCG and then SSG. The Differences in extraction methods, raw materials, and animal species were factors that influence the differences in the characteristics of gelatin. This difference also causes the WHC of the three types of gelatin to tend to be different.

Water holding capacity as in Table 4 show the percentage of BCG was relatively higher than the two types of gelatin from snakehead, but the percentage of both is still relatively higher than the WHC of cobia fish skin gelatin (97.03 ±1.53) (Amiza et al., 2015). A study was carried out by Balti et al. (2011) in cuttlefish (Sepia officinalis) showed a relatively similar percentage with WHC on SSG and SBG, which was about 150 to 200%. The high WHC can be attributed to the hydrophilic amino acid content of gelatin (Nurul and Sarbon, 2015) and the main chemical component of gelatin, namely hydroxyproline (Ninan, Jose and Abubacker, 2011; Zarai et al., 2012). Allegedly, if the hydroxyproline level was high then the ability to hold water of gelatin would also follow (Jeya et al., 2012).

Water holding was one of the important functional properties in the processing process because of its function in improving the texture and compactness of food products. This trait according to Zarai et al. (2012) refers to the ability of proteins to absorb water and maintain it against the gravitational force in the protein matrix. Furthermore, Zarai et al. (2012) that its capacity to holding water makes it suitable and convenient to be applied to various frozen meat and fish products to reduce drip loss during the thawing or cooking process which impact protein denaturation resulting in partial loss of capacity to hold of water.

The three types of gelatin observed did not show any significant difference (p >0.05) on FBC. The fat binding capacity of SBG was relatively slightly higher than in SSG and BCG. Both showed lower percentages than FBC from red snapper bone gelatin (Lutjanus campechanus) and brown spotted grouper (Epinephelus chlorostigma) which were 493.90% ±4.20 and 429.57% ±4.73 respectively (Jeya et al., 2012), but it is higher than the cobia skin gelatin (Rachycentron canadum) which was 164.0% (Amiza et al., 2015). The difference in FBC from gelatin cannot be separated from the characteristics of the gelatin itself, which among others was influenced by raw materials and extraction methods so that it may result in differences in the composition of amino acids. The ability of gelatin to bind fat was mainly facilitated by hydrophobic amino acids (Jellouli et al., 2011), the greater the ability to bind fat tends to be higher Hydrophobic amino acids include tyrosine, isoleucine, valine, leucine, proline, phenylalanine, methionine and alanine. Ninan, Jose and Abubacker (2011) state that high levels of tyrosine were thought to be responsible for the binding capacity of fat in gelatin.

Analysis using Fourier transform infrared (FTIR) spectroscopy was carried out to identify changes in the functional group and secondary structure of gelatin (Muyonga, Cole and Duodu, 2004b) based on the vibration spectra produced by the compounds contained therein at certain wavelengths. The absorption value of snakehead fish gelatin (SSG and SBG) compared to BCG was presented in Table 5.

Amide A peaks from SSG, SBG, and BCG in the absorption area of 3388.93 cm^-1, 3383.14 cm^-1, and 3442.94 cm^-1, respectively. The smaller value absorption that appears in the spectra, indicates a lower free amine group, which means less degradation. Absorption values of BCG which were in the range above 3400 cm^-1 indicate that wavenumbers were in the vibration region of free NH stretch (Ahmad and Benjakul, 2011; Kittiphattanabawon et al., 2015; Sinthusamran, Benjakul and Kishimura, 2014). The SSG and SBG absorption areas show a slightly lower wavenumber, that was in the absorption area of 3400 – 3200 cm-¹, which means there was a shift in wavenumbers to a lower frequency, this was because there were NH groups of peptides involved with hydrogen bonds (Ahmad and Benjakul, 2011; Muyonga, Cole and Duodu, 2004c). A relatively high SSG wave number compared to SBG shows that the hydrolysis process of snakehead fish skin at a temperature of 60 °C for 12 hours causes relatively more hydrogen bonds to be released than hydrogen bonds in snakehead bone at the same temperature. This indicates that the type of raw material and the relative extraction process affect the secondary structure of the gelatin protein, besides this difference according to Kittiphattanabawon et al. (2015) may also be due to differences in amino acid composition and sequence.

The amide I band between SSG and BCG were at the same wavenumber, which was slightly higher than SBG, namely 1653.00 cm^-1, 1653.00 cm^-1, and 1651.07 cm^-1, respectively. The difference in the type of raw material and the extraction method used causes the wavenumbers of the two types of snakehead fish gelatin to be different. At the same temperature (60 °C) with different extraction times, the SBG wave number (24 hours of extraction) was slightly lower than SSG (12 hours of extraction) indicating that the level of C=O reactivity in the skin was able to open and act reactively more quickly on the chain- α compared to snakehead fish bones, so this situation is according to Jridi et al. (2015) has the potential to cause the loss of the triple helix due to excessive hydrolysis of the intermolecular cross-linking. Furthermore, this situation by Yu et al. (2014) is alleged to have caused the loss of amino acid reactivity, including lysine, hydroxylysine, and histidine, which function to maintain inter and intra-molecular cross-links in the telopeptide region.

In the amide band II, the peak wavenumbers between SSG, SBG, and BCG have shown the absorption values were 1541.12 cm^-1, 1521.84 cm^-1, and 1543.05 cm^-1, respectively. The amide region II showed an abnormality in the molecular structure due to the transformation of α-helix into a random coil structure due to the partial hydrolysis process during extraction (Muyonga. Cole and Duodu. 2004b), which was characterized by the occurrence of NH flexural vibrations coupled with CN stretching vibrations (Yu et al., 2014). The temperature and length of extraction time can cause changes in the triple helix as a result of the denaturation of collagen to gelatin (Muyonga, Cole and Duodu, 2004b). The smaller the wavenumber, the smaller the triple helix damage rate observed.

The SSG and SBG wavelengths in the peak region of the amide band III showed the same absorption value, namely 1234.44 cm^-1, while BCG was at 1238.30 cm^-1, indicating a greater disturbance in the molecule, and this according to Sinthusamran, Benjakul and Kishimura (2014) associated with the loss of the triple helical structure. Amide region III was a spectrum region of 1220 – 1320 cm^-1 and is associated with CN stretching and NH deformation as well as absorption arising from vibrations of CH2 groups from the glycine backbone and proline side chains (Ahmad and Benjakul, 2011).