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Current IssueEditorial BoardOnline First ArticlesArchive

Full Research Article

Enhancing the Physicochemical Properties of Tuna Skin Gelatin Through Ultrasound-assisted Green Extraction: A Comparative Study

S
Syahriati1,*
https://orcid.org/ 0000-0001-5422-6089
I
Imran Muhtar1
R
Rahmawati Saleh1
J
Justus E. Loppies2
R
Rosniati2
S
Sitti Ramlah2
1Department of Agricultural Technology, Pangkep State Polytechnic of Agriculture, Jl. Poros Makassar Pare KM. 83 Mandalle, Pangkep,South Sulawesi(90655), Indonesia.
2Agroindustry Research Center, National Research and Innovation Agency, Serpong, South Tangerang 15314, Indonesia.

ABSTRACT

Background: Gelatin is widely used in food industry and other industries as an emulsifying and emulsifying agent. Ultrasound-assisted extraction (UAE) was used to produce gelatin from tuna skin to enhance its physicochemical properties and extraction efficiency. This study aimed to determine precise method produces gelatin with a better yield, gel strength, viscosity, color and other physicochemical properties.

Methods: Two extraction sequences were compared: ultrasound extraction followed by acid-base extraction (method A) and vice versa (method B). Using both methods, gelatin was extracted from tuna skin, with yields measured, color analyzed by lightness and ΔE and gel strength tested. Viscosity, moisture, protein contents and pH were assessed to understand gelatin quality.

Result: The result showed method B produced higher gelatin yield (13.34%) than method A (4.92%). Method B gelatin had greater gel strength (220 g vs. 200 g), brighter color (lower ÄE) and slightly higher viscosity (10 cP vs. 9 cP). SEM analysis revealed denser microstructure, supporting stronger gels. Significant differences occurred in yield, color and gel strength. Ultrasound-assisted acid-base extraction effectively produced high-quality tuna skin gelatin. This method offers potential applications in food, pharmaceutical and cosmetic industries, providing a sustainable alternative to mammalian gelatin.

INTRODUCTION

The global fish processing industry, significantly boosts seafood production. Indonesia contributes over 16% of the world’s tuna, producing more than 1.2 million tonnes annually, with exports of 198,131 tons valued at 659.99 million USD (PUSDATIN, 2018). Processing generates 30-40% by-products such as skin, bones, fins and entrails, which can be repurposed into valuable products including gelatin, collagen, bioactive peptides and lipids used in food, pharmaceuticals and cosmetics.
       
Gelatin, obtained through collagen hydrolysis, is valued for its gelling, biodegradable and non-toxic properties  (Song et al., 2024; Zhang and Wang, 2024). Mammalian sources dominate production, with porcine skin (80%) and bovine hides (15%) (Shah and Yusof, 2014). However, this raises ethical, health and religious issues such as halal certification and prion disease risks (Ahmed et al., 2020). Fish-based gelatin, particularly from tuna skin, provides a sustainable alternative, reducing land-based farming emissions (Prajaputra et al., 2024) and minimizing seafood waste (Sisa et al., 2024). Despite abundant raw material (Li et al., 2014), extraction is challenging due to collagen structure and inefficiency of conventional methods.
       
Recent technologies aim to improve gelatin extraction efficiency and sustainability. Ultrasound-assisted extraction (UAE) employs high-frequency sound waves to break collagen, reducing time, energy and chemical use (Xu et al., 2022). UAE yields gelatin with superior gel strength and thermal stability (Derkach et al., 2020; Chen et al., 2023), making it promising for fish-based applications. UAE can also help increase extraction yields, improved the physical, nutritional and functional properties (Patil et al., 2018; Adoni et al., 2025). Although less explored than enzymatic hydrolysis and supercritical CO2 extraction, UAE is simpler and more cost-effective. Enzymatic methods improve taste, emulsification and shelf life (Pakbin et al., 2022), while CO2 extraction produces high-purity gelatin with low residues (Idham et al., 2023), but both face high costs and complexity.
       
Fish gelatin has strong potential for pharmaceutical and cosmetic applications due to its biocompatibility, biodegradability and non-immunogenic properties, supporting drug delivery, controlled release and safer formulations (Al-Nimry  et al., 2021; Hussein et al., 2023). It also dissolves more easily at low temperatures than mammalian gelatin, although its characteristics vary by species, age and raw material composition (Zhang et al., 2020; Guo et al., 2023). This study investigates UAE for extracting gelatin from tuna skin to address existing research gaps. Ultrasound is known to improve protein functionality (Wang et al., 2023) and enhance gelatin stability and foaming capacity (Boughriba et al., 2022), yet applications using tuna skin remain limited. By comparing UAE with enzymatic and CO2  methods, this study evaluates the functional properties of tuna skin gelatin and assesses its alignment with Indonesian National Standard and GMIA specifications to strengthen its industrial potential.
 

MATERIALS AND METHODS

Tuna skin (Thunnus albacore) from a local fillet processor was cleaned before extraction due to its high collagen content. NaOH removed non-collagen proteins and CH3 COOH hydrolyzed collagen, producing gelatin with distilled water. Analytical-grade chemicals (Merck) supported pH, moisture, ash and protein analyses. Equipment included flasks, jars, petri dishes, stirrers, filter cloths and blenders. Extraction used an Elmasonic ultrasound system, with freeze-drying for gelatin. Physicochemical tests used Konica Minolta CR-400 (color), VR 3000 Viscometer (viscosity), Texture Analyzer TA-XT Plus (gel strength) and SEM FlexSEM 1000 II (microstructure).
 
Sample preparation and extraction procedures
 
Tuna skin was cleaned, washed with distilled water, cut into 1 x 1 cm2 pieces and stored at -20°C. Two extraction methods combined ultrasound-assisted extraction (UAE) and acid-base treatment. Method A: UAE in water (1:6 b/v, 20 kHz, 750 W, 55°C, 1 h), alkaline treatment with 0.2 M NaOH, then acid extraction with 0.20 M CH3COOH. Method B: alkaline, acid, then UAE. Extracted gelatin was centrifuged (6000 rpm, 10 min), freeze-dried (-50°C, 120 mBar, 48 h) and powdered for analysis.
 
Analysis procedures
 
Yield
 
The gelatin yield was determined by weighing the final gelatin powder after freeze-drying and comparing it with the initial raw tuna skin weight (AOAC, 2005).
 
Color
 
Gelatin powder color was measured using a Konica Minolta CR-400 Chroma Meter for L, a and b values (Cheow et al., 2007). These values indicate Lightness (L*), greenness (-a*), redness (+a*) and blueness/yellowness (±b*). Lighter gelatin is preferred for food and pharmaceutical applications.
 
Viscosity
 
The viscosity of gelatin was analyzed following (Rosti and Takagi, 2021). The powder was dissolved in distilled water at 60°C in a shaker bath. After complete dissolution, viscosity was measured using a Brookfield viscometer at 50°C at 10 rpm. Values were multiplied by the spindle conversion factor from the Bookfield manual (Brookfield Engineering Laboratories,  2002) to determine viscosity in centipoises (cP).
 
Gel strength
 
Gel strength was determined using a Texture Analyzer TA-XT Plus (Godalming, Surrey GU7 1YL, UK). A 6.67% gelatin solution was gelled at 4°C for 16 h. Bloom strength was measured by applying force until rupture.
 
Microstructure
 
Microstructural analysis of gelatin powder was conducted using SEM following Zhou et al., (2006). A 0.1 g gelatin sample was mounted on a specimen holder, cleaned and coated with gold or carbon to enhance conductivity. The sample was observed using a FlexSEM 1000 II at 500x magnification, a working distance of 6-10 mm and an accelerating voltage of 20 kV to obtain high-resolution images of the surface morphology.
 
Moisture content
 
Moisture content of tuna skin gelatin was determined gravimetrically by oven drying at 100°C for 6 h and expressed as percentage weight loss (AOAC, 2005).
 
Protein content
 
Protein content of tuna skin gelatin was analyzed using the Micro-Kjeldahl method (AOAC, 2005). A 0.2 g sample was digested with K2SO4, HgO and H2SO4 for 1-1.5 h until clear green. After cooling and dilution, the solution was distilled with NaOH, producing NH3 collected in boric acid. The distillate was titrated with HCl until green turned pink. Total nitrogen content was determined and protein content calculated using the standard formula (AOAC, 2005).
 
Gelatin pH
 
The pH of tuna skin gelatin was measured following GMIA (2019). A 6.67 g sample was dissolved in 100/mL water at 60°C, cooled to 25°C and measured with a calibrated pH meter.
 
Data analysis
 
The physical and chemical properties of tuna skin gelatin extracted using Method A (ultrasound-assisted extraction with acid-base treatment) and Method B (acid-base treatment with ultrasound-assisted extraction) were evaluated. A t-test at 95% confidence level compared the mean values of yield, moisture, ash, protein content and pH between the two methods.

RESULTS AND DISCUSSION

Yield of gelatin
 
The gelatin yield from tuna skin extracted using the acid-base method followed by ultrasound treatment (Method B) reached 13.34%, which was significantly higher than that obtained using ultrasound followed by acid–base extraction (Method A) at 4.92% (p<0.05). The higher yield in Method B indicates greater extraction efficiency, as gelatin yield reflects the effectiveness of the extraction process. This value is also higher than the 5.4% gelatin yield from Camel’s skin obtained using a base treatment with 2% Ca(OH)2 and 1% HCl followed by heating at 60°C for 6 hours (Al-Zoreky  et al., 2025). The increased yield is attributed to the acid–base treatment, which loosens the collagen matrix, followed by ultrasonic cavitation that accelerates collagen disruption and dissolution. These findings are consistent with reports by Ahmad et al., (2018), Rahman and Lamsal (2021) and Hoo et al., (2022), which indicate that ultrasound application after initial acid–base treatment enhances collagen extraction efficiency.
 
Color
 
Color indicates gelatin quality by showing purity levels. Gelatin from tuna skin using Method A had L* = 75.05, a* = 2.52, b* = 17.28 (ΔE = 24.01), while Method B showed L* = 80.32, a* = 0.46, b* = 7.97 (ΔE = 15.12), indicating higher purity and better contaminant removal. The gelatin’s appearance is shown in Fig 1.

Fig 1: Color appearance of tuna skin gelatin sheets and powder extracted by (a) ultrasound-acid-base and (b) acid-base-ultrasound method.


       
Color reflects gelatin purity and marketability. Method B produced higher Lightness (80.32) and lower ΔE (15.12) than Method A (L*: 75.05; ΔE: 24.01) (Table 1), indicating whiter, more purified gelatin with reduced redness and yellowness. Studies (Gunawan et al., 2017; Lin et al., 2015) note that gelatin color depends on raw materials, processing and impurity removal. Method B’s enhanced color results from effective acid-base pretreatment and ultrasound extraction, which facilitate pigment release and collagen breakdown.

Table 1: Gelatin color value of tuna skin (Thunnus albacore) from ultrasound extraction.


 
Viscosity
 
Viscosity is an important parameter determining the functional properties of gelatin in food and pharmaceutical applications. Gelatin extracted using method A had a viscosity of 9 cP, while method B produced 10 cP. Although both values remain below the GMIA standard (15-75 cP), they are closer to the British Standard (15-40 cP). The slightly higher viscosity in method B suggests that ultrasound-assisted extraction contributes to a denser molecular network, improving viscosity and gel strength, although the values are still lower than commercial gelatin. These differences are influenced by factors such as temperature, extraction time, acid concentration and chemical composition, indicating the need for further optimization of extraction conditions.
 
Gel strength
 
Gel strength measures gelatin firmness and texture. Method A produced gelatin with 200 g gel strength, while method B achieved 220 g. Method B’s stronger gel strength suggests acid-base extraction with ultrasound enables better collagen cross-linking. This is consistent with the findings of Hasdar et al., (2024), which found that the combination of acetic acid and ultrasound pretreatment was very effective in increasing gelatin strength. Gel strength is an essential parameter determining gelatin’s functional properties in food and pharmaceutical applications. Method B produced higher gel strength than method A, indicating that sequential acid-base extraction with ultrasound enhances cross-linking and yields stronger gels. Based on Bloom degree, gelatin is classified as low (<100 g), medium (100-200 g) and high (>200 g) (Usman et al., 2021; Adnan et al., 2024). Higher Bloom values improve stability, controlled release (Kuai et al., 2020), scaffold strength (Roldán  et al., 2024) and food texture (Obas et al., 2021). Determining factors include extraction method (Okur et al., 2020), chemical composition (Ahmad et al., 2021), molecular weight (Wang, 2024) and processing conditions (Zhang et al., 2021). SEM showed a denser structure in method B, supporting Li et al., (2024) that ultrasound enhances molecular integrity through cross-linking.
 
Microstructure
 
SEM analysis revealed distinct microstructures in the gelatin samples. Method A produced a porous, sponge-like structure with large cavities, while method B showed a denser structure with smaller cavities (Fig 2). Method B’s more uniform network contributed to better gel strength and texture, aligning with findings that ultrasound extraction enhances network compactness (He et al., 2021; Wang et al., 2023).

Fig 2: Microstructure of tuna skin gelatin (Thunnus albacore) obtained by ultrasound extraction followed by acid-base extraction method.


       
SEM analysis showed clear microstructural differences between methods. Method A produced porous gelatin with larger cavities, while method B yielded denser, compact gelatin with smaller cavities, resulting in higher gel strength. Gelatin with strong gels typically shows compact structures (Sae-leaw  et al., 2016). Cross-linking enhances collagen triple helix formation, strengthening networks (Zuev et al., 2024). Method B’s compact structure improved gel strength, confirming Li et al., (2024) that ultrasound-assisted extraction creates denser gelatin than heat extraction, producing superior texture and functional properties.
 
Chemical composition
 
The moisture content of gelatin from method A was 2.02%, while Method B produced 5.16% (Table 2), a significant difference (p<0.05). Both values complied with SNI 8622-2018 (£12%) and FAO/WHO standards (£18%), but fell below GMIA’s recommended 8-15% for food gelatin. Moisture content influences shelf life, texture and quality; excessive levels reduce lightness and cause off-flavors. Method B’s higher value is linked to acid-base extraction with ultrasound, enhancing collagen hydration and water-holding capacity, consistent with Li et al., (2024). These findings indicate process differences affect gelatin quality and that drying optimization may be needed for standard compliance.
       
In Table 2, the protein content of gelatin from method B reached 80.44%, significantly higher than that of method A which was 64.99% (p<0.05). This value approached the GMIA standard of 84-90% (GMIA, 2012), indicating greater efficiency in collagen extraction and conversion to gelatin. The higher protein level in method B resulted from sequential acid-base extraction combined with ultrasound treatment, which enhanced collagen solubilization. Protein content reflects final gelatin quality since gelatin is derived from collagen (Asmudrono et al., 2019; Gerungan et al., 2019). These findings align with previous studies (Khushboo, 2023; Ata  et al., 2023) confirming a direct correlation between protein levels and extraction efficiency.

Table 2: Chemical characteristics of tuna skin gelatin (Thunnus albacore) extracted using ultrasound.


       
The pH values of gelatin from methods A and B were 5.72 and 5.99 (Table 2), with no significant difference (p>0.05). Both values met the SNI 8622-2018 and GMIA standards of pH 3.8-7.5, indicating suitability for industrial use. Gelatin pH affects viscosity, gel strength, solubility and stability (Feng et al., 2023; Wang et al., 2023). Method B’s slightly higher pH (5.99) was associated with increased viscosity (10 cP) and gel strength (220 g), consistent with previous studies (Wang et al., 2023; Chen et al., 2023). Although the difference was minimal, the near-neutral pH values support good functional and application potential.
 
Implications and limitations
 
This study’s findings have important implications for gelatin extraction from fish skin. Method B, combining acid-base and ultrasound extraction, produces gelatin with higher yield, better colour and improved gel strength-crucial properties for food, pharmaceutical and cosmetic applications. However, the viscosity in both methods fell below GMIA standards, requiring parameter optimization. Further research into alternative techniques and industrial scale-up is needed to maintain consistent quality and yield.
 
Future research directions
 
Future studies should explore how ultrasonic power, temperature and time affect gelatin yield and quality, while developing enzymatic and sustainable extraction methods to enhance efficiency, environmental and economic sustainability.

CONCLUSION

Ultrasound-assisted extraction enhances tuna skin gelatin properties. Comparing ultrasound-acid-base (method A) and acid-base-ultrasound (method B), the latter consistently outperformed. Method B yielded higher output (13.34% vs. 4.92%), stronger gel, better color and compact microstructure. Although viscosity fell short of GMIA standards, method B produced stable gelatin for industrial use. This method effectively converts tuna skin, a sustainable by-product, into high-quality gelatin with potential in food, pharmaceuticals and cosmetics.
 

ACKNOWLEDGEMENT

The present study was supported by the Education Fund Management Institution (LPDP) for funding the Research and Innovation Program Grant for Advanced Indonesia Batch IV of the National Research and Innovation Agency, with contract numbers 138/IV/KS/11/2023 and 3876/PL22/KS/2023 of the Pangkep State Polytechnic of Agriculture.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Informed consent
 
The Committee of Experimental Animal Care approved all animal procedures for experiments and handling techniques.

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

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    Full Research Article

    Enhancing the Physicochemical Properties of Tuna Skin Gelatin Through Ultrasound-assisted Green Extraction: A Comparative Study

    S
    Syahriati1,*
    https://orcid.org/ 0000-0001-5422-6089
    I
    Imran Muhtar1
    R
    Rahmawati Saleh1
    J
    Justus E. Loppies2
    R
    Rosniati2
    S
    Sitti Ramlah2
    1Department of Agricultural Technology, Pangkep State Polytechnic of Agriculture, Jl. Poros Makassar Pare KM. 83 Mandalle, Pangkep,South Sulawesi(90655), Indonesia.
    2Agroindustry Research Center, National Research and Innovation Agency, Serpong, South Tangerang 15314, Indonesia.

    ABSTRACT

    Background: Gelatin is widely used in food industry and other industries as an emulsifying and emulsifying agent. Ultrasound-assisted extraction (UAE) was used to produce gelatin from tuna skin to enhance its physicochemical properties and extraction efficiency. This study aimed to determine precise method produces gelatin with a better yield, gel strength, viscosity, color and other physicochemical properties.

    Methods: Two extraction sequences were compared: ultrasound extraction followed by acid-base extraction (method A) and vice versa (method B). Using both methods, gelatin was extracted from tuna skin, with yields measured, color analyzed by lightness and ΔE and gel strength tested. Viscosity, moisture, protein contents and pH were assessed to understand gelatin quality.

    Result: The result showed method B produced higher gelatin yield (13.34%) than method A (4.92%). Method B gelatin had greater gel strength (220 g vs. 200 g), brighter color (lower ÄE) and slightly higher viscosity (10 cP vs. 9 cP). SEM analysis revealed denser microstructure, supporting stronger gels. Significant differences occurred in yield, color and gel strength. Ultrasound-assisted acid-base extraction effectively produced high-quality tuna skin gelatin. This method offers potential applications in food, pharmaceutical and cosmetic industries, providing a sustainable alternative to mammalian gelatin.

    INTRODUCTION

    The global fish processing industry, significantly boosts seafood production. Indonesia contributes over 16% of the world’s tuna, producing more than 1.2 million tonnes annually, with exports of 198,131 tons valued at 659.99 million USD (PUSDATIN, 2018). Processing generates 30-40% by-products such as skin, bones, fins and entrails, which can be repurposed into valuable products including gelatin, collagen, bioactive peptides and lipids used in food, pharmaceuticals and cosmetics.
           
    Gelatin, obtained through collagen hydrolysis, is valued for its gelling, biodegradable and non-toxic properties  (Song et al., 2024; Zhang and Wang, 2024). Mammalian sources dominate production, with porcine skin (80%) and bovine hides (15%) (Shah and Yusof, 2014). However, this raises ethical, health and religious issues such as halal certification and prion disease risks (Ahmed et al., 2020). Fish-based gelatin, particularly from tuna skin, provides a sustainable alternative, reducing land-based farming emissions (Prajaputra et al., 2024) and minimizing seafood waste (Sisa et al., 2024). Despite abundant raw material (Li et al., 2014), extraction is challenging due to collagen structure and inefficiency of conventional methods.
           
    Recent technologies aim to improve gelatin extraction efficiency and sustainability. Ultrasound-assisted extraction (UAE) employs high-frequency sound waves to break collagen, reducing time, energy and chemical use (Xu et al., 2022). UAE yields gelatin with superior gel strength and thermal stability (Derkach et al., 2020; Chen et al., 2023), making it promising for fish-based applications. UAE can also help increase extraction yields, improved the physical, nutritional and functional properties (Patil et al., 2018; Adoni et al., 2025). Although less explored than enzymatic hydrolysis and supercritical CO2 extraction, UAE is simpler and more cost-effective. Enzymatic methods improve taste, emulsification and shelf life (Pakbin et al., 2022), while CO2 extraction produces high-purity gelatin with low residues (Idham et al., 2023), but both face high costs and complexity.
           
    Fish gelatin has strong potential for pharmaceutical and cosmetic applications due to its biocompatibility, biodegradability and non-immunogenic properties, supporting drug delivery, controlled release and safer formulations (Al-Nimry  et al., 2021; Hussein et al., 2023). It also dissolves more easily at low temperatures than mammalian gelatin, although its characteristics vary by species, age and raw material composition (Zhang et al., 2020; Guo et al., 2023). This study investigates UAE for extracting gelatin from tuna skin to address existing research gaps. Ultrasound is known to improve protein functionality (Wang et al., 2023) and enhance gelatin stability and foaming capacity (Boughriba et al., 2022), yet applications using tuna skin remain limited. By comparing UAE with enzymatic and CO2  methods, this study evaluates the functional properties of tuna skin gelatin and assesses its alignment with Indonesian National Standard and GMIA specifications to strengthen its industrial potential.
     

    MATERIALS AND METHODS

    Tuna skin (Thunnus albacore) from a local fillet processor was cleaned before extraction due to its high collagen content. NaOH removed non-collagen proteins and CH3 COOH hydrolyzed collagen, producing gelatin with distilled water. Analytical-grade chemicals (Merck) supported pH, moisture, ash and protein analyses. Equipment included flasks, jars, petri dishes, stirrers, filter cloths and blenders. Extraction used an Elmasonic ultrasound system, with freeze-drying for gelatin. Physicochemical tests used Konica Minolta CR-400 (color), VR 3000 Viscometer (viscosity), Texture Analyzer TA-XT Plus (gel strength) and SEM FlexSEM 1000 II (microstructure).
     
    Sample preparation and extraction procedures
     
    Tuna skin was cleaned, washed with distilled water, cut into 1 x 1 cm2 pieces and stored at -20°C. Two extraction methods combined ultrasound-assisted extraction (UAE) and acid-base treatment. Method A: UAE in water (1:6 b/v, 20 kHz, 750 W, 55°C, 1 h), alkaline treatment with 0.2 M NaOH, then acid extraction with 0.20 M CH3COOH. Method B: alkaline, acid, then UAE. Extracted gelatin was centrifuged (6000 rpm, 10 min), freeze-dried (-50°C, 120 mBar, 48 h) and powdered for analysis.
     
    Analysis procedures
     
    Yield
     
    The gelatin yield was determined by weighing the final gelatin powder after freeze-drying and comparing it with the initial raw tuna skin weight (AOAC, 2005).
     
    Color
     
    Gelatin powder color was measured using a Konica Minolta CR-400 Chroma Meter for L, a and b values (Cheow et al., 2007). These values indicate Lightness (L*), greenness (-a*), redness (+a*) and blueness/yellowness (±b*). Lighter gelatin is preferred for food and pharmaceutical applications.
     
    Viscosity
     
    The viscosity of gelatin was analyzed following (Rosti and Takagi, 2021). The powder was dissolved in distilled water at 60°C in a shaker bath. After complete dissolution, viscosity was measured using a Brookfield viscometer at 50°C at 10 rpm. Values were multiplied by the spindle conversion factor from the Bookfield manual (Brookfield Engineering Laboratories,  2002) to determine viscosity in centipoises (cP).
     
    Gel strength
     
    Gel strength was determined using a Texture Analyzer TA-XT Plus (Godalming, Surrey GU7 1YL, UK). A 6.67% gelatin solution was gelled at 4°C for 16 h. Bloom strength was measured by applying force until rupture.
     
    Microstructure
     
    Microstructural analysis of gelatin powder was conducted using SEM following Zhou et al., (2006). A 0.1 g gelatin sample was mounted on a specimen holder, cleaned and coated with gold or carbon to enhance conductivity. The sample was observed using a FlexSEM 1000 II at 500x magnification, a working distance of 6-10 mm and an accelerating voltage of 20 kV to obtain high-resolution images of the surface morphology.
     
    Moisture content
     
    Moisture content of tuna skin gelatin was determined gravimetrically by oven drying at 100°C for 6 h and expressed as percentage weight loss (AOAC, 2005).
     
    Protein content
     
    Protein content of tuna skin gelatin was analyzed using the Micro-Kjeldahl method (AOAC, 2005). A 0.2 g sample was digested with K2SO4, HgO and H2SO4 for 1-1.5 h until clear green. After cooling and dilution, the solution was distilled with NaOH, producing NH3 collected in boric acid. The distillate was titrated with HCl until green turned pink. Total nitrogen content was determined and protein content calculated using the standard formula (AOAC, 2005).
     
    Gelatin pH
     
    The pH of tuna skin gelatin was measured following GMIA (2019). A 6.67 g sample was dissolved in 100/mL water at 60°C, cooled to 25°C and measured with a calibrated pH meter.
     
    Data analysis
     
    The physical and chemical properties of tuna skin gelatin extracted using Method A (ultrasound-assisted extraction with acid-base treatment) and Method B (acid-base treatment with ultrasound-assisted extraction) were evaluated. A t-test at 95% confidence level compared the mean values of yield, moisture, ash, protein content and pH between the two methods.

    RESULTS AND DISCUSSION

    Yield of gelatin
     
    The gelatin yield from tuna skin extracted using the acid-base method followed by ultrasound treatment (Method B) reached 13.34%, which was significantly higher than that obtained using ultrasound followed by acid–base extraction (Method A) at 4.92% (p<0.05). The higher yield in Method B indicates greater extraction efficiency, as gelatin yield reflects the effectiveness of the extraction process. This value is also higher than the 5.4% gelatin yield from Camel’s skin obtained using a base treatment with 2% Ca(OH)2 and 1% HCl followed by heating at 60°C for 6 hours (Al-Zoreky  et al., 2025). The increased yield is attributed to the acid–base treatment, which loosens the collagen matrix, followed by ultrasonic cavitation that accelerates collagen disruption and dissolution. These findings are consistent with reports by Ahmad et al., (2018), Rahman and Lamsal (2021) and Hoo et al., (2022), which indicate that ultrasound application after initial acid–base treatment enhances collagen extraction efficiency.
     
    Color
     
    Color indicates gelatin quality by showing purity levels. Gelatin from tuna skin using Method A had L* = 75.05, a* = 2.52, b* = 17.28 (ΔE = 24.01), while Method B showed L* = 80.32, a* = 0.46, b* = 7.97 (ΔE = 15.12), indicating higher purity and better contaminant removal. The gelatin’s appearance is shown in Fig 1.

    Fig 1: Color appearance of tuna skin gelatin sheets and powder extracted by (a) ultrasound-acid-base and (b) acid-base-ultrasound method.


           
    Color reflects gelatin purity and marketability. Method B produced higher Lightness (80.32) and lower ΔE (15.12) than Method A (L*: 75.05; ΔE: 24.01) (Table 1), indicating whiter, more purified gelatin with reduced redness and yellowness. Studies (Gunawan et al., 2017; Lin et al., 2015) note that gelatin color depends on raw materials, processing and impurity removal. Method B’s enhanced color results from effective acid-base pretreatment and ultrasound extraction, which facilitate pigment release and collagen breakdown.

    Table 1: Gelatin color value of tuna skin (Thunnus albacore) from ultrasound extraction.


     
    Viscosity
     
    Viscosity is an important parameter determining the functional properties of gelatin in food and pharmaceutical applications. Gelatin extracted using method A had a viscosity of 9 cP, while method B produced 10 cP. Although both values remain below the GMIA standard (15-75 cP), they are closer to the British Standard (15-40 cP). The slightly higher viscosity in method B suggests that ultrasound-assisted extraction contributes to a denser molecular network, improving viscosity and gel strength, although the values are still lower than commercial gelatin. These differences are influenced by factors such as temperature, extraction time, acid concentration and chemical composition, indicating the need for further optimization of extraction conditions.
     
    Gel strength
     
    Gel strength measures gelatin firmness and texture. Method A produced gelatin with 200 g gel strength, while method B achieved 220 g. Method B’s stronger gel strength suggests acid-base extraction with ultrasound enables better collagen cross-linking. This is consistent with the findings of Hasdar et al., (2024), which found that the combination of acetic acid and ultrasound pretreatment was very effective in increasing gelatin strength. Gel strength is an essential parameter determining gelatin’s functional properties in food and pharmaceutical applications. Method B produced higher gel strength than method A, indicating that sequential acid-base extraction with ultrasound enhances cross-linking and yields stronger gels. Based on Bloom degree, gelatin is classified as low (<100 g), medium (100-200 g) and high (>200 g) (Usman et al., 2021; Adnan et al., 2024). Higher Bloom values improve stability, controlled release (Kuai et al., 2020), scaffold strength (Roldán  et al., 2024) and food texture (Obas et al., 2021). Determining factors include extraction method (Okur et al., 2020), chemical composition (Ahmad et al., 2021), molecular weight (Wang, 2024) and processing conditions (Zhang et al., 2021). SEM showed a denser structure in method B, supporting Li et al., (2024) that ultrasound enhances molecular integrity through cross-linking.
     
    Microstructure
     
    SEM analysis revealed distinct microstructures in the gelatin samples. Method A produced a porous, sponge-like structure with large cavities, while method B showed a denser structure with smaller cavities (Fig 2). Method B’s more uniform network contributed to better gel strength and texture, aligning with findings that ultrasound extraction enhances network compactness (He et al., 2021; Wang et al., 2023).

    Fig 2: Microstructure of tuna skin gelatin (Thunnus albacore) obtained by ultrasound extraction followed by acid-base extraction method.


           
    SEM analysis showed clear microstructural differences between methods. Method A produced porous gelatin with larger cavities, while method B yielded denser, compact gelatin with smaller cavities, resulting in higher gel strength. Gelatin with strong gels typically shows compact structures (Sae-leaw  et al., 2016). Cross-linking enhances collagen triple helix formation, strengthening networks (Zuev et al., 2024). Method B’s compact structure improved gel strength, confirming Li et al., (2024) that ultrasound-assisted extraction creates denser gelatin than heat extraction, producing superior texture and functional properties.
     
    Chemical composition
     
    The moisture content of gelatin from method A was 2.02%, while Method B produced 5.16% (Table 2), a significant difference (p<0.05). Both values complied with SNI 8622-2018 (£12%) and FAO/WHO standards (£18%), but fell below GMIA’s recommended 8-15% for food gelatin. Moisture content influences shelf life, texture and quality; excessive levels reduce lightness and cause off-flavors. Method B’s higher value is linked to acid-base extraction with ultrasound, enhancing collagen hydration and water-holding capacity, consistent with Li et al., (2024). These findings indicate process differences affect gelatin quality and that drying optimization may be needed for standard compliance.
           
    In Table 2, the protein content of gelatin from method B reached 80.44%, significantly higher than that of method A which was 64.99% (p<0.05). This value approached the GMIA standard of 84-90% (GMIA, 2012), indicating greater efficiency in collagen extraction and conversion to gelatin. The higher protein level in method B resulted from sequential acid-base extraction combined with ultrasound treatment, which enhanced collagen solubilization. Protein content reflects final gelatin quality since gelatin is derived from collagen (Asmudrono et al., 2019; Gerungan et al., 2019). These findings align with previous studies (Khushboo, 2023; Ata  et al., 2023) confirming a direct correlation between protein levels and extraction efficiency.

    Table 2: Chemical characteristics of tuna skin gelatin (Thunnus albacore) extracted using ultrasound.


           
    The pH values of gelatin from methods A and B were 5.72 and 5.99 (Table 2), with no significant difference (p>0.05). Both values met the SNI 8622-2018 and GMIA standards of pH 3.8-7.5, indicating suitability for industrial use. Gelatin pH affects viscosity, gel strength, solubility and stability (Feng et al., 2023; Wang et al., 2023). Method B’s slightly higher pH (5.99) was associated with increased viscosity (10 cP) and gel strength (220 g), consistent with previous studies (Wang et al., 2023; Chen et al., 2023). Although the difference was minimal, the near-neutral pH values support good functional and application potential.
     
    Implications and limitations
     
    This study’s findings have important implications for gelatin extraction from fish skin. Method B, combining acid-base and ultrasound extraction, produces gelatin with higher yield, better colour and improved gel strength-crucial properties for food, pharmaceutical and cosmetic applications. However, the viscosity in both methods fell below GMIA standards, requiring parameter optimization. Further research into alternative techniques and industrial scale-up is needed to maintain consistent quality and yield.
     
    Future research directions
     
    Future studies should explore how ultrasonic power, temperature and time affect gelatin yield and quality, while developing enzymatic and sustainable extraction methods to enhance efficiency, environmental and economic sustainability.

    CONCLUSION

    Ultrasound-assisted extraction enhances tuna skin gelatin properties. Comparing ultrasound-acid-base (method A) and acid-base-ultrasound (method B), the latter consistently outperformed. Method B yielded higher output (13.34% vs. 4.92%), stronger gel, better color and compact microstructure. Although viscosity fell short of GMIA standards, method B produced stable gelatin for industrial use. This method effectively converts tuna skin, a sustainable by-product, into high-quality gelatin with potential in food, pharmaceuticals and cosmetics.
     

    ACKNOWLEDGEMENT

    The present study was supported by the Education Fund Management Institution (LPDP) for funding the Research and Innovation Program Grant for Advanced Indonesia Batch IV of the National Research and Innovation Agency, with contract numbers 138/IV/KS/11/2023 and 3876/PL22/KS/2023 of the Pangkep State Polytechnic of Agriculture.
     
    Disclaimers
     
    The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
     
    Informed consent
     
    The Committee of Experimental Animal Care approved all animal procedures for experiments and handling techniques.

    CONFLICT OF INTEREST

    The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

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