Extraction and Characterization of Gelatin from Skin By-Products of Seabream, Seabass and Rainbow Trout Reared in Aquaculture.

The expansion of fish filleting, driven by the increasing demand for convenience food, concomitantly generates a rising amount of skinning by-products. Current trends point to a growing share of aquaculture in fish production, so we have chosen three established aquaculture species to study the properties of gelatin extracted from their skin: rainbow trout, commonly filleted; and seabass and seabream, marketed whole until very recently. In the first case, trout skin yields only 1.6% gelatin accompanied by the lowest gel strength (96 g bloom), while yield for the other two species exceeds 6%, and gel strength reaches 181 and 229 g bloom for seabass and seabream, respectively. These results are in line with the proportion of total imino acids analyzed in the gelatin samples. Molecular weight profiling shows similarities among gelatins, but seabass and seabream gelatins appear more structured, with higher proportion of ß-chains and high molecular weight aggregates, which may influence the rheological properties observed. These results present skin by-products of seabream, and to a minor extent seabass, as suitable raw materials to produce gelatin through valorization processes.

Silver carp scale gelatins for the stabilization of fish oil-loaded emulsions.

Herein, three types of silver carp scale gelatins were extracted, and their molecular weight distribution, structural properties, functional properties and emulsifying properties were investigated and discussed. Acetic acid-extracted gelatin (AAG), hot water-extracted gelatin (HWG), and pepsin enzyme-extracted gelatin (PEG) showed similar and four clear bands in sodium dodecyl sulfate-polyacrylamide gel electrophoresis pattern, whereas they showed different ß chain amounts and ß-sheet percentages. The water-holding capacity values (g/g of gelatin) were: AAG (16.8 ± 1.1) > HWG (14.0 ± 0.7) ≈ PEG (13.5 ± 1.6). The fat-binding capacity values (g/g of gelatin) were: AAG (11.8 ± 0.3) > HWG (9.5 ± 1.3) > PEG (5.3 ± 0.4). Emulsion droplet sizes and creaming index values decreased with the increase of gelatin concentrations for all the fish oil-loaded emulsions stabilized by three types of gelatins. Compared with PEG, AAG and HWG show similar and higher emulsion stability at high gelatin concentration (10 mg/mL). The stabilization mechanism of fish oil-loaded silver carp scale gelatin-stabilized emulsions involved an "extraction method-protein molecular weight distribution-protein molecular structure-molecular interaction-emulsibility-droplet structure-emulsion stability" route. This work would be beneficial for the research on the relationship of structure and function of gelatin and to the comprehensive utilization of aquatic products.

Physicochemical and Antioxidant Properties of Gelatin and Gelatin Hydrolysates Obtained from Extrusion-Pretreated Fish (Oreochromis sp.) Scales.

Fish gelatin and its hydrolysates exhibit a variety of biological characteristics, which include antihypertensive and antioxidant properties. In this study, fish gelatins were extracted from extrusion-pretreated tilapia scales, and then subjected to analyses to determine the physicochemical properties and antioxidant activity of the extracted gelatins. Our findings indicate that TSG2 (preconditioned with 1.26% citric acid) possessed the greatest extraction yield, as well as higher antioxidant activities compared with the other extracted gelatins. Hence, TSG2 was subjected to further hydrolyzation using different proteases and ultrafiltration conditions, which yielded four gelatin hydrolysates: TSGH1, TSGH2, TSGH3, and TSGH4. The results showed that TSGH4 (Pepsin + Pancreatin and ultrafiltration < 3000 Da) had a higher yield and greater antioxidant activity in comparison with the other gelatin hydrolysates. As such, TSGH4 was subjected to further fractionation using a Superdex peptide column and two-stage reverse-phase column HPLC chromatography, yielding a subfraction TSGH4-6-2-b, which possessed the highest 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity compared with the other fractions. Further LC-ESI/MS/MS analysis of TSGH4-6-2-b suggested two novel peptides (GYDEY and EPGKSGEQGAPGEAGAP), which could have potential as naturally-occurring peptides with antioxidant properties. These promising results suggest that these antioxidant peptides could have applications in food products, nutraceuticals, and cosmetics.

Enhanced gelling performance of oxhide gelatin prepared from cowhide scrap by high pressure-assisted extraction.

In this study, the feasibility of preparing oxhide gelatin from cowhide scrap by high pressure assisted-liquid extraction was verified. Different processing conditions, including high pressure time (15 to 25 min), pressure (250 to 350 MPa), and liquid-to-solid ratio (1:3 to 1:5), were optimized through response surface methodology. Under the optimum manufacturing conditions, when the high-pressure processing (HPP) time was 22 min, the pressure was 289 MPa, and the liquid-to-solid ratio was 1:4, the highest extraction yield (36%) and gel strength (224 g) were achieved. Based on DSC, XRD, FTIR, SEM, gelling and melting temperatures, HPP led to the structural modification of the gelatinized collagen, which enhanced the rearrangement of the gel structure during the gelation process and made it have better gelling properties. In addition, compared with the commercial sample, they do not differ significantly in the relaxation time and peak area of prepared oxhide gelatin. These findings provide new insights into the practicability of HPP during the preparation of oxhide gelatin, which can noticeably reduce the processing time and be applied to industrial production. PRACTICAL APPLICATION: Compared with traditional processing, a high pressure-assisted extraction process can noticeably reduce the processing time while producing cowhide gelation with similar physicochemical and functional properties. Meanwhile, high pressure processing (HPP) led to the structural destruction of the cowhide and gelatinized collagen, which enhanced the rearrangement of the gelatin structure during the gelation process and made it have better gelling properties. Importantly, high pressure-assisted extraction can facilitate the use of a low-cost raw material and improve the preparation efficiency of oxhide gelatin, which shows great potential in large-scale and efficient industrial production and the quality control of oxhide gelatin.

Structure-function engineering of novel fish gelatin-derived multifunctional peptides using high-resolution peptidomics and bioinformatics.

The multifunctional properties of fish gelatin hydrolysates have not been completely elucidated. Here, the biological characterization of these peptides was performed to engineer multifunctional peptides. Bioactive peptides were produced from mackerel byproducts via successive enzymatic hydrolysis reactions using subtilisin A and actinidin as microbial and herbal proteases. The antibacterial activity against both gram-negative and -positive food-borne pathogens, including Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Klebsiella pneumoniae, as well as the inhibitory potential of angiotensin-converting enzyme (ACE) and dipeptidyl peptidase IV (DPP-IV), was accessed in vitro. The synthesized peptides demonstrated multifunctional properties, which were further confirmed by in silico protocols. The ACE and DPP-IV inhibitory (IC50) values of P1, P2, and P3 were 0.92 and 0.87, 0.51 and 0.93, 0.78 and 1.16 mg mL-1, respectively. Moreover, the binding energy was sufficient for all three peptides to inhibit both ACE and DPP-IV enzymes with excellent three-dimensional conformation (RMSD = 0.000) for all six docking mechanisms.

Effect of drying methods on the solubility and amphiphilicity of room temperature soluble gelatin extracted by microwave-rapid freezing-thawing coupling.

The effect of three drying methods (hot air, freeze and spray drying) on the solubility and amphiphilicity of gelatin were investigated and compared. Results showed spray drying gelatin (SDG) and hot air drying gelatin (HDG) showed the lowest and best solubility, respectively. This phenomenon was attributed to the degree of subunits degradation and hydrophobicity. The HDG showed an obvious degradation during the hot air drying and displayed the strongest hydrophilicity, while SDG showed a slight degradation and strongest hydrophobicity. The results of wettability showed that SDG had a better amphiphilicity (92.48°) in comparison with HDG (57.7°) and freeze drying gelatin (VDG, 77.53°), which can effectively reduce the interfacial tension of gelatin, thus significantly improving the stability of foam and emulsion (p < 0.05). These results suggested the drying methods can adjust the amphiphilicity of gelatin, and the SDG displayed a better amphiphilicity, showing good potential applications in foam and emulsion.

Improved solubility and interface properties of pigskin gelatin by microwave irradiation.

In this study, the microwave irradiation as a green approach was applied to improve the properties (mainly solubility and interface properties) of pigskin gelatin. The results showed that the solubility of pigskin gelatin was improved obviously at room temperature (25 °C) due to the destruction of polymer subunits. Furthermore, the exposure of more hydrophobic groups in microwave-irradiated gelatin increased its hydrophobicity, consequently improving the amphiphilic property and the interfacial properties of gelatin. The results of interface behavior showed that the interfacial tension of microwave-irradiated gelatin was reduced obviously with the extension of irradiation time (0-30 min), which is more beneficial to adsorption of gelatin molecules at the interface, thus resulting in a significant increase of adsorption rate (AP) from 56.13% (0 min) to 91.87% (30 min). Correspondingly, the foaming and emulsifying properties of gelatin were also improved significantly (p < 0.05). This study would promote the development of food-grade foam and emulsion based on pigskin gelatin by adjusting solubility and interface properties.

A novel extraction approach and unique physicochemical properties of gelatin from the swim bladder of sturgeon.

BACKGROUND: Gelatin is traditionally produced from mammals and widely applied in the food industry. The production is tedious, time-consuming and environment-unfriendly, while the application is restricted because of zoonosis risk and religious sentiment. RESULTS: Gelatin was extracted by hot water from sturgeon swim bladder after defatting with alcohol and hexane. The yield reached to 94.15% under the optimized conditions of 50 °C, 30 min and 10 mL g-1 . Its amino acid and subunit profiles were similar to type I collagen. Compared to commercial porcine, bovine and piscine gelatins, it exhibited higher whiteness (3.38), emulsion activity (171.76 m2 g-1 ), gel strength (853.23 g), water-holding capacity (92.37%) and viscoelasticity (0.03). But the transmittance (40.56% at 450 nm and 59.07% at 620 nm), emulsion stability (30.09 min), foam expansion (203.00) and stability (26.92), gelling (16.88 °C) and melting temperature (21.80 °C) were lower. While the pH (6.87) and viscosity (28.60 mPa s) were moderate. Moreover, it made better hydrogels and nanofibers. CONCLUSION: Gelatin was extracted from sturgeon swim bladder using a clean and efficient approach, and exhibited unique properties and great potential for the food industry. © 2020 Society of Chemical Industry.

Microstructure and characteristic properties of dogfish skin gelatin gels prepared by freeze/spray-drying methods.

The effects of two pretreatments (microwaves or oven-drying) on the dogfish (Squalus acanthias) skin as well as two drying processes (freeze-drying or spray-drying) on the extracted gelatins were studied. Thus six types of gelatins were obtained, three of which were freeze-dried (FG) and the others were spray-dried (SG), from the untreated skin (US), microwaves-pretreated skin (MS) and oven-pretreated skin (OS). The highest yield (8.67%) was obtained for the OSFG, while the lowest one (3.06%) was measured for the OSSG. Interestingly, all gelatins exhibited relatively high protein (84.02-89.53%), and low lipid (0.50-1.71%) and ash (3.05-7.17%) contents. In addition, gelatins were analyzed by the Fourier transform infrared and the spectra displayed important differences in some specific peaks, particularly in the amide I, amide II and amide III. The gelatins extracted from the untreated skin, regardless the drying method, presented the highest foaming capacity. The textural profile analysis showed that USSG was the hardest (213.6 g) and the chewier (23.8 N × mm) gelatin. Moreover, analysis of thermal properties showed that USSG also has the highest glass-transition temperature. The interesting properties of gelatin extracted from dogfish skin encourage their future use as a functional ingredient in industrial food formulations.

Distinguishing tissue origin of bovine gelatin in processed products using LC/MS technique in combination with chemometrics tools.

Gelatin, as a by-product of the meat industry, is extracted from bone and skin of mainly bovine and porcine origins. It is used widely in the food, drug, and cosmetic industries. Authenticity testing methods can be used to confirm whether labelled ingredients are present in the product. Generally, studies on gelatin are concerned mainly with determining species, but detecting tissue origin is also important from religious, health, and commercial perspectives. In the present study, for the first time, liquid chromatography/mass spectrometry (HPLC/MS) was used to differentiate bovine bone gelatin from gelatin derived from bovine skin. Tryptic-digested gelatins were measured using HPLC/MS and, subsequently, two powerful chemometrics approaches (i.e., PCA and PLS-DA) were used to classify samples as either skin or bone gelatins. Origin of bovine gelatins in different test samples were predicted accurately using this method. The results showed both the stability and reliability of the proposed procedure.

Characterization and Antioxidant Activity of Collagen, Gelatin, and the Derived Peptides from Yellowfin Tuna (Thunnus albacares) Skin.

Skin waste from tuna processing needs to be utilized, such as extraction of its collagen and gelatin. Their functional properties can be improved by enzymatic hydrolysis for conversion to peptides. Thus, the research objectives were to examine the characteristics and antioxidant activity of collagen, gelatin, and the derived peptide from yellowfin tuna skin. Collagen was extracted using 0.75 M acetic acid at 4 °C, while gelatin was prepared using 0.25% citric acid and extracted at 65 °C. Hydrolysis was carried out with 2% Alcalase, followed by fractionation with a molecular weight cut off sieve for both collagen and gelatin. Collagen yield was 22.6% with pH value of 6.63 and whiteness of 96.7%. Gelatin yield was 20.0% with pH value of 4.94 and whiteness of 51.0%. Hydrolysis for three hours resulted in 52.7% and 45.2% degree of hydrolysis for collagen and gelatin, respectively. The molecular weights of collagen peptides ranged from 2.94 to 11.93 kDa, while those of gelatin peptides ranged from 3.54 to 16,620 kDa. Antioxidant activities of these peptides were higher than those before hydrolysis. The high antioxidant activity (IC50) of collagen peptides were found in <3, 3-10, and 10-30 kDa fractions as well as in the gelatin peptides.

Chicken Heads as a Promising By-Product for Preparation of Food Gelatins.

Every year, the poultry industry produces a large number of by-products such as chicken heads containing a considerable proportion of proteins, particularly collagen. To prepare gelatin is one of the possibilities to advantageously utilize these by-products as raw materials. The aim of the paper was to process chicken heads into gelatins. An innovative method for conditioning starting raw material was using the proteolytic enzyme. Three technological factors influencing the yield and properties of extracted gelatins were monitored including the amount of enzyme used in the conditioning of the raw material (0.4% and 1.6%), the time of the conditioning (18 and 48 h), and the first gelatin extraction time (1 and 4 h). The gelatin yield was between 20% and 36%. The gelatin gel strength ranged from 113 to 355 Bloom. The viscosity of the gelatin solution was determined between 1.4 and 9.5 mPa.s. The content of inorganic solids varied from 2.3% to 3.9% and the melting point of the gelatin gel was recorded between 34.5 and 42.2 °C. This study has shown that gelatin obtained from chicken heads has a promising potential with diverse possible applications in the food industry, pharmacy, and cosmetics.

The anti-skin-aging effect of oral administration of gelatin from the swim bladder of Amur sturgeon (Acipenser schrenckii).

Gelatin was extracted from the swim bladder of Amur sturgeon with hot water at 50 °C with acceptable yield (76.54%) and it showed type I collagen features. The degree of hydrolysis reached 70.42%, and 26.55% of collagen peptides (380.76 Da) survived, after simulating digestion, absorption and peripheral blood circulation in vitro. In situ single-pass intestinal perfusion showed that the AMW of the collagen peptides was about 1100 Da in the intestine and they were mainly absorbed in the jejunum (61.11%). A 12-month feeding experiment using rats demonstrated that gelatin improved the histological structure, increased the thickness of the dermis (18.45%) and the density of collagen fibers (22.17%), and decreased the ratio of type III to type I collagen (43.44%) of chronologically aged skin at 3.85 g per kg bw per d. Moreover, the antioxidant enzyme activities in skin were significantly enhanced, while the malondialdehyde content was reduced by 31.99%. These results provided new insights into the development of new nutraceuticals and functional foods for the anti-skin-aging effect.

Physicochemical characterization and study of molar mass of industrial gelatins by AsFlFFF-UV/MALS and chemometric approach.

Industrial gelatins have different physicochemical properties that mainly depend of the raw materials origin and the extraction conditions. These properties are closely related to the molar mass distribution of these gelatins. Several methods exist to characterize molar mass distribution of polymer, including the Asymmetrical Flow Field Flow Fractionation method. The goal of this study is to analyze the relationship between physicochemical properties and the gelatins molar mass distribution obtained by Asymmetrical Flow Field Flow Fractionation. In this study, 49 gelatins samples extracted from pig skin are characterized in terms of gel strength and viscosity and their molar mass distribution are analyzed by Asymmetrical Flow Field Flow Fractionation coupled to an Ultraviolet and Multi Angle Light Scattering detector. This analytical method is an interesting tool for studying, simultaneously, the primary chains and the high-molar-mass fraction corresponding to the polymer chains. Correlation analysis between molar mass distribution data from the different fractions highlights the importance of high molar mass polymer chains to explain the gel strength and viscosity of gelatins. These results are confirmed by an additional chemometric approach based on the UV absorbance of gelatin fractograms to predict gel strength (r2Cal = 0.85) and viscosity (r2Cal = 0.79).

Enhancement of Cell Adhesion, Cell Growth, Wound Healing, and Oxidative Protection by Gelatins Extracted from Extrusion-Pretreated Tilapia (Oreochromis sp.) Fish Scale.

Gelatin has been broadly utilized in the food, pharmaceutical, photographic, cosmetic and packaging industries, and there is also huge potential for novel applications of gelatin in the fields of biotechnology and biomedicine. In the present study, we extracted gelatin from fish processing waste, i.e., scale of tilapia, by a combined method of extrusion-pretreatment and hot water extraction. The extrusion-pretreatment process increases the extraction yield of gelatin. Three gelatins (FS2: preconditioning with double-distilled water (ddH2O) before extrusion; FS12: preconditioning with citric acid solution before extrusion; FS14: preconditioning with acetic acid solution before extrusion) were obtained and all of them enhanced cell adhesion, cell growth, and wound healing in HaCaT cells and protected HaCaT cells from H2O2-induced cellular damage. Among FS2, FS12, and FS14, FS12 exhibited the most pronounced enhancement of cell adhesion, cell growth, and wound healing in HaCaT cells, and thus it may have potential as an effective natural raw material in cell therapies for cutaneous wounds and for reducing H2O2-induced oxidative damage of cells. In additional experiments, it was found that phosphorylations of Akt and mTOR are involved in the signaling pathway activated by FS2, FS12, and FS14 in HaCaT cells.

Characterization and Antioxidant and Angiotensin I-Converting Enzyme (ACE)-Inhibitory Activities of Gelatin Hydrolysates Prepared from Extrusion-Pretreated Milkfish (Chanos chanos) Scale.

Fish gelatin hydrolysates have been shown to possess various biological activities due to their unique Gly-Pro-Y and Gly-X-Hyp sequences. In the current study, fish gelatin was extracted from non-extruded milkfish scale (FSG1) or extrusion-pretreated milkfish scale (FSG2); extracted gelatins were hydrolyzed with different combinations of Flavourzyme and Alcalase to give four different hydrolysates, namely: FSGH1 (FSG1 hydrolyzed with Flavourzyme), FSGH2 (FSG1 hydrolyzed with Alcalase + Flavourzyme), FSGH3 (FSG2 hydrolyzed with Flavourzyme), and FSGH4 (FSG2 hydrolyzed with Alcalase + Flavourzyme). The extrusion-pretreatment process enhanced the extraction yield of gelatin from fish scale. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Fourier transform infrared (FTIR) analyses showed the extracts FSG1 and FSG2 possessed characteristics of gelatin. Moreover, the physicochemical characteristics of FSGH1⁻FSGH4 were examined by analyses of their degree of hydrolysis, amino acid composition, UV spectrum, FTIR spectrum, molecular weight, and RP-HPLC profile. Additional biological functional analyses showed that all of the studied gelatin hydrolysates FSGH1⁻FSGH4 possessed antioxidant activity dose-dependently as revealed by DPPH scavenging, ABTS scavenging, and reducing power analyses. In addition, FSGH2 and FSGH4 showed higher angiotensin-I-converting enzyme (ACE)-inhibitory activity as compared to FSGH1 and FSGH3. Taken together, FSGH2 and FSGH4 showed high antioxidant activity and potent anti-ACE activity. Due to the potential antioxidant and antihypertensive properties of FSGH2 and FSGH4, further research is needed to explore their possible use as natural supplementary raw materials in food and nutraceutical products.

Autolysis of bovine skin, its endogenous proteases, protease inhibitors and their effects on quality characteristics of extracted gelatin.

The autolysis of pretreated bovine skin (PBS) (treated with 0.1 M NaOH and 1% HCl), its endogenous proteases, inhibitors and their effects on quality attributes of gelatin were examined. PBS was subjected to different temperatures (20-90 °C) and pH (2-9) and treated with different protease inhibitors. Maximum autolytic activity of PBS was observed at 40 °C and pH 5. Ethylene-bis (oxyethylenenitrilo) tetraacetic acid (EGTA) was the most effective in impeding the degradation of γ-, ß- and α- chains of PBS protein indicating that metallocollagenases were the predominant endogenous proteases in bovine skin. Gelatin was extracted in the absence (GAE) and presence (GPE) of EGTA, and EGTA with papain enzyme (GPEP). GPEP had a higher yield and lower gel strength than GEA and GPE. Metallocollagenases partook in the degradation of gelatin thereby affecting its functional properties. Pretreating PBS with or without EGTA, and papain influenced the quality attributes of gelatin.

Atlantic salmon (Salmo salar) co-product-derived protein hydrolysates: A source of antidiabetic peptides.

Large quantities of low-value protein rich co-products, such as salmon skin and trimmings, are generated annually. These co-products can be upgraded to high-value functional ingredients. The aim of this study was to assess the antidiabetic potential of salmon skin gelatin and trimmings-derived protein hydrolysates in vitro. The gelatin hydrolysate generated with Alcalase 2.4L and Flavourzyme 500L exhibited significantly higher (p < 0.001) insulin and GLP-1 secretory activity from pancreatic BRIN-BD11 and enteroendocrine GLUTag cells, respectively, when tested at 2.5 mg/mL compared to hydrolysates generated with Alcalase 2.4L or Promod 144MG. The gelatin hydrolysate generated with Alcalase 2.4L and Flavourzyme 500L showed significantly more potent (p < 0.01) DPP-IV inhibitory activity than those generated with Alcalase 2.4L or Promod 144MG. No significant difference was observed in the insulinotropic activity mediated by any of the trimmings-derived hydrolysates when tested at 2.5 mg/mL. However, the trimmings hydrolysate generated with Alcalase 2.4L and Flavourzyme 500L exhibited significantly higher DPP-IV inhibitory (p < 0.05:Alcalase 2.4L and p < 0.01:Promod 144MG) and GLP-1 (p < 0.001, 2.5 mg/mL) secretory activity than those generated with Alcalase 2.4L or Promod 144MG. The salmon trimmings hydrolysate generated with Alcalase 2.4L and Flavourzyme 500L when subjected to simulated gastrointestinal digestion (SGID) was shown to retain its GLP-1 secretory and DPP-IV inhibitory activities, in addition to improving its insulin secretory activity. However, the gelatin hydrolysate generated with Alcalase 2.4L and Flavourzyme 500L was shown to lose GLP-1 secretory activity following SGID. A significant increase in membrane potential (p < 0.001) and intracellular calcium (p < 0.001) by both co-product hydrolysates generated with Alcalase 2.4L and Flavourzyme 500L suggest that both hydrolysates mediate their insulinotropic activity through the KATP channel-dependent pathway. Additionally, by stimulating a significant increase in intracellular cAMP release (p < 0.05) it is likely that the trimmings-derived hydrolysate may also mediate insulin secretion through the protein kinase A pathway. The results presented herein demonstrate that salmon co-product hydrolysates exhibit promising in vitro antidiabetic activity.

Investigation on complex coacervation between fish skin gelatin from cold-water fish and gum arabic: Phase behavior, thermodynamic, and structural properties.

The study is aimed to investigate phase behavior, thermodynamic, and structural properties based on complex coacervation between fish skin gelatin (FSG) from cold-water fish and gum arabic (GA). Phase separation behavior between FSG and GA was investigated as a function of pH through varying mixing ratios from 4:1 to 1:4 at 25 °C and 1.0 wt% of total biopolymer concentration. The turbidity of FSG-GA mixture reached the maximum (1.743) at the 1:2 of mixing ratio and pHopt 3.5, and stabilized at zero. Then physicochemical properties of FSG-GA coacervates at pHopt 3.5 and FSG-GA mixtures at pH 6.0 (>pHc) were evaluated. Scanning electron microscope (SEM) and X-ray diffraction (XRD) showed that the interactions between FSG and GA occurred at pHopt 3.5 and were very weak at pH 6.0 (>pHc). The isothermal titration calorimetry (ITC) results including the negative Gibbs free energy change (ΔG = -18.71 ±â€¯1.300 kJ/mol), binding enthalpy (ΔH = -41.81 ±â€¯1.300 kJ/mol) and binding entropy (TΔS = -23.10 kJ/mol) indicated that the complexation between FSG and GA was spontaneous and driven by negative enthalpy owing to the electrostatic interaction and hydrogen bondings. The zeta potential (ZP) of FSG-GA coacervates at pHopt 3.5 was -9.00 ±â€¯0.79 mV that was not close to electrically neutral, indicating other interactions besides electronic interaction. Hydrogen bondings in FSG-GA mixtures at pH 6.0 and 3.5 were found to be stronger than pure FSG at pH 6.0 and 3.5 owing to that the amide II peaks shifted to high wavenumbers. Electronic interaction was proven to exist in FSG-GA mixtures at pH 6.0 through the vanishment of asymmetric COO- stretching. However, the electronic interaction in FSG-GA coacervates at pHopt 3.5 was obviously stronger than FSG-GA mixtures at pH 6.0, resulting from the vanishment of asymmetric and symmetric COO- stretching vibration and the positively charged FSG and GA. The intrinsic fluorescence represented that the introduction of GA changed the microenvironment of tyrosine residues in FSG, which may be owing to the unfolding of the tertiary conformation. Moreover, the decrease of pH could promote the formation of random coils of FSG through circular dichroism (CD). Therefore the addition of GA into FSG and decrease of pH might enhance the conformation freedom of FSG, which would bring about favorable entropic effects and contribute to the complexation.