Journal of Chemistry
Volume 2019 (2019), Article ID 5462471, 11 pages
https://doi.org/10.1155/2019/5462471

Research Article

Enhancement of Hydrophobicity of Fish Skin Gelatin via Molecular Modification with Oxidized Linoleic Acid

Wipawee Theerawitayaart,¹ Thummanoon Prodpran,¹ and Soottawat Benjakul²

¹Department of Material Product Technology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand
²Department of Food Technology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand

Correspondence should be addressed to Thummanoon Prodpran; thummanoon.p@psu.ac.th

Received 27 August 2018; Accepted 16 January 2019; Published 6 February 2019

Academic Editor: Beatriz P. P. Oliveira

Copyright © 2019 Wipawee Theerawitayaart et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

Fish gelatin possesses hydrophilicity in nature since it contains a few hydrophobic amino acids and a large portion of hydrophilic amino acids. Low hydrophobicity of fish gelatin results in poor water vapor barrier and water resistance of gelatin films. This can limit its usage as packaging material. To overcome this drawback, the molecular attachment of hydrophobic domains can effectively improve the hydrophobicity of fish gelatin. In this study, fish skin gelatin modified with oxidized linoleic acid (OLA) prepared under various conditions using different molar ratios of OLA/free amino group content was characterized. When OLA was prepared at 60 and 80°C for various oxidation times (0–24 h), the peroxide value (PV) increased continuously up to 9 and 12 h when reaction was performed at 80 and 60°C, respectively. Consequently, the PV decreased until the end of the reaction (24 h). Thiobarbituric acid reactive substances (TBARS) of OLA were increased sharply up to 12 h, regardless of reaction temperatures. Thus, primary and secondary lipid oxidation products mainly occurred within the first 12 h. As gelatin was modified with different OLA at various OLA/free amino group molar ratios, the one modified with OLA prepared at 60°C for 24 h at a molar ratio of 10 : 1 had the highest increases in surface hydrophobicity and carbonyl content with the coincidentally lowest free amino group content, compared with control gelatin (without OLA modification). Fourier transforms infrared (FTIR) spectra also reconfirmed the presence of fatty acid covalently attached to resulting gelatin. Therefore, OLA could be used to modify gelatin and increase its hydrophobicity.

1. Introduction

Gelatin is obtained by thermal denaturation or partial hydrolysis of collagen from the skin and bone. Nowadays, fish gelatin has gained increasing attention as the material for pharmaceutical, medical, and food industries [1]. Fish skin gelatin has several advantages, e.g., the lack of outbreaks of bovine spongiform encephalopathy (BSE) and religious constraint (Judaism and Islam) [2]. Nevertheless, gelatin contains a large portion of hydrophilic amino acids (lysine, serine, arginine, hydroxyproline, and aspartic and glutamic acids) with a low content of hydrophobic amino acids (proline and leucine) [3]. As a consequence, films or packaging based on gelatin exhibits poor water vapor barrier property, thereby limiting their applications [4].

Hydrophobicity of gelatin can be enhanced by molecular incorporation of hydrophobic substances such as fatty acids into the gelatin molecules [5]. Attachment of fatty acid into protein including gelatin could improve the surface hydrophobicity, affecting solubility and hydrophilic/hydrophobic nature of the resulting modified gelatin. The covalent interaction between lipid and protein could be induced by using the oxidized lipids [6]. Oxidation of polyunsaturated fatty acid such as linoleic acid leads to the formation of radicals, lipid hydroperoxides, and various secondary lipid oxidation products, especially reactive aldehydes and ketones including malondialdehyde, 4-hydroxynonenal, and 4-hydroxyhexenal (Scheme 1) [7, 8]. These oxidation products could covalently modify side chains and the polypeptide backbone of protein molecules [6, 7]. The reaction between free amino groups of proteins and oxidized fatty acids has been successfully used as an effective means to improve surface hydrophobicity of protein [9]. Fatty acids oxidized under various conditions might modify the gelatin in different fashions. This might be associated with different types or amounts of reactive lipid oxidation products. However, a little information on the impact of oxidized linoleic acid (OLA) prepared with different conditions on fish gelatin exists. The aim of this study was to improve surface hydrophobicity of fish gelatin via molecular modification with oxidized linoleic acid (OLA) under various conditions.

Scheme 1: Oxidation of linoleic acid (LA) into primary and secondary oxidation products [8].

2. Materials and Methods

2.1. Fish Gelatin and Chemicals

Fish gelatin produced from tilapia skin (∼240 bloom) was obtained from Lapi Gelatine S.p.A (Empoli, Italy). Linoleic acid, cumene hydroperoxide, 1-anilinonaphthalene-8-sulfonic acid (ANS), sodium dodecyl sulfate (SDS), 2,4,6-trinitrobenzenesulfonic acid (TNBS), sodium sulfite, L-leucine, and malonaldehyde (MDA) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Methanol, trichloroacetic acid (TCA), butanol, hydrochloric acid, barium chloride (BaCl₂), and ferrous sulfate (FeSO₄) were procured from Merck (Darmstadt, Germany). Ammonium thiocyanate (NH₄SCN), ethanol, chloroform, and 2-propanol were obtained from Lab-Scan (Bangkok, Thailand). 2-dinitrophenylhydrazine (DNPH) was purchased from Wako Pure Chemical Industries Ltd. (Chuo-Ku, Osaka, Japan). All chemicals were of analytical grade.

2.2. Preparation of Oxidized Linoleic Acid (OLA) under Different Conditions

Oxidized linoleic acid (OLA) was prepared according to the method described by Aewsiri et al. [6] with a few modifications. Thirty mL of linoleic acid was added with 30 mL of water (HPLC grade). The mixture was then homogenized using a homogeniser (IKA Labortechnik homogenizer, Selangor, Malaysia) at a speed of 9,500 rpm for 2 min. The homogenate was obtained by centrifugation using a centrifuge (model z323k, Hermle Labor Technik, Wehingen, Germany) at 3,000 × for 15 min to remove water-soluble oxidized lipids. To oxidize linoleic acid, the prepared fatty acid was flushed with air constantly and incubated at different temperatures (60 and 80°C) in a controlled-temperature water bath for different times (0, 1, 3, 6, 9, 12, and 24 h). OLA samples were subjected to analyses as following.

2.2.1. Peroxide Value (PV)

PV, an indicator of the initial stages of oxidation, was determined according to the method of Shantha and Decker [10] with a slight modification. OLA samples (0.3 mL) were mixed with 1.5 mL of chloroform/methanol (2 : 1, v/v) mixture. The mixture was vortexed for 10 sec, and 15 µL of 3.94 M ammonium thiocyanate and 15 µL of ferrous iron solution (prepared by mixing 0.132 M BaCl₂ and 0.144 M FeSO₄ at a ratio of 1 : 1, v/v) were added. After 20 min of incubation at room temperature, the absorbance was measured at 510 nm. The concentration of hydroperoxides was calculated from a cumene hydroperoxide standard curve (0–500 mg/mL). The PV was expressed as mg hydroperoxide/kg sample.

2.2.2. Thiobarbituric Acid Reactive Substances (TBARS)

TBARS, representing the secondary oxidation products, were determined according to the method of Buege and Aust [11]. The sample (1 mL) was added with TBA reagent (4 mL) (0.375% thiobarbituric acid, 15% trichloroacetic acid, and 0.25 M HCl). The mixture was heated in boiling water for 10 min, followed by cooling in running tap water. The mixture was centrifuged at 3600 × for 20 min at room temperature. The absorbance of the supernatant obtained was measured at 532 nm. The TBARS value was calculated from the standard curve of malonaldehyde (MDA) (2–10 ppm), and TBARS was expressed as mg MDA/kg sample.

2.2.3. Fourier Transform Infrared (FTIR) Spectroscopic Analysis

Linoleic acid was incubated at 60°C for different times (0, 6, 12, and 24 h). The OLA samples were transferred into an amber vial, flushed with nitrogen, and sealed tightly. The samples were stored at −40°C until analysis. FTIR spectra of OLA were obtained using a Bruker Model Equinox 55 FTIR spectrometer (Bruker Co., Ettlingen, Germany). A small quantity of the OLA was accumulated with a Pasteur pipette between KBr disks, creating a thin film. The absorption of IR in the region of 650–4000 cm⁻¹ was determined using 32 scans with a resolution of 4 cm⁻¹. These spectra were subtracted from the reference spectrum of air.

2.3. Preparation of Fish Skin Gelatin Modified with OLA Prepared under Different Conditions

Fish skin gelatin was modified with OLA according to the method of Aewsiri et al. [6] to obtain gelatin-OLA complexes via covalent interaction. First, OLA was prepared at 60°C for different times (6, 12, and 24 h). OLA was dissolved with ethanol at a ratio of 1/130 and 1/260 (v/v) to obtain the concentrations of 20 and 40 mM, respectively. Gelatin solution was separately prepared by dissolving gelatin powder in distilled water to obtain a final concentration of 1.1% protein (w/v) as determined by the biuret method [12]. To modify gelatin, the OLA solutions (1 mL) were added to gelatin solution (90 mL) at OLA-to-free amino group molar ratios of 5 : 1 and 10 : 1. The mixtures were then adjusted to 100 mL with distilled water to obtain the final concentration of 1% gelatin. The mixtures were stirred using a magnetic stirrer for 3 h at room temperature. Thereafter, the sample were dialyzed with a dialysis bag (MW cut-off = 14000 Da) with 20 volumes of 1% ethanol for 12 h at room temperature to remove free OLA (unbound to proteins). The dialysis was further performed using 20 volumes of distilled water for 12 h to remove ethanol. The dialysates were filtered through a filter paper no. 1 (Whatman, Germany), freeze-dried, and stored at −20°C prior to analyses. The control was prepared in the same manner, except that OLA was omitted. All gelatin samples were subjected to analyses.

2.3.1. Determination of Surface Hydrophobicity

Surface hydrophobicity was measured as per the method of Benjakul et al. [13] using 1-anilinonaphthalene-8-sulfonic acid (ANS) as a probe with a slight modification. Samples were diluted to 1, 2, 3, and 4 mg/mL with 10 mM sodium phosphate buffer (pH 6.0). To 2 mL of diluted solutions, 20 µL of 8 mM ANS in 0.1 M sodium phosphate buffer (pH 7.0) was added. The fluorescence intensity of the mixture was measured at an excitation wavelength of 374 nm and an emission wavelength of 485 nm. The initial slope of plot of fluorescence intensity vs. protein concentration was assigned to as S₀ANS.

2.3.2. Determination of Carbonyl Content

The carbonyl content was determined using 10 mM 2,4-dinitrophenylhydrazine (DNPH) following the method of Liu et al. [14]. To 0.5 mL of the sample solution (4 mg protein/mL), 2.0 mL of 10 mM 2,4-dinitrophenylhydrazine (DNPH) in 2 M HCl was added. The mixture was allowed to stand for 1 h at room temperature. Thereafter, 2 mL of 20 mM potassium phosphate (pH 2.3) containing 0.6 M guanidine hydrochloride was added, and the mixture was mixed well until completely dissolved. The absorbance was read at 370 nm. The carbonyl content was calculated from a molar absorptivity of 22,400 M⁻¹·cm⁻¹ [15]. Carbonyl content was expressed as nmol/mg protein.

2.3.3. Determination of Free Amino Group Content

The free amino group content was measured according to the method of Benjakul and Morrissey [16]. Reaction mixtures consisted of 125 µL of diluted samples and 2.0 mL of 0.2 M TNBS solution. The mixture was incubated at 50°C for 30 min in a temperature-controlled water bath (Memmert, Schwabach, Germany) and placed in the dark condition. To terminate the reaction, 2.0 mL of 0.1 M sodium sulfite was added. After cooling for 15 min at room temperature, the absorbance was read at 420 nm. The free amino group content was calculated from a L-Leucine standard curve (0.5–5.0 mM). The free amino group content was expressed as µmol/g protein.

2.3.4. Fourier Transform Infrared (FTIR) Spectroscopic Analysis

Fish skin gelatin without and with OLA modification was conditioned in a desiccator containing P₂O₅ for 2 weeks at room temperature to obtain the most dehydrated gelatin before analysis. Gelatin samples were scanned with an FTIR spectrometer, equipped with a horizontal ATR Trough plate crystal cell (45° ZnSe; 80 mm long, 10 mm wide, and 4 mm thick) (PIKE Technology Inc., Madison, WI, USA) at 25°C as described by Nuthong et al. [17]. The spectra were corrected at 32 scans with the resolution of 4 cm⁻¹.

2.3.5. SDS-Polyacrylamide Gel Electrophoresis (SDS–PAGE)

Protein patterns of gelatin with and without OLA modification were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to the method of Laemmli [18] using 4% stacking gel and 10% running gel. Gelatin solutions were then mixed with sample buffer (20% (v/v) glycerol, 0.5 M Tris-HCl, pH 6.8 containing 4% (w/v) SDS, and 10% (v/v) β-ME) at the ratio of 1 : 1 (v/v). Samples (15 µg protein) as determined by the Biuret method [12] were loaded onto the gel, and electrophoresis was carried out at a constant current of 15 mA using a Mini Protein II unit (Bio-Rad Laboratories, Inc., Richmond, CA, USA). Following electrophoresis, the gel was stained with 0.05% (w/v) Coomassie blue R-250 in 15% (v/v) methanol and 5% (v/v) acetic acid. After staining, the gel was destained with 30% (v/v) methanol and 10% (v/v) acetic acid. High molecular weight range protein markers were used to estimate the molecular weight of protein.

2.4. Statistical Analysis

All experiments were run in triplicates. Data were subjected to analysis of variance (ANOVA), and differences between means were tested by Duncan’s multiple range test [19] using the SPSS program (SPSS 17.0 for windows; SPSS Inc., Chicago, IL, USA).

3. Results and Discussion

3.1. Characteristics of OLA Prepared under Various Conditions

3.1.1. PV and TBARS

The changes in PV and TBARS value of linoleic acid oxidized at different temperatures for various times are illustrated in Figures 1(a) and 1(b), respectively. The PV demonstrates the primary lipid oxidation products, mainly peroxides, while TBARS value represents the secondary lipid oxidation products, including aldehydes, ketones, and epoxides as well as other carbonyl and hydroxyl compounds [20]. Increase in the PV of samples oxidized at 60°C was obtained when the oxidation time increased up to 9 h (). Subsequently, the PV decreased continuously for up to 24 h of reaction. At the higher reaction temperature (80°C), the increase in the PV was observed for up to 6 h (). Thereafter, a gradual decrease was found until the end of the reaction (24 h). At the same oxidation time, the higher PV was obtained in OLA prepared at lower temperature (60°C). The result suggested that the primary oxidation product, especially hydroperoxide, could be formed to a higher extent, but the rate of decomposition proceeded. As a result, the primary products were retained at a lower extent.

(a)

(b)

Figure 1: Changes in peroxide value (a) and TBARS value (b) of OLA prepared at different temperatures for various times. The bar represents the standard deviation ().

For TBARS values, the drastic increase in the TBARS value was observed for OLA prepared at both temperatures up to 12 h (). Subsequently, the sample prepared at 60°C had a slight increase in TBARS values. Nevertheless, the sample oxidized at 80°C had no changes in TBARS values (). After 24 h of reaction, the higher TBARS value was found in the sample incubated at 60°C. Therefore, both reaction temperatures and times played a major role in the formation and the loss of secondary oxidation products. In general, higher loss was attained when the reaction was carried out at a higher temperature (80°C). Both lipid oxidation products, primary and secondary products, developed under different reaction conditions in OLA could undergo interaction with gelatin to different degrees [6].

3.1.2. FTIR Spectra

Figure 2 shows the FTIR spectra of linoleic acid (LA) and oxidized linoleic acid (OLA) prepared at 60°C for various times (6, 12, and 24 h). The assignment of functional groups responsible for IR absorption peaks is presented in Table 1. The obtained IR spectrum of LA showed similar characteristic peaks as generally reported for nonoxidized fatty acid [21–23]. The spectrum of nonoxidized fatty acid consisted of the major bands with the wavenumbers of 3009 cm⁻¹ (C-H stretching vibrations of the cis-double bond (=CH)); 2954 cm⁻¹ (symmetric stretching vibration shoulder of CH₃); 2926 and 2854 cm⁻¹ (asymmetric and symmetric stretching vibrations of CH₂, respectively); 1715 cm⁻¹ (C=O stretching vibrations); 1465 and 1458 cm⁻¹ (bending vibrations of the CH₂ and CH₃, respectively); 1398 cm⁻¹ (bending in plane vibrations of the CH cis-olefinic group); 1377 cm⁻¹ (bending vibrations of the CH₂ group); and 1238, 1163, 1120, and 1099 cm⁻¹ (C-O stretching vibrations) [24].

(a)

(b)

Figure 2: FTIR spectra in the wavenumber region of 3500–2400 cm⁻¹ (a) and 1900–400 cm⁻¹ (b) of linoleic acid (LA) and OLA prepared at 60°C for different oxidation times.

Table 1: Functional group assignment on major absorption bands occurring in the infrared spectra of nonoxidized linoleic acid (LA) and oxidized linoleic acid (OLA).

Wavenumber (cm⁻¹)
Type of vibration and functional group

LA
OLA

3350–3120

Stretching of OH in fatty acid

3600–3200
Stretching of OH in aldehyde, peroxide coupled with OH of fatty acid

3009
3009
CH stretching of cis-double bond (=CH)

2954
2954
Asymmetric stretching vibration shoulder of the aliphatic CH₃ group

2926, 2854
2926, 2854
Asymmetric and symmetric stretching, respectively, of aliphatic CH₂ group

—
1745
Stretching vibration of C=O of aldehyde and ester

1715
1715
Stretching of C=O of fatty acid

1654
1654
C=C stretching vibration

1465, 1458
1465, 1458
Bending vibration of aliphatic CH₂, CH₃, respectively

1420
1420
Rocking vibration of CH bonds of cis-distributed olefins

1238, 1175–1055
Stretching vibration of C-O ester groups

1180
C-O-O vibration of hydroperoxide

990–980
Bending of C-O of ester overlapping with CH bending of trans-double bond

975
975
Bending of C-O of acid

723
723
Overlapping of the CH₂ rocking vibration and the out-of-plane vibration of cis-distributed olefins

680
680
CH bending of cis-double bond

The IR spectra of oxidized linoleic acid (OLA) obtained at various times exhibited noticeable differences as compared to those of nonoxidized LA, indicating the formation of functional groups as a result of oxidation. The absorption band in the range 3600–3200 cm⁻¹ (representing stretching vibrations of OH and OOH [23, 25]) of OLA showed increased intensity as compared to that of nonoxidized LA. This was mostly associated with the formation of hydroperoxide and secondary products containing an OH moiety from the oxidation of LA. The amplitude of the vibrational peak at 3009 cm⁻¹ of OLA at 12 and 24 h of oxidation was lower than that of nonoxidized LA. However, the OLA at 6 h, which was the early stage of oxidation, showed higher amplitude. The amplitude of this peak seemed to decrease with oxidation time, except for the OLA at 6 h. This indicated that the extent of oxidation reaction generally increased with time. As the oxidation proceeded, the loss of conjugated cis-double bonds occurred. This more likely led to isomerization of cis-double bonds to conjugated trans-double bonds or could generate secondary oxidation products, which were not formed during the initial stage of oxidation [25]. Moreover, the spectral region between 1800 and 1000 cm⁻¹ (Figure 2(b)) underwent significant changes during the oxidation of LA. The new absorption band observed as a shoulder at 1745 cm⁻¹ in OLA spectra could be assigned to stretching vibrations of the C=O moiety in aldehyde, ketone, or ester, which are the secondary products from lipid oxidation [23]. The presence of this peak could result in widening or broadening of the carbonyl band at 1715 cm⁻¹ of fatty acid. In addition, the peaks around 1160–1175 and 1238 cm⁻¹, associated with the stretching vibrations of the C-O in ester moiety [24] as well as 1055 cm⁻¹ assigned to C-O bending were noticed. The absorption at around 1180 cm⁻¹ due to C-O-O vibrations of hydroperoxides (ROOH) was also detected; this vibrational peak overlapped with C-O stretching vibrations [26]. It can be observed from the spectra that the amplitude of peaks at 1745 and 1238–1055 cm⁻¹ increased with increasing oxidation time. This indicated the higher proportion of saturated aldehyde and saturated acyl groups of LA when oxidized at longer time [25]. Moreover, the absorption peaks appearing approximately at 975 and 680 cm⁻¹ in IR spectra were related to =C-H out-of-plane bending of trans and cis isomers, respectively [22]. As the oxidation time increased, the intensity of the band at 680 cm⁻¹ decreased, while that of the 975 cm⁻¹ band increased. This result also indicated that isomerization of cis-double bonds to conjugated trans-double bonds more likely took place upon the progress of oxidation of LA. Therefore, the IR result reconfirmed that the oxidation carried out under conditions in this study was able to change the structure of linoleic acid, as indicated by the presence and increase in intensity of bands corresponding to saturated aldehyde groups and saturated acyl groups in OLA.

3.2. Characteristics of Gelatin Molecularly Modified with OLA

3.2.1. Surface Hydrophobicity

Surface hydrophobicity (S₀ANS) of native gelatin samples and those modified with OLA prepared from various oxidation times at different OLA/free amino group molar ratios is shown in Table 2. S₀ANS of all modified gelatin samples increased as the molar ratio and oxidation time of OLA used increased (). Increase in S₀ANS indicated that the hydrophobic domains of modified gelatin were increased, more likely due to OLA being attached to gelatin. The highest S₀ANS was observed for gelatin modified with OLA oxidized for 24 h using a ratio of OLA-to-free amino group of 10 : 1. It was noted that the gelatin modified with OLA showed the higher S₀ANS than the control gelatin. This might be due to the higher interaction between the free amino group of gelatin and reactive groups, especially carbonyl groups located in OLA. Similar results were obtained when soy protein was modified with acrolein (secondary lipid oxidation product), in which surface hydrophobicity increased [27]. The hydrophobicity of modified protein was increased with increasing number of carbonyl compounds on the protein surface [27]. When linoleic acid was oxidized, it became more electrophilic, which was then able to attach to the nucleophilic domain of gelatin. Those covalently attached domains mainly contributed to the increased surface hydrophobicity of modified gelatin.

Table 2: Surface hydrophobicity, free amino group content, and carbonyl content of fish gelatin modified without and with OLA prepared at various oxidation times using different molar ratios of OLA/free amino group of gelatin.

Molar ratio of OLA/free amino group
Oxidation time of OLA (h)
Surface hydrophobicity
Free amino group content (µmol/g protein)
Carbonyl content (nmol/mg protein)

Control
—
2.06 ± 0.01a
4.39 ± 0.04a
2.36 ± 0.01a

At molar ratio 5 : 1
6
2.72 ± 0.08b
4.27 ± 0.03ab
2.59 ± 0.05b

12
2.94 ± 0.16c
4.14 ± 0.07b
2.76 ± 0.01c

24
3.16 ± 0.09d
3.94 ± 0.06c
2.96 ± 0.01d

At molar ratio 10 : 1
6
2.97 ± 0.14c
4.14 ± 0.07b
3.04 ± 0.03e

12
3.22 ± 0.07d
4.15 ± 0.13b
2.95 ± 0.01d

24
3.36 ± 0.02e
3.86 ± 0.12c
3.55 ± 0.01f

Mean ± SD (). Different superscript letters in the same column indicate significant differences (). Control: gelatin without modification.

3.2.2. Free Amino Group Content

Free amino group contents of gelatins modified with OLA prepared at 60°C for various oxidation times and using different OLA-to-free amino group molar ratios are shown in Table 2. The free amino group content of modified gelatin generally decreased with oxidation time of OLA and the molar ratio (OLA/free amino group of gelatin) used for modification (). Nevertheless, gelatin modified with OLA oxidized for 6 h with a OLA/NH₂ group molar ratio of 5 : 1 had no changes in the free amino group content (). The lowest free amino group content was observed for gelatin modified with OLA oxidized for 24 h (), regardless of the OLA/NH₂ molar ratio used. OLA might interact with gelatin molecules via ε-amino group of lysine in the gelatin [6], as shown in Scheme 2. More specifically, the reaction might involve the nucleophilic addition between the amino group of gelatin and the aldehyde carbonyl group of OLA [26]. The extent of gelatin-OLA reaction was higher by using OLA oxidized at longer time that contained a higher amount of aldehyde functional groups. As a consequence, gelatin modified using OLA with longer oxidation time had the lower free amino group content () (Table 2). Liu and Xiong [28] also reported that free amino group might react with the carbonyl compounds. The result suggested that OLA having higher oxidation more likely interacted with gelatin to a higher extent. Therefore, the oxidation time of OLA and the molar ratio of OLA-to-free amino group of gelatin were important factors affecting the covalent attachment of OLA onto gelatin.

(a)

(b)

Scheme 2: Reaction between secondary oxidation products of OLA and gelatin (a) and molecular model for gelatin chain without and with OLA modification (b).

3.2.3. Carbonyl Content

Carbonyl contents of gelatins and those subjected to modification with different OLA at various ratios is presented in Table 2. The incorporation of OLA into gelatin resulted in the higher carbonyl content of resulting gelatin in comparison to the control gelatin. Increase in the carbonyl content was obtained when gelatin was modified with OLA prepared with the longer oxidation time. Higher carbonyl content was also found in gelatin as the OLA/gelatin molar ratio increased (). This result suggested that gelatin could interact with OLA via free amino groups, particularly at ε-amino group of lysine [6]. Moreover, lipid oxidation products in OLA were postulated to accelerate the oxidation of gelatin, as indicated by the increase in carbonyl content. The increase in carbonyl content in gelatin modified with OLA might be mediated by Schiff’s base process via reactive amino groups [29] (Scheme 2(a)). Wu et al. [27] also reported that the soy protein modified with aldehyde groups had decreases in lysine, cysteine, and histidine residues, as induced by the increased protein oxidation. Therefore, gelatin could be modified by OLA via carbonyl groups, mainly via Schiff’s base but the degree of modification was dependent on the degree of oxidation of OLA used. Additionally, the OLA/free amino group of gelatin ratio was another factor determining the modification of gelatin.

3.2.4. FTIR Spectra

FTIR spectra of gelatin without and with modification by using various OLA at the ratios of OLA-to-free amino group of 5 : 1 and 10 : 1 are depicted in Figure 3. All gelatin samples exhibited the major characteristic bands of protein at ∼3280 cm⁻¹ (amide-A, illustrating NH-stretching coupled with hydrogen bonding), ∼2934 cm⁻¹ (amide-B, illustrating CH-stretching and -NH₃⁺), ∼1634 cm⁻¹ (amide-I, representing the C=O stretching/hydrogen bonding coupled with COO⁻), ∼1523 cm⁻¹ (amide-II, illustrating the NH bending coupled with CN stretching), and ∼1234 cm⁻¹ (amide-III, representing the CN and NH in plane bending of amide bonds or CH₂ of glycine) [6]. The absorption peaks of amide-I, amide-II, and amide-III of the gelatin appeared at 1634, 1523, and 1234 cm⁻¹, respectively. Amide-I and amide-II peaks increased and shifted to a higher wavenumber when the gelatins were modified with various OLA compared to the control gelatin. The shift was more pronounced when OLA prepared at longer oxidation time and a higher OLA/free amino group ratio was used. The incorporation of OLA into gelatin might lower interaction of these C=O bonds or NH along the chains of gelatin. It was noted that the amplitude of amide bands increased when OLA was used for modifications. OLA with higher oxidation caused increase in amplitude of those bands in the resulting gelatin. The shift to a higher wavenumber of amide-I peak could also be associated with the presence of an imine moiety (C=N) formed via Schiff’s base reaction between gelatin and OLA [26] (Scheme 2(a)). The absorption peak of C=N was reported to occur around 1690 cm⁻¹ (stretching), which presented at a higher wavenumber than C=O of amide. The vibration of the C=N group could merge or couple with that of C=O group of amide, resulting in broadening and shift of amide-I band to a higher wavenumber. This result could be indicative for the covalent attachment of OLA onto the gelatin chain.

(a)

(b)

Figure 3: FTIR spectra in the wavenumber region of 3600–2800 cm⁻¹ (a) and 1800–1000 cm⁻¹ (b) of fish skin gelatin modified with OLA prepared using different oxidation times and using various OLA-to-free amino group molar ratios.

For the amide-A peak, this peak represents the vibration of the NH group coupled with hydrogen bonding. The shift to lower wavenumbers indicated that the NH group of a peptide interacted with adjacent molecules via hydrogen bonds [30]. From the IR spectra, the amide-A peak of gelatin without modification appeared at a lower wavenumber than did that of OLA-modified gelatins. This was mostly due to the higher intermolecular interaction via hydrogen bonding between nonmodified gelatin molecules. However, the amide-A peak generally appeared at a higher wavenumber when gelatin was modified with OLA. This observation might be associated with that the long hydrocarbon chain of fatty acid of OLA attached to gelatin molecule could hinder the interaction via hydrogen bonding among adjacent gelatin molecules, as postulated in Scheme 2(b). Therefore, functional groups of fish gelatin were affected to some extent by molecular modification with OLA, and the OLA with different rates of oxidation as well as OLA-to-free amino group molar ratio affected the molecular property of the resulting gelatin.

3.2.5. Protein Patterns of Modified Gelatin and Control Gelatin

Protein patterns of fish skin gelatin modified with OLA having different oxidation rates and various OLA/NH₂ group molar ratios in comparison with gelatin are shown in Figure 4. All modified gelatin samples showed similar pattern to the control gelatin (without OLA modification). All samples had α₁- and α₂-chains as the main components. α₁- and α₂-chains have been reported as the dominant components in fish skin gelatin [31]. For all gelatins modified with OLA under different conditions, there were no differences in protein patterns. It was suggested that OLA mainly attached to gelatin without induction of intermolecular cross-linking between protein chains. Additionally, OLA with low molecular weight might not affect the molecular weight of modified gelatin. Consequently, OLA modification had no influence on the protein pattern of fish skin gelatin.

Figure 4: Protein patterns of fish gelatin modified with OLA prepared at different oxidation times and using various OLA-to-free amino group molar ratios. M: high MW protein marker and C: control gelatin (without modification).

4. Conclusions

The covalent attachment of OLA onto fish skin gelatin increased surface hydrophobicity, as indicated by the increased carbonyl group content and decreased free amino group content. The degree of modification depended on the degree of oxidation of OLA used as well as molar ratios of OLA/free amino group of gelatin. The treatments used for modification did not affect the protein pattern of fish skin gelatin. This inferred that the modification resulted in the incorporation of linoleic acid into gelatin without induction of covalent bonded cross-linking of gelatin. Gelatin modified with OLA (oxidized at 60°C for 24 h) at a molar ratio of 10 : 1 for 3 h at room temperature was suggested to improve surface hydrophobicity of fish gelatin.

Data Availability

No data were used to support this study.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Acknowledgments

This work was supported by Thai Government and Prince of Songkla University (grant number AGR600248s). The Grant-in-Aid for thesis from Graduate School, Prince of Songkla University, to Miss Wipawee Theerawitayaart was also acknowledged.

References

A. Jongjareonrak, S. Benjakul, W. Visessanguan, and M. Tanaka, “Antioxidative activity and properties of fish skin gelatin films incorporated with BHT and α-tocopherol,” Food Hydrocolloids, vol. 22, no. 3, pp. 449–458, 2008.

D. M. Asher, “The transmissible spongiform encephalopathy agents: concerns and responses of United States regulatory agencies in maintaining the safety of biologics,” Developments in Biological Standardization, vol. 100, pp. 103–118, 1999.

R. Núñez-Flores, B. Giménez, F. Fernández-Martín, M. E. López-Caballero, M. P. Montero, and M. C. Gómez-Guillén, “Role of lignosulphonate in properties of fish gelatin films,” Food Hydrocolloids, vol. 27, no. 1, pp. 60–71, 2012.

A. A. Karim and R. Bhat, “Fish gelatin: properties, challenges, and prospects as an alternative to mammalian gelatins,” Food Hydrocolloids, vol. 23, no. 3, pp. 563–576, 2009.

A. Kato and S. Nakai, “Hydrophobicity determined by a fluorescence probe method and its correlation with surface properties of proteins,” Biochimica et Biophysica Acta (BBA)-Protein Structure, vol. 624, no. 1, pp. 13–20, 1980.

T. Aewsiri, S. Benjakul, W. Visessanguan, P. A. Wierenga, and H. Gruppen, “Surface activity and molecular characteristics of cuttlefish skin gelatin modified by oxidized linoleic acid,” International Journal of Biological Macromolecules, vol. 48, no. 4, pp. 650–660, 2011.

E. R. Stadman and B. S. Berlett, “Reactive oxygen-mediated protein oxidation in aging and disease,” Chemical Research in Toxicology, vol. 10, no. 5, pp. 485–494, 1997.

K. M. Schaich, “Lipid co-oxidation of proteins: one size dose not fit all,” InForm, vol. 25, pp. 134–139, 2014.

P. A. Wierenga, M. B. J. Meinders, M. R. Egmond, F. A. G. J. Voragen, and H. H. J. de Jongh, “Protein exposed hydrophobicity reduces the kinetic barrier for adsorption of ovalbumin to the Air–Water interface,” Langmuir, vol. 19, no. 21, pp. 8964–8970, 2003.

N. C. Shantha and E. A. Decker, “Rapid, sensitive, iron-based spectrophotometric methods for determination of perorlride values of food lipids,” Journal of AOAC International, vol. 77, no. 2, pp. 421–424, 1994.

J. A. Buege and S. D. Aust, “[30] Microsomal lipid peroxidation,” Methods in Enzymology, Elsevier, New York, NY, USA, 1978, vol. 52 of Biomembranes-Part C: Biological Oxidations.

H. W. Robinson and C. G. Hodgen, “The biuret reaction in the determination of serum protein. I. A study of the condition necessary for the production of the stable color which bears a quantitative relationship to the protein concentration,” Journal of Biological Chemistry, vol. 135, pp. 707–725, 1940.

S. Benjakul, T. A. Seymour, M. T. Morrissey, and H. An, “Physicochemical changes in Pacific whiting muscle proteins during iced storage,” Journal of Food Science, vol. 62, no. 4, pp. 729–733, 1997.

G. Liu, Y. L. Xiong, and D. A. Butterfield, “Chemical, physical, and gel-forming properties of oxidized myofibrils and whey- and soy-protein isolates,” Journal of Food Science, vol. 65, no. 5, pp. 811–818, 2000.

R. L. Levine, D. Garland, C. N. Oliver et al., “[49] Determination of carbonyl content in oxidatively modified proteins,” in Methods in Enzymology, pp. 464–478, Elsevier, New York, NY, USA, 1990, vol. 186 of Oxygen Radicals in Biological Systems Part B: Oxygen Radicals and Antioxidants.

S. Benjakul and M. T. Morrissey, “Protein hydrolysates from Pacific whiting solid wastes,” Journal of Agricultural and Food Chemistry, vol. 45, no. 9, pp. 3423–3430, 1997.

P. Nuthong, S. Benjakul, and T. Prodpran, “Characterization of porcine plasma protein-based films as affected by pretreatment and cross-linking agents,” International Journal of Biological Macromolecules, vol. 44, no. 2, pp. 143–148, 2009.

U. K. Laemmli, “Cleavage of structural proteins during the assembly of the head of bacteriophage T4,” Nature, vol. 227, no. 5259, pp. 680–685, 1970.

R. Steel and J. Torrie, Principle and Procedure of Statistic, McGraw-Hill, New York, NY, USA, 1980.

J. Kanner, “Dietary advanced lipid oxidation endproducts are risk factors to human health,” Molecular Nutrition & Food Research, vol. 51, no. 9, pp. 1094–1101, 2007.

H. W. Gardner, R. Kleiman, and D. Weisleder, “Homolytic decomposition of linoleic acid hydroperoxide: identification of fatty acid products,” Lipids, vol. 9, no. 9, pp. 696–706, 1974.

T. Sago, H. Ishii, H. Hagihara, N. Takada, and H. Suda, “Analysis of chemiluminescence spectra in oxidative degradation of oleic acid,” Chemical Physics Letters, vol. 565, pp. 138–142, 2013.

T. P. Wrobel, L. Mateuszuk, S. Chlopicki, K. Malek, and M. Baranska, “Imaging of lipids in atherosclerotic lesion in aorta from ApoE/LDLR−/− mice by FT-IR spectroscopy and Hierarchical Cluster Analysis,” The Analyst, vol. 136, no. 24, pp. 5247–5255, 2011.

K. Czamara, K. Majzner, M. Z. Pacia, K. Kochan, A. Kaczor, and M. Baranska, “Raman spectroscopy of lipids: a review,” Journal of Raman Spectroscopy, vol. 46, no. 1, pp. 4–20, 2014.

M. D. Guillén and N. Cabo, “Characterization of edible oils and lard by Fourier transform infrared spectroscopy. Relationships between composition and frequency of concrete bands in the fingerprint region,” Journal of the American Oil Chemists’ Society, vol. 74, no. 10, pp. 1281–1286, 1997.

F. I. Simplicio, A. B. Seabra, G. F. P. D. Souza, and M. G. D. Oliveira, “In vitro inhibition of linoleic acid peroxidation by primary S-nitrosothiols,” Journal of the Brazilian Chemical Society, vol. 21, no. 10, pp. 1885–1895, 2010.

W. Wu, X. Wu, and Y. Hua, “Structural modification of soy protein by the lipid peroxidation product acrolein,” LWT-Food Science and Technology, vol. 43, no. 1, pp. 133–140, 2010.

G. Liu and Y. L. Xiong, “Oxidatively induced chemical changes and interactions of mixed myosin, ?-lactoglobulin and soy 7S globulin,” Journal of the Science of Food and Agriculture, vol. 80, no. 11, pp. 1601–1607, 2000.

T. Özben, Free Radicals, Oxidative Stress, and Antioxidants: Pathological and Physiological Significance, Springer Science and Business Media, New York, NY, USA, 2013.

B. B. Doyle, E. G. Bendit, and E. R. Blout, “Infrared spectroscopy of collagen and collagen-like polypeptides,” Biopolymers, vol. 14, no. 5, pp. 937–957, 1975.

S. Benjakul, K. Oungbho, W. Visessanguan, Y. Thiansilakul, and S. Roytrakul, “Characteristics of gelatin from the skins of bigeye snapper, Priacanthus tayenus and Priacanthus macracanthus,” Food Chemistry, vol. 116, no. 2, pp. 445–451, 2009.