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.     

Characterization of fish gelatin from surimi processing wastes: Thermal analysis and effect of transglutaminase on gel properties

Fish gelatin extraction from wastes of fish Herring species (Tenualosa ilisha) was carried out by a series of pretreatment with 0.2 M Ca(OH)₂ followed by 0.1 M citric acid and final water extraction at 50 °C for 3 h. The resulting fish gelatin preparation was evaluated for its dynamic viscoelastic properties, gelling and melting temperatures and gel strength. The gelling and melting temperatures of gelatin samples (at 6.67%, w/v) were obtained from differential scanning calorimetry and rheological studies. The melting temperature of extracted fish gelatin (EFG) obtained ranged from 16.2 to 16.7 °C compared to that of commercial fish gelatin gel (CFG), from 23.7 to 25.6 °C and halal bovine gelatin (HBG), from 26.5 to 28.7 °C. On the other hand, gelling temperatures of EFG, CFG and HBG ranged from 5.1 to 5.2 °C, 11.9 to 17.46 °C, and 12.6 to 19.33 °C, respectively. EFG gave gels with a considerably lower G′ values than CFG and HBG. The bloom strength of EFG gels at 6.67% (w/v) was 69.03 g which was much lower than HBG (336.2 g) and CFG (435.9 g). Enzyme transglutaminase was added in the amounts of 0.5, 1.0, 3.0 and 5.0 mg/g gelatin to modify the gel properties of the extracted fish gelatin. The modified EFG gels obtained had higher gel strengths of 101.1 g and 90.56 g with added transglutaminase of 1.0 and 3.0 mg/g, respectively. However with addition of 5.0 mg/g enzyme the gel strength increased only up to 75.06 g. SDS-PAGE of extracted gelatin gel showed protein band intensities for α1-chains and 53 kDa but in gels added with higher concentration of transglutaminase, these protein band intensities seemed to disappear.     

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.     

Antioxidative activity and emulsifying properties of cuttlefish skin gelatin modified by oxidised phenolic compounds

Antioxidative activity and emulsifying properties of cuttlefish skin gelatin modified by different oxidised phenolic compounds including caffeic acid, ferulic acid and tannic acid at different concentrations were investigated. Oxidised phenolic compounds were covalently attached to gelatin as indicated by the decrease in amino groups. Fourier transform infrared spectroscopic studies indicated the presence of an aromatic ring and a hydroxyl group in gelatin after modification. Modified gelatin had the increased antioxidative activity but the decreased surface hydrophobicity. Gelatin modified with 5% oxidised tannic acid had no change in emulsifying properties. Emulsion stability and oxidative stability of menhaden oil-in-water emulsion stabilised by 0.5% and 1.0% gelatin without and with modification by 5% oxidised tannic acid were studied. Both gelatins at a higher concentration (1.0%) yielded an emulsion with a smaller particle size. Modified gelatin inhibited the formation of TBARS in the emulsion more effectively than the control gelatin throughout the 12 days of storage.     

Properties of film from cuttlefish (Sepia pharaonis) skin gelatin incorporated with cinnamon,clove and star anise extracts

Properties of film from cuttlefish (Sepia pharaonis) ventral skin gelatin without and with partial hydrolysis (1.2% degree of hydrolysis) incorporated with 1% ethanolic extract of cinnamon (CME), clove (CLE) and star anise (SAE) were determined. Films with different herb extracts (without and with oxidation) had higher tensile strength (TS) but lower elongation at break (EAB), compared with the control film (without addition of herb extracts) (p < 0.05). Lower water vapor permeability (WVP) and L^∗-value but higher b^∗- and ΔE^∗-values were observed when the extracts were incorporated (p < 0.05). Electrophoretic study revealed that cross-linking was pronounced in films containing different herb extracts. Oxidized extracts yielded films with higher TS and WVP than those without oxidized herb extracts (p < 0.05). Generally, similar properties were noticeable for films from gelatin with and without partial hydrolysis. Nevertheless, higher mechanical properties were obtained for the latter. FTIR spectra indicated that protein–polyphenol interactions were involved in the film. Thermo-gravimetric analysis revealed that films incorporated with SAE or SAE with oxidation (OSAE) exhibited lower heat susceptibility and weight loss in the temperature range of 50–600 °C, compared with control film. Films with SAE and OSAE had smoother surface for gelatin without hydrolysis; however, coarser surface was observed in film from gelatin with partial hydrolysis. Thus, the incorporation of different herb extracts directly affected the properties of film from cuttlefish skin gelatin with and without hydrolysis.     

Indigenous proteases in the skin of unicorn leatherjacket (Alutherus monoceros) and their influence on characteristic and functional properties of gelatin

Indigenous proteases in the skin of unicorn leatherjacket (Alutherus monoceros) were characterised using autolytic study. Maximised autolysis was found at pH 7 and 50 °C. Autolysis was markedly inhibited by 0.04 mM soybean trypsin inhibitor (SBTI), suggesting that heat activated serine protease was predominant in the skin. The impact of indigenous proteases on the properties of gelatin extracted from unicorn leatherjacket skin was investigated. Gelatin was extracted from unicorn leatherjacket skin using distilled water at 50 °C for 12 h in the presence and absence of 0.04 mM SBTI. In the presence of SBTI, the degradation was markedly inhibited, but a lower gelatin extraction yield was obtained (P < 0.05). Extracted gelatins contained α₁ and α₂ chains as the predominant components with some degradation peptides. FTIR spectra indicated a greater loss of molecular order of the triple helix and a higher degradation was found in gelatin extracted in the absence of 0.04 mM SBTI. The net charge of gelatin samples extracted with and without 0.04 mM SBTI became zero at pHs of 8.45 and 7.31, respectively, as determined by ζ-potential titration. Higher gel strength (320.68 ± 3.02 g) was obtained in gelatin extracted with SBTI, compared with that of gelatin extracted without SBTI (288.63 ± 1.44 g). High emulsifying activity index but lower emulsifying stability index was observed in the former. Therefore, heat-activated serine protease was involved in the degradation of gelatin molecules, thereby affecting the yield, proteinaceous components and properties of gelatin from unicorn leatherjacket skin.     

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.     

Emulsifying Property and Antioxidative Activity of Cuttlefish Skin Gelatin Modified with Oxidized Linoleic Acid and Oxidized Tannic Acid

Cuttlefish skin gelatins modified with oxidized linoleic acid (OLA) and oxidized tannic acid (OTA) were characterized and determined for emulsifying properties and antioxidative activity. Modification of gelatin with 5% OTA increased the total phenolic content and 1,1-diphenyl-2-picrylhydrazyl, 2,2′-azinobis(3-ethylbenzothiazoline-6-sulphonic acid) radical scavenging activity and ferric reducing antioxidant power of gelatin–OTA. Incorporation of OLA into gelatin (OLA-to-free amino group molar ratio of 10:1) increased surface hydrophobicity of gelatin from 17.39 to 32.38 and reduce surface tension at air/water interface of gelatin solution from 53 to 32 mN/m. Gelatin–OLA had the increase in emulsion activity index, compared with gelatin without modification and was capable of producing a fine emulsion (d ₃₂?=?0.79 μm, d ₄₃?=?0.82 μm). Modification of gelatin–OLA complex with OTA at different concentrations (2.5%, 5%, and 10%) increased antioxidative activity but decrease emulsifying properties. However, gelatin–OLA modified with 5% OTA had higher emulsifying properties than the commercial gelatin (bovine gelatin). The presence of an alkyl group and a hydroxyl group in gelatin after modification with OLA and OTA, respectively, was revealed by Fourier transform infrared study. Coincidental decrease in free amino group was also noticeable in modified gelatin. Menhaden oil-in-water emulsion stabilized by gelatin modified with OLA and 5% OTA was more resistant to lipid oxidation and phase separation as evidenced by the lower thiobarbituric acid reactive substances value and smaller oil droplet size, compared with that stabilized by commercial bovine gelatin. Thus, the modification of gelatin by both OLA and OTA was able to improve antioxidative and emulsifying properties of cuttlefish skin gelatin.     

Amino acid composition and functional properties of collagen polypeptide from Yak (Bos grunniens) bone

Collagen polypeptide was prepared from Yak bone by applying papain as a catalyst. Its relative molecular weight distribution and amino acid composition were determined. Moreover, its main functional properties including water and oil absorption, water-holding capacity, emulsifying ability and stability, foaming ability and foam stability were investigated. The polypeptide had a molecular weight distribution of 11.7-43 kDa, in which most distributed at 11.7-25.3 kDa. It was high in glycine, alanine, proline, hydroxyproline and glutamic acid but low in methionine, tyrosine and histidine, the total amino acid amount being 88.06 g/100 g. The polypeptide showed good water adsorption, water-holding capacity, oil absorption, emulsifying and foaming properties. It is hoped to be applied in food and cosmetic.     

Antioxidant and functional properties of gelatin hydrolysates obtained from skin of sole and squid

Antioxidant and functional properties were evaluated for gelatin hydrolysates obtained from sole and squid skin gelatin by Alcalase, with a degree of hydrolysis of ∼35% and ∼50%, respectively. Both hydrolysates mainly consisted of peptides below 6.5 kDa, together with peptidic material from around 16 to 6.5 kDa. Moreover, the squid hydrolysate showed a peptide band of around 26 kDa. Antioxidant properties of both gelatins were highly increased by hydrolysis, especially ABTS and metal chelating abilities. The squid hydrolysate showed the highest antioxidant capacity by FRAP, ABTS and metal chelating assays in spite of the lower content in hydrophobic amino acids. Both gelatin hydrolysates had a good solubility (over 95%). The emulsifying activity index (EAI) decreased with increasing concentration. Conversely, the foam expansion increased with increasing concentration. However, both foam and emulsion stabilities were not apparently affected by the concentration of hydrolysate. In the case of the sole hydrolysate, which showed a lower degree Pro and Lys hydroxylation, foam stability was very poor, and 50% of foam expansion was lost after 5 min at all concentrations.     

Digestive acid protease from zebra blenny (Salaria basilisca): Characteristics and application in gelatin extraction

The present study reports on the characterization and evaluation of a crude acidic protease from the viscera of zebra blenny (Salaria basilisca) for use in gelatin extraction. Using hemoglobin, zymogram analysis revealed the presence of at least one clear band. The crude acid protease was noted to be optimally active at pH 3.0 and 50 °C and highly stable over a pH range of 2.0 to 7.0. The enzymatic extract lost about 87% of its activity after incubation with pepstatin A for 30 min at 4 °C. The acidic protease from the viscera of zebra blenny was noted to be effective in the extraction of gelatin from the skin of zebra blenny, with an extraction yield of 14.65% based on the wet weight of zebra blenny skin. The extracted zebra blenny skin gelatin (ZBSG) was characterized based on its chemical composition, polypeptide pattern, gel strength, textural parameters, and functional properties. ZBSG had high protein (90.6%) and low ash (3.1%) and fat (0.6%) contents. It contained α₁ and α₂-chains as the major constituents and determined as belonging to type I. The bloom strength of solidified gelatin was 151.3 g. The findings from Fourier Transformed Infrared (FT-IR) spectroscopy suggested the presence of helical arrangements of ZBSG. The latter showed excellent concentration-dependent functional properties. While emulsion activity index (EAI) and emulsion stability index (ESI) decreased, foam expansion (FE) and foam stability (FS) increased as the concentration of gelatin increased. Overall, zebra blenny endogenous acid protease could open new promising opportunities for the extraction of gelatin.     

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.     

Antioxidative activity and emulsifying properties of cuttlefish skin gelatin–tannic acid complex as influenced by types of interaction

The non-covalent interaction between cuttlefish skin gelatin and tannic acid was observed in gelatin modified with unoxidized tannic acid at pH 7, whereas covalent interaction was found in gelatin modified with oxidized tannic acid at pH 9. Degree of tannic acid incorporation into gelatin via non-covalent interaction was more pronounced than that found via covalent interaction as evidenced by lowered free amino group content and increased total phenolic content and hydroxyl group and aromatic ring determined by FTIR. Gelatin modified with oxidized tannic acid had the slight decrease in surface hydrophobicity, with no changes in particle size distribution of the emulsions. Modification of gelatin with tannic acid, especially via non-covalent interaction, increased in vitro antioxidative activity, compared with the control gelatin. Gelatin modified with tannic acid via covalent interaction rendered the emulsion with high stability and could inhibit lipid oxidation of menhaden oil-in-water emulsion effectively throughout the storage of 12 days.

Industrial relevance

Cuttlefish skin gelatin modified with tannic acid possessing both emulsifying activity and the improved antioxidative activity can be used as a natural and safe additive in food industry. Therefore, cuttlefish skin, a by-product from seafood processing industry, can be produced as the high value added product with wider applications.     

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.     

Improvement of foaming properties of cuttlefish skin gelatin by modification with N-hydroxysuccinimide esters of fatty acid

Conformation and foaming properties of cuttlefish skin gelatin modified by N-hydroxysuccinimide esters of different saturated fatty acids including capric acid (C10:0), lauric acid (C12:0) and myristic acid (C14:0) at different molar ratios (0.25, 0.50, 1.00 and 2.00) were investigated. Covalent attachment of fatty acids into gelatin was observed as evidenced by the decrease in amino groups. Fourier transform infrared spectroscopic study indicated the presence of alkyl group of modified gelatin. The higher increase in surface activity with coincidental increase in surface hydrophobicity was observed in gelatin modified with fatty acid ester having a longer chain, especially at the higher molar ratio. The increase in foam expansion was related with the improved surface activity mediated by the modification by N-hydroxysuccinimide esters of fatty acid.     

Thermal characterisation of gelatin extracted from yellowfin tuna skin and commercial mammalian gelatin

Glass transition and other thermal characteristics of gelatin from different sources were studied by differential scanning calorimetry (DSC) and modulated DSC (MDSC). The initial glass transition temperatures of equilibrated gelatin samples at 11.3% relative humidity, determined from reversible heat flow thermogram of MDSC, were 23, 75 and 59 °C, respectively, for tuna skin, bovine and porcine gelatin. When gelatin samples were equilibrated at higher relative humidity of 52.9%, glass transition temperature of fish skin and bovine gelatin decreased to −3 and 57 °C, respectively. Further increase of equilibration relative humidity to 75.3% showed increased value in the case of tuna skin, whereas bovine and porcine did not show any significant change. DSC and MDSC results indicated that tuna gelatin showed lower glass transition compared to mammalian source gelatin equilibrated at the same constant relative humidity. In general glass transition measured by DSC was found lower than the values measured by MDSC. The results in this study showed that the degree of plasticization varied with the source of gelatin as well as their extraction methods.     

Optimum conditions for extracting collagen from the tunica albuginea of immunologically castrated pig testes and the functional properties of the isolated collagen

This study evaluated alternative methods for extracting collagen from the tunica albuginea of pig testes and characterized the functional properties of the isolated collagen. Using the statistical tools of factorial design (2^4-1) and a central composite rotatable design (2³), it was concluded that the best conditions were 0.83 mol L^− 1 acetic acid, 0.24% pepsin and 28 h of hydrolysis to isolate 82.54 g of collagen per 100 g of sample. This purified collagen had improved functional properties in relation to bovine skin collagen, including water solubility, water-holding capacity, emulsifying capacity and emulsion stability. These results suggest that isolated collagen from the tunica albuginea can be used in pharmaceutical and food products.     

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 of gelatin from the skins of bigeye snapper, Priacanthus tayenus and Priacanthus macracanthus

Gelatins extracted from the skins containing fine scales of two species of bigeye snapper, Priacanthus tayenus (GT) and Priacanthus macracanthus (GM), were characterised. Both gelatins had the protein as the major component with high content of imino acids (proline & hydroxyproline) (186.29–187.42 mg/g). GT and GM contained calcium at levels of 6.53 and 2.92 g/kg, respectively. Both gelatins contained α1 and α2 chains as the predominant components and some degradation peptides. The absorption bands of both gelatins in Fourier transform infrared (FTIR) spectra were mainly situated in the amide band region (amide I and amide II). GT and GM had a relative solubility greater than 90% in the wide pH ranges (1–10). The bloom strength of GM (254.10 g) was higher than that of GT (227.73 g) (P < 0.05), but was slightly lower than that of commercial bovine gelatin (293.22 g) (P < 0.05). Finer gel structure with smaller strands and voids was observed in GM gel, in comparison with that observed in GT counterpart.     

Preparation and functional characterisation of fish skin gelatin and comparison with commercial gelatin

Gelatin was extracted from the skin of farmed giant catfish (GC) and tilapia (TP) at a yield of 19.50% and 23.34% (wet wt). It was high in protein (84–88%) but low in fat (0.09–1.24%) and ash content (0.15–0.17%). The GC exhibited lower emulsifying activity (24–35%), but greater foam ability (98–110%), water holding capacity (477–844%) and fat binding capacity (2541–3314%) than commercial beef skin gelatin (BF) (P < 0.05). GC and TP showed comparable functional properties to BF. SDS‐PAGE patterns of TP gelatin showed high band intensity for the α‐ and β‐components, while the lowest band intensity of the major component was found in the BF. From the study, it can be concluded that the farmed freshwater fish skin GC and TP is a prospective source for producing a significant gelatin yield with desirable functionalities. Because of these, fish skin gelatin could be more effectively and widely used in food industries as a good food ingredient.