Characteristics of gelatin from the skin of unicorn leatherjacket (Aluterus monoceros) as influenced by acid pretreatment and extraction time

Gelatins from the skin of unicorn leatherjacket (Aluterus monoceros) pretreated with different acids (0.2 M acetic acid or 0.2 M phosphoric acid) and extracted with distilled water at 45 °C for various times (4 and 8 h) were characterized. Yields of 5.23–9.18 or 6.12–11.54% (wet weight basis) were obtained for gelatins extracted from the skin pretreated with 0.2 M acetic acid or 0.2 M phosphoric acid, respectively. Extracted gelatins contained α₁ and α₂ chains as the predominant components and some degradation peptides. The absorption bands of gelatins in FTIR spectra were mainly situated in the amide band region (amide I, amide II and amide ???) and showed the significant loss of molecular order of triple helix. Gelatin samples had a relative solubility greater than 90% in the wide pH ranges (1–10). The gel strength of gelatin from skin pretreated with phosphoric acid (GPA) was higher than that of gelatin from skin pretreated with acetic acid (GAA). Both GPA and GAA had the lower gel strength than that of commercial bovine gelatin (P < 0.05). Net charge of GAA and GPA became zero at pHs of 6.64–7.15 and 6.78–7.26, respectively, as determined by zeta potential titration. Emulsifying and foaming properties of GAA and GPA increased with increasing concentrations (1–3%, w/v). Those properties were governed by pretreatments and extraction time. Thus gelatin can be successfully extracted from unicorn leatherjacket skin using the appropriate acid pretreatment and extraction time.     

Physico-mechanical and antimicrobial properties of gelatin film from the skin of unicorn leatherjacket incorporated with essential oils

Gelatin films incorporated with bergamot (BO) and lemongrass oil (LO) at various concentrations as glycerol substitute were prepared and characterised. Incorporation of BO and LO at 5–25% (w/w protein) resulted in the decreases in both tensile strength (TS) and elongation at break (EAB) of the films. Water vapour permeability (WVP) were decreased in LO incorporated films, while it was increased in film added with BO at level higher than 5% (P < 0.05). Film solubility and transparency values decreased, and the films had the lowered light transmission in the visible range when BO and LO were incorporated. Films incorporated with LO showed inhibitory effect in a concentration dependent manner against Escherichia coli, Listeria monocytogenes, Staphylococcus aureus and Salmonella typhimurium, but BO added film inhibited only L. monocytogenes and S. aureus. Films containing both BO and LO did not inhibit Pseudomonas aeruginosa. Significant change of molecular organisation and higher intermolecular interactions among gelatin molecules were found in the film structure as determined by FTIR. Thermo-gravimetric analysis (TGA) demonstrated that films added with BO and LO exhibited enhanced heat stability with higher degradation temperature, compared with control film. Scanning electron microscopic (SEM) images revealed the presence of micro-pores in the essential oil incorporated films, which contributed to physical properties of the resulting films. Thus, gelatin films incorporated with BO and LO can be used as active packaging, but the properties could be modified, depending on essential oil added.     

Extraction and characterisation of pepsin-solubilised collagen from the skin of unicorn leatherjacket (Aluterus monocerous)

Pepsin-solubilised collagen (PSC) was extracted from the skin of unicorn leatherjacket (Aluterus monocerous), using 0.5 M acetic acid in the presence of pepsin from albacore tuna, yellowfin tuna or porcine pepsin at a level of 20 units/g of defatted skin. Yields of 8.48 ± 0.3%, 8.40 ± 0.3% and 7.56 ± 0.4% (wet weight basis) were obtained for PSC extracted with the aid of albacore tuna pepsin (APSC), yellowfin tuna pepsin (YPSC) and porcine pepsin (PPSC), respectively. All PSCs were classified as Type I collagen containing two α₁-chains and one α₂-chain with no disulphide bond. The peptide maps of different PSCs hydrolysed by V8 protease and lysyl endopeptidase were different. ATR-FTIR spectra analysis revealed that PSC molecules had the compact triple helical structure stabilised mainly by the hydrogen bond. T_max of all PSCs (31.68–31.98 °C) shifted to lower values (29.33–29.40 °C) when dispersed in 0.05 M acetic acid, indicating the conformational changes in the collagen structure induced by acid. Relative viscosity of 0.03% PSC in 0.1 M acetic acid solution decreased progressively as the temperature increased from 4 to 52 °C, indicating thermal destabilisation or denaturation of PSC molecules. All PSCs were soluble in the pH range of 1–6 and sharply decreased at neutral pH. Decreases in solubility were observed in the presence of NaCl, especially at concentrations above 2% (w/v). Therefore, the skin of unicorn leatherjacket could serve as a potential source of collagen.     

Impact of divalent salts and bovine gelatin on gel properties of phosphorylated gelatin from the skin of unicorn leatherjacket

The impact of zinc chloride (ZnCl₂) and calcium chloride (CaCl₂) as well as bovine gelatin (BG) on the gel strength of phosphorylated fish gelatin (PFG) from the skin of unicorn leatherjacket was investigated. The gel strength of PFG increased with increasing concentrations of ZnCl₂ and CaCl₂ (2.5–40 μmol L⁻¹). A higher gel strength was observed with CaCl₂, compared with ZnCl₂. The gel strength of PFG with 20 μmol L⁻¹ CaCl₂ increased by 15.7%, compared to the control gel. Nevertheless, at higher concentration (40 μmol L⁻¹) of both salts, gel strength of PFG decreased. Hardness of gels decreased with increasing PFG content (P < 0.05). Nevertheless, no differences in hardness were found amongst gels with BG/PFG ratios of 4:0 and 3:1 (P ≥ 0.05). Thus, PFG could be used in combination with CaCl₂ to substitute for BG at a level of 25%.     

Extraction and functional properties of gelatin from the skin of cuttlefish (Sepia officinalis) using smooth hound crude acid protease-aided process

The characteristics and functional properties of gelatin from skin cuttlefish (Sepia officinalis) were investigated and compared to those of halal bovine gelatin (HBG). The gelatin extraction efficiency was improved by an acid-swelling process in the presence of smooth hound crude acid protease extract (SHCAP). The yields of gelatins from cuttlefish skin after 48 h with acid and with crude acid protease (15 units/g alkaline-treated skin) were 2.21% and 7.84%, respectively. The gelatin from skin cuttlefish had high protein (91.35%) but low fat (0.28%) contents. Compared to HBG, the cuttlefish-skin gelatin (CSG) has different amino acids composition than halal bovine gelatin. CSG contained slightly low hydroxyproline and proline (180‰) than HBG (219‰), whereas the content of serine was higher (49‰ versus 29‰). The gel strength of the gelatin gel from CSG (181 g) was lower than that of HBG (259 g) (p < 0.05) possibly due to lower hydroxyproline content. Cuttlefish-skin gelatin exhibited a similar emulsifying activity but greater emulsifying and foam stability than the halal bovine gelatin (p < 0.05). Foam formation ability, foam stability and water-holding capacity of CSG were slightly lower than those of the HBG, but fat-binding capacity was higher in the cuttlefish gelatin.     

Rheological and functional properties of gelatin from the skin of Bigeye snapper (Priacanthus hamrur) fish: Influence of gelatin on the gel-forming ability of fish mince

The rheological and functional properties of gelatin from the skin of bigeye snapper (Priacanthus hamrur) fish were assessed. The protein content of dried gelatin was 94.6% and moisture content was 4.2%. The amino acid profile of gelatin revealed high proportion of glycine and imino acids. The bloom strength of solidified gelatin was 108 g. The average molecular weight of fish skin gelatin was 282 kDa as determined by gel filtration technique. The emulsion capacity (EC) of gelatin at a concentration of 0.05% (w/v) was 1.91 ml oil/mg protein and with increase in concentration, the EC values decreased. The gelling and melting temperatures of gelatin were 10 and 16.8 °C, respectively as obtained by small deformation measurements. The flow behavior of gelatin solution as a function of concentration and temperature revealed non-Newtonian behavior with pseudoplastic phenomenon. The Casson and Herschel–Bulkley models were suitable to study the flow behavior. The yield stress was maximum at 10 °C with the concentration of 30 mg/ml. Thermal gelation behavior of threadfin bream (Nemipterus japonicus) mince in presence of different concentration of gelatin was assessed. Gelatin at a concentration of 0.5% yielded higher storage modulus (G′) value than control. Frequency sweep of heat set gel with gelatin revealed strong network formation.     

Characteristics and functional properties of gelatin from zebra blenny (Salaria basilisca) skin

Gelatin was extracted from alkali-pretreated skin of zebra blenny (Salaria basilisca) using commercial pepsin with a yield of 18 g/100 g of skin sample. The polypeptides pattern, gel strength, viscosity, textural parameters and functional properties of the zebra blenny skin gelatin (ZBSG) were investigated. Amino acid analysis revealed that ZBSG contained almost all essential amino acids, with glycine being the most predominant one. ZBSG was identified as a type I gelatin, containing α₁ and α₂-chains as the major constituents. Its gel strength and viscosity were 170.2 g and 5.95 cP, respectively. Fourier transformed infrared spectroscopy (FT-IR) spectra showed helical arrangements in its structure. Its solubility and functional properties were concentration-dependent. While foam expansion (FE) and foam stability (FS) increased with the increase of concentration, emulsifying activity index (EAI) and emulsion stability index (ESI) were noted to decrease. ZBSG also showed strong clarification ability particularly for apple juice, without affecting nutritional value.     

Chemical composition and characteristics of skin gelatin from grey triggerfish (Balistes capriscus)

Gelatin was extracted from the skin of grey triggerfish (Balistes capriscus) by the acid extraction process with a yield of 5.67 g/100 g skin sample on the basis of wet weight. The chemical composition and functional properties of gelatin were investigated. The gelatin had high protein (89.94 g/100 g) but low fat (0.28 g/100 g) contents. Differences in the amino acid composition between grey triggerfish skin gelatin (GSG) and halal bovine gelatin (HBG) were observed. GSG contained a lower number of imino acids (hydroxyproline and proline) (176 residues per 1000 residues) than HBG (219 residues per 1000 residues), whereas the content of serine was higher (40 versus 29 residues per 1000 residues, respectively). The gel strength of the GSG (168.3 g) was lower than that of HBG (259 g) (p < 0.05) possibly due to lower hydroxyproline content. Grey triggerfish skin gelatin exhibited a slightly lower emulsifying activity and water-holding capacity but greater emulsifying and foam stability, foam formation ability and fat-binding capacity than the halal bovine gelatin (p < 0.05). SDS-PAGE of GSG showed high band intensity for the major protein components, especially, α- and β-components and a similar molecular weight distribution to that of standard calf skin collagen type I.     

Extraction and properties of gelatin from channel catfish (Ietalurus punetaus) skin

Response surface method was used to determine the optimum operating conditions for extracting the gelatin from channel catfish skin. The optimal conditions for maximum gel strength are 68.8 h for the time of treatment with calcium hydroxide solution, 43.2 °C for the extraction temperature, 5.73 h for the extraction time with hot water. The gelatin from channel catfish skin showed a high gel strength, 276±5 g. Compare to porcine skin gelatin, the gelatin from channel catfish skin has different amino acids composition and a lower thermo-stability.     

Chemical compositions and characterisation of skin gelatin from farmed giant catfish (Pangasianodon gigas)

Gelatin was extracted from the skin of farmed giant catfish (Pangasianodon gigas) with a yield of 20.1 g/100 g skin sample on the basis of wet weight. The chemical composition and properties of gelatin were characterised. The gelatin had high protein (89.1 g/100 g) but low fat (0.75 g/100 g) content and contained a high number of imino acids (proline and hydroxyproline) (211 residues per 1000 residues). Giant catfish skin gelatin had a slightly different amino acid composition than calf skin gelatin. The bloom strength of the gelatin gel from giant catfish skin gelatin (153 g) was greater than that of calf skin gelatin (135 g) (P < 0.05). Viscosity, foam capacity and foam stability of gelatin from giant catfish skins were in general greater than those of the gelatin from calf skin tested. SDS-PAGE of giant catfish skin gelatin showed a high band intensity for the major protein components, especially, α-, β- and γ-components and was similar to that of standard calf skin collagen type I.     

Characteristics and functional properties of gelatin from splendid squid (Loligo formosana) skin as affected by extraction temperatures

Gelatin was extracted from the skin of splendid squid (Loligo formosana) at different temperatures (50, 60, 70 and 80 °C) with extraction yield of 8.8%, 21.8%, 28.2%, and 45.3% (dry weight basis) for G50, G60, G70 and G80, respectively. Gelatin from the skin of splendid squid had a high protein content (∼90%) with low moisture (8.63–11.09%), fat (0.22–0.31%) and ash contents (0.17–0.68%). Gelatin extracted at higher temperature (G80) had a relatively higher free amino group content than gelatin extracted at lower temperatures (G50, G60 and G70) (P < 0.05). All gelatins contained α- and β-chains as the predominant components. Amino acid analysis of gelatin revealed the high proline and hydroxyproline contents for G50 and G60. FTIR spectra of obtained gelatins revealed the significant loss of molecular order of the triple-helix. The gel strength of gelatin extracted at lower temperature (G50) was higher than that of gelatins extracted at higher temperatures including G60, G70 and G80, respectively. The net charge of G50, G60, G70 and G80 became zero at pHs of 6.84, 5.94, 5.49, and 4.86, respectively, as determined by zeta potential titration. Gelatin extracted at higher temperature (G80) had the lower L* value but higher a* and b* values, compared with those extracted at lower temperatures (P < 0.05). Emulsion activity index decreased, whilst emulsion stability index, foam expansion and stability increased as the concentration (1–3%) increased (P < 0.05). Those properties were governed by extraction temperatures of gelatin. Thus gelatin can be successfully extracted from splendid squid skin using the appropriate extraction temperature.     

Characterization of gelatin films prepared from under-utilized blue shark (Prionace glauca) skin

Gelatin film from blue shark (Prionace glauca) skin was investigated in order to utilize what is one of the most serious marine wastes in Japan. Film properties from shark skin such as tensile strength (TS), elongation at break (EAB) were evaluated. The TS of gelatin film from shark skin was affected by the protein concentration (1, 2 and 3%) of the film-forming solution (FFS). TS of the film from a 2% protein FFS was the highest. EAB and water vapor permeability (WVP) increased with increasing FFS protein concentration. WVP of shark skin gelatin was evidently low as compared to gelatin films from other fish. An increase in the FFS protein concentration decreased transparency at almost all wavelengths. Furthermore, opacity at 280 nm was characteristically high as compared to films from bony fish skin. The addition of glycerol improved flexibility and enhanced the UV barrier property at 280 nm. However, transparency at the visible range and WVP increased with increasing glycerol content.From the above, it was suggested that shark skin gelatin film technology can be applied to pharmaceutical products or rich-fat food due to its excellent water and UV barrier properties.     

Angiotensin-I-converting enzyme (ACE)-inhibitory and antihypertensive properties of squid skin gelatin hydrolysates

Gelatin extracted from squid (Dosidicus eschrichitii Steenstrup) skin was hydrolysed with pepsin to prepare Angiotensin-I-Converting Enzyme (ACE) inhibitory peptide. The ACE-inhibitory activity was measured by spectrophotometric assay. The hydrolysate was fractionated into three ranges of molecular weight (6 kDa < HSSG-I < 10 kDa, 2 kDa < HSSG-II < 6 kDa, HSSG-III < 2 kDa) using an ultrafiltration unit. The HSSG-III showed the most potent ACE inhibitory activity in vitro with IC₅₀ of 0.33 mg/ml. Renovascular hypertensive rats (RHR) model was made with two-kidney one clip assay, and antihypertensive effects were studied in RHR treated with HSSG-III for 30 days by oral administration. Arterial blood pressure were measured respectively. The HSSG-III remarkably reduced the arterial blood pressure of RHR. These results suggested that hydrolysate of squid skin gelatin obtained by treatment with pepsin was a good source of peptides with ACE-inhibitory activity and had an antihypertensive effect by oral administration.     

Effects of partial hydrolysis and plasticizer content on the properties of film from cuttlefish (Sepia pharaonis) skin gelatin

Properties of film from cuttlefish (Sepia pharaonis) ventral skin gelatin with different degree of hydrolysis (DH: 0.40, 0.80 and 1.20%) added with glycerol as plasticizer at various levels (10, 15 and 20%, based on protein) were investigated. Films prepared from gelatin with all DH had the lower tensile strength (TS) and elongation at break (EAB) but higher water vapor permeability (WVP), compared with the control film (without hydrolysis) (p < 0.05). At the same glycerol content, both TS and EAB decreased, while WVP increased (p < 0.05) with increasing %DH. At the same DH, TS generally decreased as glycerol content increased (p < 0.05), however glycerol content had no effect on EAB when gelatins with 0.80 and 1.20% DH were used (p > 0.05). DH and glycerol content had no marked impact on color and the difference in color (ΔE^∗) of resulting films. Electrophoretic study revealed that degradation of gelatin and their corresponding films was more pronounced with increased %DH, resulting in the lower mechanical properties of films. Based on FTIR spectra, with the increasing %DH as well as glycerol content, higher amplitudes for amide-A and amide-B peaks were observed, compared with film from gelatin without hydrolysis (control film) due to the increased –NH₂ group caused by hydrolysis and the lower interaction of –NH₂ group in the presence of higher glycerol. Thermo-gravimetric analysis indicated that film prepared from gelatin with 1.20% DH exhibited the higher heat susceptibility and weight loss in the temperature range of 50–600 °C, compared with control film. Thus, both chain length of gelatin and glycerol content directly affected the properties of cuttlefish skin gelatin films.     

Effect of heat treatment of film-forming solution on the properties of film from cuttlefish (Sepia pharaonis) skin gelatin

Effects of heat treatment at different temperatures (40–90 °C) of film-forming solution (FFS) containing 3% gelatin from cuttlefish (Sepia pharaonis) ventral skin and 25% glycerol (based on protein) on properties and molecular characteristics of resulting films were investigated. The film prepared from FFS heated at 60 and 70 °C showed the highest tensile strength (TS) with the highest melting transition temperature (T_max) (p < 0.05). Nevertheless, film from FFS heated at 90 °C had the highest elongation at break (EAB) with the highest glass transition temperature (T_g) (p < 0.05). With increasing heating temperatures, water vapor permeability (WVP) of films decreased (p < 0.05), but no differences in L*-value and transparency value were observed (p > 0.05). Based on FTIR spectra, the lower formation of hydrogen bonding was found in film prepared from FFS with heat treatment. Electrophoretic study revealed that degradation of gelatin was more pronounced in FFS and resulting film when heat treatment was conducted at temperature above 70 °C. Thus, heat treatment of FFS directly affected the properties of resulting films.     

Functional properties of gelatin from cuttlefish (Sepia pharaonis) skin as affected by bleaching using hydrogen peroxide

Functional properties of gelatin from dorsal and ventral skin of cuttlefish with and without bleaching by H₂O₂ at different concentrations (2% and 5% (w/v)) for 24 and 48 h were studied. Gelatin from skin bleached with 5% H₂O₂ for 48 h showed the highest yield (49.65% and 72.88% for dorsal and ventral skin, respectively). Bleaching not only improved the colour of gelatin gel by increasing the L^∗-value and decreasing a^∗-value but also enhanced the bloom strength, and the emulsifying and foaming properties of the resulting gelatin. Gelatin from bleached skin contained protein with a molecular weight of 97 kDa and had an increased carbonyl content. Fourier transform infrared spectroscopic study showed higher intermolecular interactions and denaturation of gelatin from bleached skin than that of the control. These results indicated that hydrogen peroxide most likely induced the oxidation of gelatin, resulting in the formation of gelatin cross-links, giving improved functional properties.     

Effects of hydrogen peroxide and Fenton’s reagent on the properties of film from cuttlefish (Sepia pharaonis) skin gelatin

Properties of film from cuttlefish (Sepia pharaonis) ventral skin gelatin without and with partial hydrolysis (1.2% degree of hydrolysis), as influenced by H₂O₂ and Fenton’s reagent at different levels, were investigated. Films treated with H₂O₂ (0.01–0.04 M) and Fenton’s reagent [H₂O₂ (0.01–0.04 M) + FeSO₄ (0.001–0.004 M)] had higher tensile strengths (TS) but similar or lower elongations at break (EAB), compared with the control film (p < 0.05). Slight differences in water vapour permeability (WVP) were observed for all films. Films treated with Fenton’s reagent had a lower L^∗-value but higher a^∗-, b^∗- and ΔE^∗-values, while films treated with H₂O₂ had lower b^∗-values (p < 0.05), than had the control film. Cross-linking was pronounced in films treated with H₂O₂ or Fenton’s reagent and was associated with increased heat stability. Films treated with Fenton’s reagent had the lowest solubility in water (p < 0.05). However, fragmentation more likely took place when Fenton’s reagent (at a higher level) was used. Generally, similar results were noticeable between films from gelatin with and without partial hydrolysis. Thus, H₂O₂ and Fenton’s reagent directly affected the properties of film from cuttlefish skin gelatin, regardless of hydrolysis.     

Physico-chemical and film forming properties of giant squid (Dosidicus gigas) gelatin

Giant squid (Dosidicus gigas) inner and outer tunics were subjected to hydrolysis with pepsin prior to gelatin extraction (G1 gelatin) by a mild-acid procedure. Furthermore, a second gelatin extraction (G2 gelatin) was done using the collagenous residues that remained from the first extraction. Pepsin allows the collagen solubilisation and the extraction yield to increase by yielding extracts high in α-chains. G1 exhibited good gel forming ability but G2 showed poor viscoelastic behaviour and low gel strength, in agreement with the results for the molecular weight distribution, which showed a considerably higher content of low molecular weight components. In spite of these differences, both G1 and G2 showed good filmogenic ability and similar properties were found including the absence of colour, opacity, low water vapour permeability and high puncture deformation. Nevertheless, films made from G1 had a higher puncture force than films made from G2 as a result of the different molecular weight distribution.