Property changes of tilapia (Oreochromis mossambicus) gelatin of low bloom value in response to a chemical modification using laccase and tea polyphenols

Abstract

Lower Bloom value of gelatin will cause insufficient gel strength. Both laccase and tea polyphenols were explored in this study to modify Bloom value, structure, and other properties of tilapia gelatin with low Bloom value (70.3 g). Using the selected conditions, two modified products thus prepared had higher Bloom values (90.1 and 112.5 g). Meanwhile, the results revealed that the two modified products were conjugated and crosslinked by tea polyphenols via four key reaction steps, contained protein aggregates of higher molecular weights, and had typical triple-helix and more ordered secondary structure. Compared with tilapia gelatin, the two modified products had reduced in vitro digestibility, decreased surface hydrophobicity, enlarged gelling and melting temperatures, larger particle sizes and zeta-potentials, and enhanced foaming properties. Moreover, higher increase of Bloom value of tilapia gelatin consistently caused much changes in these assessed properties. Collectively, this crosslinking strategy is applicable in valorizing gelatin quality and value.

Highlights

• •Tilapia gelatin treated by laccase and tea polyphenols has higher Bloom value.
• •The modified gelatin remains triple-helix and has much ordered secondary structure.
• •This modification causes reduced in vitro digestibility and surface hydrophobicity.
• •Gelling and melting temperature, colloidal, and foaming properties are also enhanced.

1. Introduction

Gelatin is a partially hydrolyzed form of collagen obtained mainly from the connective tissues (e.g. skin and bones) of animals, consists of a polydisperse mixture of water-soluble protein constitutes, usually exhibits molecular weights of 1.5–250 kDa, and has the collagen-specific glycine-X-Y repeating units (X and Y represent the unspecified amino acids). Gelatin is rich in glycine, proline, and hydroxyproline but lacks tryptophan, while high proportion of imino acids contributes to conformational stability of gelatin. Gelatin products are usually classified into the type A (acid-treated, isoelectric point 8–9) and type B (alkaline-treated, isoelectric point 4.8–5.2) gelatin, depending on the used processing conditions. As one of the widely used food ingredients, functional properties of gelatin are largely controlled by its molecular weight and imino acid content. Typical properties of gelatin in food is mainly associated with its gelling ability (i.e. gelation) (Glomm et al., 2025). Upon cooling, the disordered polypeptide chains of gelatin molecules in solution will partly reassociate into the triple-helix junctions and then generate the so-called thermo-reversible gels. However, gelatin gels can be melted upon heating. Gel strength, expressed as the Bloom value under the set conditions, is a critical quality indicator of various gelatin products (Gallego, Vázquez, Rodríguez, & Soto, 2025). Gelatin with higher Bloom value can form stronger and thermally stable gel networks and is applicable in jelly, yogurt, and restructured meat products. Thus, the producers of gelatin pay special attention to Bloom values of their gelatin products.

Gelatin also has other important and applicable properties in the food industry, for example, its interfacial properties. Gelatin molecules possess amphiphilic structure; subsequently, the hydrophobic and hydrophilic domains of gelatin can interact with respective lipids/gas and liquid/aqueous phases, generating interfacial films that are vital to the formation and stability of emulsions or foams. Gelatin thus can stabilize O/W emulsions across a broader pH range (Camaño-Echavarría et al., 2025), or enhance interfacial visco-elasticity and improve foam stability in the food products including ice cream (Etxabide et al., 2025). Totally, these gelatin properties are important to the food products including confectionery, yogurt, frozen desserts, and meat products. However, to overcome intrinsic limitation of gelatin products or improve gelatin property, researchers thus pay attention in gelatin modification, using various strategies to alter both gelatin structure and property.

Among these explored strategies of gelatin modification, the application of traditional chemical crosslinkers like glutaraldehyde is restricted by the concern over toxic residues (Zhu et al., 2024); however, enzymatic gelatin crosslinking has its significant promise, due to its mild reaction conditions, high catalytic specificity, and less safety risk. Transglutaminase (TGase) (EC 2.3.2.13) can induce the formation of ε-(γ-glutamyl)lysine isopeptide bonds in protein molecules. It was clarified that the TGase-induced crosslinking for gelatin-casein system enhanced rheological property and water-holding capacity (Wu, Liu, Liu, & Wang, 2017), increased intermolecular covalent interaction in gelatin hydrogels and caused higher thermal stability, elasticity, and water-holding capacity (Chen et al., 2022; Yang et al., 2024), and led to lower water vapor permeability in gelatin films (de Oliveira et al., 2024). Moreover, TGase and the −NH₂-contaning saccharides (e.g. glucosamine and oligochitosan) could be used to crosslink and glycate proteins, yielding glycated proteins with property changes (Song & Zhao, 2014). Laccase (EC 1.10.3.2), known as urushiol oxidases or p-diphenol oxidases, can oxidize phenolic compounds to form free radicals via its copper-based active site and using oxygen as electron acceptor. In the presence of phenolic compounds, the laccase-catalyzed protein crosslinking is effective in altering protein properties. For instance, it was evident that gelatin could react with tea polyphenols in the presence of laccase, resulting in enhanced heat resistance (Zhu et al., 2025). When casein was modified by laccase and ferulic acid (a phenolic acid), the crosslinked casein had enhanced emulsifying property (Sato, Perrechil, Costa, Santana, & Cunha, 2015). Moreover, casein could be crosslinked using laccase and catechins to enhance both mechanical property and antioxidation (Haratifar & Corredig, 2014). Clearly, this laccase-mediated crosslinking might be a possible way to improve gelatin properties, when considering the valorization of gelatin products with quality defect (Rigueto et al., 2023). It thus deserves an investigation using both laccase and tea polyphenols to improve gelatin quality including its important Bloom value, because such novel strategy is useful but still not clarified and developed.

During the industrial production of tilapia (Oreochromis mossambicus) gelatin, raw skin and bones are usually subjected to longer extracting times or many extracting numbers to ensure higher gelatin recovery. However, such treatment leads to excessive and undesired collagen decomposition. Tilapia gelatin thus prepared shows a poor quality feature known as low Bloom value, and has lower value. To achieve a valorization for the tilapia gelatin of low Bloom value, an enzymatic modification using laccase and tea polyphenols was thus explored in this study to enhance its Bloom value and other properties. The tilapia gelatin was thus investigated for its crosslinking conditions including laccase addition, tea polyphenols usage, and reaction time, using Bloom value as an indicator. After that, two modified products (namely modified gelatin I and modified gelatin II) were prepared and evaluated for their total polyphenol contents, structural features, in vitro digestibility, colloidal property reflected as particle size and zeta-potential, gelling and melting temperatures, surface hydrophobicity, and foaming property, using the unmodified gelatin as a control. This study aimed to explore an novel enzymatic approach to valorize gelatin quality and value, confirming application potential of this approach in the modification of gelatin or other proteins.

2. Materials and methods

2.1. Materials and chemicals

Tilapia gelatin, measured with protein content of 995.5 g/kg and Bloom value of 70.3 g, was supplied by Guangdong Mingyang Gelatin Co., Ltd. (Maoming, Guangdong Province, China). Laccase and tea polyphenols were purchased from Wuhan Saviore Biotechnology Co., Ltd. (Wuhan, China) and Zhejiang Oriental Tea Technology Co., Ltd. (Quzhou, Zhejiang Province, China), respectively. Coomassie brilliant blue R-250 was obtained from Sigma-Aldrich (St. Louis, MO, USA), while protein markers (molecular weights of 10–180 kDa) were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Refined soybean oil was provided by COFCO Beihai Grain & Oil Industry (Tianjin) Co., Ltd. (Tianjin, China). All chemicals used in this study were of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China), while the water used in whole study was distilled water.

2.2. Selection of crosslinking condition and preparation of the samples

Crosslinking conditions of tilapia gelatin, including laccase addition, tea polyphenols usage, and reaction time, were selected through an investigation using the orthogonal array design, while the three conditions were all investigated at three levels. Briefly, tilapia gelatin was dissolved in water to generate a gelatin solution of 100 g/L (pH 7.5), mixed with tea polyphenols (0.5, 2.5, and 5.0 g/kg gelatin), and then added with laccase (5, 10, and 15 kU/kg gelatin). The mixture was kept at 45 °C with a gentle agitating for the set reaction times (1, 2, and 3 h). For the control gelatin, none of laccase and tea polyphenols was used. After finishing the reaction, the mixture was heated at 90 °C to inactive laccase, cooled, and then lyophilized. The product thus obtained was washed with 75 % (v/v) ethanol for three times to remove the unreacted or free tea polyphenols, dried at 80 °C to remove ethanol residue, yielding the modified products. All modified products were evaluated for their Bloom values, which were used in the condition selection.

Based on the results from the orthogonal array design, two modified products with different Bloom values were prepared. In detail, modified gelatin I was prepared using laccase addition of 10 kU/kg gelatin, tea polyphenols usage of 2.5 g/kg gelatin, and reaction time of 3 h, while modified gelatin II was prepared using laccase addition of 15 kU/kg gelatin, tea polyphenols usage of 5 g/kg gelatin, but a shorter reaction time of 2 h. The two modified products were also measured for their Bloom values.

2.3. Analyses of protein content, total polyphenol content, and bloom value

In this study, protein content was determined using the Kjeldahl method and a specified conversion factor of 5.50, as recommended by the method of AOAC 984.13 (AOAC, 2005). Meanwhile, total polyphenol content was determined as previously described (Ma, Zhang, & Zhao, 2022), using the Folin-Ciocalteu reagent, an UV–visible spectrophotometer (UV-2600, Shimadzu Corporation, Kyoto, Japan), and gallic acid as a standard. The results were expressed as gallic acid equivalent (g)/kg protein.

When measuring Bloom value, the sample of 7.5 g was weighed into a standard Bloom jar, added with 105 mL water, and kept at 20 °C for 1 h to ensure complete gelatin swelling. The sample was heated at 65 °C for 15 min to achieve full gelatin dissolution. The jar was thus sealed with rubber stopper and held at 10 °C for 17 h to ensure full gelatin gelation. After that, a TA.XT texture analyzer (Brookfield CT3 Texture Analyzer, Middleboro, MA, USA), equipped with a 1.27 cm diameter cylindrical probe, was employed to detect Bloom value via using a crosshead speed of 1.0 mm/s and a trigger force of 0.1 N. The maximum force required for the probe to penetrate 4 mm into the gel surface was recorded as the Bloom value, as previous described (Liu, Lai, Muhoza, & Xia, 2021).

2.4. SDS-PAGE analysis

SDS-PAGE analysis was performed according to the reported method (Cheng, Wang, Zhang, Zhai, & Hou, 2021). The sample was prepared at a protein content of 5 g/L, while the protein markers with molecular weights of 10–180 kDa were also used in this analysis. After finishing electrophoresis, the gels were fixed in methanol/acetic acid/water (2:1:7, v/v/v) for 50 min, stained with Coomassie brilliant blue R-250 solution for 2.5 h, and destained in methanol/acetic acid/water (1:1:8, v/v/v) until clear bands were observed. The gels were then imaged using a gel documentation system (DYCZ-GelDoc, Beijing Municipality, Beijing, China) to generate the electrophoresis picture.

2.5. Determination of infrared, fluorescence, and circular dichroism spectra

To obtain Fourier transform infrared (FT-IR) spectrum, a mixture containing the sample of 2 mg and sieved KBr of 200 mg was used to prepare a transparent pellet. The pellet was mounted in the sample holder of a Fourier transform infrared spectrometer (Nicolet iN10, Thermo Fisher Scientific Inc., Waltham, MA, USA) and scanned at 600–2000 cm⁻¹ using a resolution of 1 cm⁻¹ and 32 scans, as previously described (Kuai, Liu, Ma, Goff, & Zhong, 2020).

To obtain fluorescence spectrum, the sample was dispersed in the phosphate-buffered saline (PBS) of 10 mmol/L to reach final protein concentration of 0.2 g/L and pH 7.0, and measured at a F-7000 spectrofluorometer (Hitachi High-Technologies, Tokyo, Japan) using excitation wavelength of 279 nm, emission range of 300–500 nm, and slit width of 5 nm.

Circular dichroism (CD) spectrum was recorded using a J-810 spectropolarimeter (Jasco, Tokyo, Japan) and 0.1 g/L protein concentration. CD spectra of the samples were collected at 20 °C and 4 °C over a wavelength range of 190–240 nm, using spectral resolution of 0.5 nm, scanning speed of 100 nm/min, response time of 0.25 s, and bandwidth of 1 nm, as previously described (Kuai et al., 2020).

2.6. Evaluation of in vitro digestibility

In vitro digestibility was determined by the simulated gastric and intestinal digestion, using the reported procedures (Ma et al., 2022). The sample was prepared at a protein concentration of 10 g/L in 50 mmol/L PBS (pH 2.0). In the peptic digestion, sample solution of 10 mL was incubated with 2 mg pepsin (EC 3.4.23.1) at 37 °C for 2 h, while the digestion was terminated by adding 10 mL trichloroacetic acid (200 g/L), followed by a centrifugation at 10,000 ×g for 20 min to collect the supernatant. In the peptic-tryptic digestion, sample solution of 10 mL was firstly digested with 2 mg pepsin at 37 °C for 1 h, and heated at 90 °C for 5 min to terminate pepsin. The digested solution was then adjusted to pH 8.0, lyophilized, re-dissolved in 10 mL PBS (50 mmol/L, pH 8.0), and incubated with 6 mg trypsin (EC 3.4.21.4) at 37 °C for 1 h. The tryptic digestion was also stopped by adding trichloroacetic acid (200 g/L), while the mixture was centrifuged at 10,000 ×g for 20 min to obtain the supernatant.

The collected supernatant was divided into two portions, which were subjected into both UV spectrophotometric and protein assays as previously described (Ma et al., 2022). One portion was diluted fourfold with water and measured at 280 nm using the UV–visible spectrophotometer. UV absorbance value was normalized against a fully hydrolyzed reference (set as 100 % digestibility) to calculate in vitro digestibility of the sample. Another portion was analyzed for nitrogen content using the Kjeldahl method, and then estimated for in vitro digestibility as previously described (Ma et al., 2022).

2.7. Measurements of particle size and zeta-potential

Average particle size and zeta-potential of the sample were determined by the static light scattering at 25 °C, while the refractive index of water and protein particles were set at 1.33 and 1.42, respectively (Mehrabi-Khozani, Sarabandi, & Rezaei, 2025). The sample was dispersed in 10 mmol/L PBS (pH 7.0) to a final protein concentration of 1 g/L, and then measured at the NanoTrac Wave II (Microtrac MRB, Montgomeryville, PA, USA).

2.8. Determination of gelling and melting temperatures

Each sample was dissolved in water to generate a solution of 66.7 g/L, and determined using a rheometer (Kinexus Pro+, Malvern Panalytical Ltd., Worcestershire, UK) that consisted of a 60 mm cone-plate geometry with a gap and tilt angle of 1.0 mm and 0.5 °C, respectively. This determination was conducted at a frequency of 1 Hz with an applied strain of 0.5 %, detecting both storage modulus (G′) and loss modulus (G′′) values. Sample solution was transferred to the rheometer and cooled to induce gel formation. After that, the gels were heated to 40 °C, and the temperature was decreased from 40 °C to 4 °C at a rate of 2 °C/min to monitor cooling behavior of the solution. Annealing was conducted by maintaining the gels at 4 °C for 1 h to ensure network stabilization, while a heating stage using a temperature sweep from 4 °C to 40 °C at 2 °C/min was applied to monitor melting behavior of the gels. Gelling and melting temperatures were thereof calculated when tan δ (G′′/G′) = 1, as a previous study suggested (Sanprasert et al., 2025).

2.9. Analyses of surface hydrophobicity and foaming property

The sample solutions (0.2–1.0 g/L, 2 mL) in 50 mmol/L PBS (pH 7.0) were mixed with 20 μL 1-anilino-8-naphthalene sulfonic acid solution (8.0 mmol/L). The mixtures were incubated in the dark for 30 min. Fluorescence intensity was measured using the fluorescence spectrophotometer, excitation wavelength of 390 nm, and emission wavelength of 470 nm. Surface hydrophobicity of the sample was determined from the initial slope of a curve (fluorescence intensity plotted against protein concentration), as a reference study described (Wang & Wang, 2025).

Foaming capacity and stability of the sample were measured by a volume method. Briefly, 10 mL sample solution (66.7 g/L) was homogenized using a Type FA2E high-speed homogenizer (FLUKO, Shanghai, China) at 12,000 rotation/min for 1 min. After that, the sample was moved into a graduated cylinder (50 mL), while total volume (protein solution plus foam) was immediately recorded. After standing for 10 min, total volume (protein solution plus foam) was recorded again. Foaming capacity and stability were thus estimated as previously described (Du et al., 2025).

2.10. Statistical analysis

All experiments and analyses were performed three times, while the data were reported as means or means ± standard deviations. The one-way analysis of variance was used to evaluate the differences between mean values of multiple groups at the significance level of p < 0.05, using the SPSS 20.0 software (SPSS Inc., Chicago, IL, USA).

3. Results and discussion

3.1. The laccase-induced gelatin crosslinking and increase of bloom value

Using the orthogonal array design and selected crosslinking conditions, nine modified gelatin products were prepared and measured with higher Bloom values than the unmodified (i.e. control) gelatin (77.6–112.5 versus 70.3 g) (Table 1). The used modification treatments thus caused about 10 %–60 % increases in Bloom value. Based on these data (Table 1), a regression equation (Equation I) was thus established, reflecting the relationship between Bloom value of the modified gelatin and the used three crosslinking conditions (i.e. laccase addition, tea polyphenols usage, and reaction time) (R² = 0.947). This equation suggested that when higher values of the three reaction conditions were used in gelatin modification, they consistently led to higher Bloom value for the modified product. However, data analysis results also pointed out that both laccase addition (p < 0.01) and tea polyphenols usage (p < 0.05) were the most important factors for gelatin crosslinking, while reaction time (p > 0.05) was less important in gelatin crosslinking.

$$\mathit{Bloom\; value}\left(g\right)=56.92+2.67\times \mathit{laccase\; addition}\left(\mathit{kU}/\mathit{kg}\mathit{gelatin}\right)+2.05\times \mathit{tea}\mathit{polyphenols\; usage}\left(g/\mathit{kg}\mathit{gelatin}\right)+0.85\times \mathit{reaction\; time}\left(h\right)$$ (I)

Table 1.

Bloom values of the modified products in response to the used laccase addition, reaction time, and tea polyphenols (TP) addition.

Experimental number	Laccase addition (kU/kg gelatin)	Reaction time (h)	TP addition (g/kg gelatin)	Bloom value (g)
1	5	1	5	80.8
2	5	2	0.5	78.0
3	5	3	2.5	77.6
4	10	1	0.5	81.3
5	10	2	2.5	89.2
6	10	3	5	93.6
7	15	1	2.5	104.0
8	15	2	5	112.5
9	15	3	0.5	100.0

If using laccase addition of 10 kU/kg gelatin, tea polyphenols usage of 2.5 g/kg gelatin, and reaction time of 3 h to perform gelatin crosslinking, the targeted modified gelatin I was estimated with Bloom value of 91.3 g. When using laccase addition of 15 kU/kg gelatin, tea polyphenols usage of 5 g/kg gelatin, and reaction time of 2 h to perform gelatin cross-linking, the targeted modified gelatin II was estimated with Bloom value of 108.9 g. Actual evaluation results showed that modified gelatin I and modified gelatin II had respective Bloom values of 90.1 and 112.5 g. This fact suggested briefly that this equation could be used to predict Bloom value of the modified gelatin in response to the applied crosslinking conditions. Compared with modified gelatin I, modified gelatin II was modified with much higher enzyme addition and tea polyphenols usage but a shorter reaction time, totally had greater crosslinking extent, and thus was measured with larger Bloom value.

Meanwhile, the obtained SDS-PAGE analysis results indicated that the unmodified gelatin, modified gelatin I and modified gelatin II had some differences in the distribution of peptide fragments (Fig. 1a). Compared with the unmodified gelatin (lane 1), both modified gelatin I (lane 3) and modified gelatin II (lane 2) showed darker staining in the upper band region of the stacking gels, indicating the presence of some protein aggregates with much higher molecular weights. In addition, both modified gelatin I and modified gelatin II also had weaker staining in the band region with molecular weights of 15–70 kDa than the unmodified gelatin. Thus, it was confirmed by the SDS-PAGE results that laccase in the presence of tea polyphenols induced gelatin crosslinking, causing the formation of protein aggregates with higher molecular weights and then endowing the modified products with enlarged Bloom vales.

Fig. 1.

Peptide distribution profiles of protein markers (lane M), tilapia gelatin (lane 1), modified gelatin II (lane 2), and modified gelatin I (lane 3) after the performed SDS-PAGE analysis (a), as well as a suggested reaction mechanism for the laccase-induced gelatin crosslinking (b).

Laccase has low substrate specificity and thus can oxidize a large number of phenolic molecules including those from tea polyphenols. During the gelatin crosslinking, the oxidative action of laccase converted phenolic –OH groups of the suitable components in tea polyphenols (e.g. epigallocatechin) into the highly reactive electrophilic intermediates (i.e. quinones), based on the identified catalysis mechanism of laccase (Isaschar-Ovdat & Fishman, 2018). After then, these intermediates reacted with the nucleophilic −NH₂ groups from gelatin molecules, yielding the formation of covalent C—N bonds and subsequently covalent crosslinking of gelatin molecules. A reaction mechanism of the laccase-induced gelatin crosslinking in the presence of tea polyphenols, which involves in four key reaction steps namely two oxidative reactions and two addition reactions, was proposed accordingly (Fig. 1b). The modified products thus contained protein aggregates with higher molecular weights. Whenever the modified products received greater crosslinking extent, they possessed more protein aggregates and thereof showed higher Bloom values (modified gelatin II versus modified gelatin I). Shared a conclusion similarity to this study, a previous study indicated that laccase in the presence of feurlic acid could induce casein crosslinking and then cause an improvement in gelling and rheological properties (Ercili-Cura et al., 2009). Additionally, another study also stated that laccase modification of pea protein by chlorogenic acid (a phenolic acid) yielded the formation of protein aggregates with high molecular weights (Yi, Chen, Wen, & Fan, 2024). It was reasonable that laccase and tea polyphenols were able to induce gelatin crosslinking through the suggested mechanism (Fig. 1b), forming some gelatin aggregates in the two modified products and subsequently altering their Bloom values and other properties.

3.2. Effect of laccase-induced crosslinking on structural features of tilapia gelatin

Our analysis results also showed that the unmodified gelatin had very low total polyphenol content (0.07 g gallic acid equivalent/kg protein), while modified gelatin I and modified gelatin II showed much higher total polyphenol contents of 0.512 and 0.746 g gallic acid equivalent/kg protein, respectively. Increased total polyphenol contents in modified gelatin I and modified gelatin II suggested the covalent conjugation of tea polyphenols into gelatin molecules directly. Meanwhile, the obtained FT-IR spectra showed that the unmodified gelatin, modified gelatin I, and modified gelatin II totally shared similar IR features in the investigated wavenumber range (Fig. 2a). However, it was also observed that both modified gelatin I and modified gelatin II had a slight higher absorption peak about 1080 cm⁻¹ than the unmodified gelatin, as the arrows indicated. Based on fundamental IR knowledge, the stretching vibration of C—O bonds causes IR absorption near 1100 cm⁻¹. This meant that both modified gelatin I and modified gelatin II had more C—O bonds in their molecules than the unmodified gelatin. This fact suggested a covalent conjugation of tea polyphenols into gelatin molecules, because the unreacted tea polyphenols were removed by the conducted ethanol washing. Moreover, the fluorescence analysis results revealed that modified gelatin I and especially modified gelatin II exhibited much higher fluorescence intensity than the unmodified gelatin (Fig. 2b), demonstrating that the two modified products (especially modified gelatin II) had more aromatic rings (contributed by the conjugated tea polyphenols). In consistent with the analysis results of total polyphenol content, both FT-IR and fluorescence analysis results also confirmed the laccase-induced conjugation of tea polyphenols into gelatin molecules.

Fig. 2.

Fourier transform-infrared (a) and fluorescence spectra (b) of tilapia gelatin, modified gelatin I, and modified gelatin II.

The results from CD analysis at two temperatures (Fig. 3) declared that modified gelatin I and especially modified gelatin II had the same and different structural features, compared with the unmodified gelatin. All three samples at 4 °C had triple-helix structure, because they all showed negative ellipticity at 190–200 nm but positive ellipticity around 225 nm (Fig. 3a), which are the classic indicators of triple-helix structure of gelatin molecules. If the samples were evaluated at 20 °C, their triple-helix structure was decomposed; meanwhile, they were detected with negative ellipticity only (Fig. 3b). At the environment of 20 °C, the unmodified gelatin and modified gelatin II had the respective highest and lowest negative ellipticity around 200 nm, the typical index of random coil (i.e. disordered secondary) structure. That is, the unmodified gelatin had more disordered structure, while modified gelatin II had less disordered secondary structure. Also, modified gelatin II was measured with lower negative ellipticity and thus had less disordered secondary structure than modified gelatin I. It was thus concluded that the used crosslinking caused less disordered (i.e. much ordered) secondary structure in two modified products, and higher crosslinking extent led to much ordered secondary structure.

Fig. 3.

Circular dichroism spectra of tilapia gelatin, modified gelatin I, and modified gelatin II measured at 4 °C (a) and 20 °C (b).

In theory, laccase in the presence of oxygen induces the oxidization of a wide variety of phenolic compounds (Kurniawati & Nicell, 2009), including diphenols and polyphenols. When tea polyphenols were converted into reactive quinones, they could react covalently with the –NH₂ groups from protein molecules. The modified products thereby received a covalent conjugation of tea polyphenols, and had higher levels of C—O bonds and aromatic rings in their structure than unmodified gelatin. Increased total polyphenol content, higher IR absorption peak at 1080 cm⁻¹, and greater fluorescence intensity around 310 nm were thus observed in the two modified products. Additionally, a previous study pointed out that the horseradish peroxidase-induced crosslinking of two milk protein products caused an ordered secondary structure (Zhang, Wang, Liu, & Zhao, 2016), while another study indicated that an application of chemical modification of this type in bovine gelatin also led to gelatin crosslinking and much ordered secondary structure (Han & Zhao, 2016). These results thus supported present finding; that is, the present modification induced the conjugation of tea polyphenols and endowed the two modified products with an ordered secondary structure.

3.3. Effect of laccase-induced crosslinking on in vitro digestion of tilapia gelatin

When the unmodified gelatin, modified gelatin I, and modified gelatin II were evaluated for in vitro digestion, the results suggested that the laccase-induced crosslinking led to reduced in vitro digestibility for modified gelatin I especially modified gelatin II (Table 2). When the Kjeldahl method was used in protein assaying, modified gelatin I and modified gelatin II showed 40.2 %–55.1 % peptic digestibility or 60.0 %–70.4 % peptic-tryptic digestibility; meanwhile, the unmodified gelatin had 73.3 % peptic digestibility or 85.2 % peptic-tryptic digestibility. Higher crosslinking extent also led to lower in vitro digestion (modified gelatin II versus modified gelatin I). Moreover, the data comparison results clearly pointed out that the digestibility values obtained from the Kjeldahl method were always lower than those obtained from the UV spectrophotometric method. It was regarded that the UV spectrophotometric method was not suitable in evaluating the in vitro digestibility of the phenol/polyphenol-conjugated proteins, because phenolic substances theoretically have stronger UV absorption at 280 nm and thus cause an unnegligible interference on the UV analysis results of protein digestion. Subsequently, the UV analysis results gave higher (but incorrect) protein amount in the peptic or peptic-tryptic digests, while in vitro digestibility value was overestimated. The present results proved again that the Kjeldahl method can detect protein amount without polyphenol interference precisely, and is suitable in evaluating digestibility values of the phenol/polyphenol-conjugated proteins; otherwise, an application of the UV spectrophotometric method might yield incorrect result and probably wrong conclusion.

Table 2.

In vitro digestibility (%) of tilapia gelatin, modified gelatin I, and modified gelatin II measured with the Kjeldahl or UV methods.

	Digestive method	In vitro digestibility (Kjeldahl method)	In vitro digestibility (UV method)
Tilapia gelatin	Peptic digestion	73.3 ± 1.1d	76.6 ± 1.0C
	Peptic-tryptic digestion	85.2 ± 1.8e	90.1 ± 1.4D
Modified gelatin I	Peptic digestion	55.1 ± 1.0b	65.2 ± 1.3B
	Peptic-tryptic digestion	70.4 ± 1.6d	77.0 ± 1.1C
Modified gelatin II	Peptic digestion	40.2 ± 0.8a	49.2 ± 0.9 A
	Peptic-tryptic digestion	60.0 ± 1.1c	66.1 ± 1.3B

Different lowercase or capital letters after the values as superscripts in the same column indicate that one-way ANOVA of the means differs significantly (p < 0.05).

When proteins are crosslinked with polyphenols, the conjugated polyphenols will block off some digestive sites in protein structure, which are susceptible to digestive enzymes. Subsequently, the proteins have less decomposition of peptide bonds upon their exposure to pepsin, trypsin, and other proteases, and thereof exhibit reduced in vitro digestion. For instance, both myoglobin and whey proteins conjugated by phenols or polyphenols were measured with reduced enzymatic digestion (Kroll, Rawel, & Seidelmann, 2000; Rawel, Kroll, & Hohl, 2001). Similarly, it was also reported that the flavonol-conjugated casein had decreased in vitro digestion than original casein (Ma et al., 2022). More importantly, Ma and coauthors confirmed that the UV spectrophotometric method was not applicable in measuring in vitro digestibility of the conjugated casein (Ma et al., 2022). The present results thus shared conclusion consistence with these reported results. Fortunately, gelatin has poor nutrition value in the body, and is not the main source of dietary proteins. Reduced digestibility of the modified gelatin might not impact nutrition value of the processed foods containing the modified gelatin. Moreover, reduced digestibility of the modified gelatin is advantageous for its application in developing the controlled-release matrices, encapsulation system, and functional coatings, in which a slower rate of protein degradation is desirable.

3.4. Effect of laccase-induced crosslinking on particle size and zeta-potential of tilapia gelatin in solution

Analysis results showed that the laccase-induce crosslinking led to enlarged particle sizes and zeta-potentials for modified gelatin I and modified gelatin II (Table 3). Compared with the unmodified gelatin, modified gelatin I and modified gelatin II had larger average particle sizes (139.1 versus 202.0 and 264.6 nm) and greater zeta-potentials (−12.92 versus − 20.11 and − 34.51 mV). Consistently, modified gelatin II had higher index values than modified gelatin I. This fact suggested that an application of this enzymatic modification on tilapia gelatin promoted intermolecular aggregation, and then caused larger particle sizes and greater zeta-potentials. The two modified products thus received enhanced colloidal property. Reasonably, modified gelatin II had higher crosslinking extent and thus was measured with higher index values. The present results thus confirmed that this laccase-induced modification was effective in improving colloidal property of tilapia gelatin.

Table 3.

Measured particle sizes and zeta potentials of tilapia gelatin, modified gelatin I, and modified gelatin II.

	Tilapia gelatin	Modified gelatin I	Modified gelatin II
Average particle size (nm)	139.1 ± 3.3c	202.0 ± 3.0b	264.6 ± 1.0a
Zeta potential (mV)	−(12.92 ± 0.76)C	−(20.11 ± 1.13)B	−(34.51 ± 1.26)A

Different lowercase or capital letters after the values as superscripts in the same row indicate that one-way ANOVA of the means differs significantly (p < 0.05).

In general, chemical modification of food proteins can alter their colloidal property, especially using particle size and zeta-potential as two evaluation indicators. For example, a crosslinked bovine gelatin by horseradish peroxidase showed larger zeta-potential value than bovine gelatin (Han & Zhao, 2016), while a crosslinked soy protein by TGase had increased average particle sizes (101.0 versus 82.9 nm) and enlarged zeta-potential (−30.7 versus − 27.7 mV) than the unmodified soy protein (Fu & Zhao, 2017). Additionally, Han and coauthor also proved that the cross-linking of gelatin by horseradish peroxidase induced higher colloidal stability (Han & Zhao, 2016). Clearly, these mentioned results supported strongly that the two modified products (especially modified gelatin II) displayed better colloidal behavior in solution, reflected by their enlarged particle sizes and zeta-potentials.

3.5. Effect of laccase-induced crosslinking on gelling and melting temperatures of tilapia gelatin

The unmodified gelatin, modified gelatin I, and modified gelatin II in solutions were also monitored for their viscoelastic behavior upon a cooling or heating process. Overall, the monitored moduli (i.e. G′ and G′′) showed clear response to the applied temperature changes, and were determined with different transition sites (Fig. 4). Based on these results, it was estimated that the unmodified gelatin, modified gelatin I, and modified gelatin II in solutions had gelling temperatures of 14.2 °C, 16.0 °C, and 17.1 °C, or melting temperatures of 19.3 °C, 21.3 °C, and 22.0 °C, respectively. That is, the used modification caused nearly 2–3 °C increases in gelling and melting temperatures. It was thus confirmed that the laccase-induced gelatin crosslinking resulted in stronger interactions between gelatin molecules and increased thermal stability of gelatin gels, ensuring higher temperature transition in the thermo-responsive sol-gel products. In addition, higher crosslinking extent led to higher thermal stability for gelatin gels. It was speculated that why the two modified products had higher Bloom values was also contributed by the enhanced interactions between gelatin molecules.

Fig. 4.

Viscoelastic properties of tilapia gelatin (a, b), modified gelatin I (c, d), and modified gelatin II solutions (e, f) upon cooling (a, c, e) and heating (b, d, f). G′, storage modulus; G′′, loss modulus.

Chemically, protein crosslinking means a generation of intermolecular and intramolecular covalent bonds, yielding enhanced molecular interactions. Crosslinked proteins especially gelatin thus had higher gelling and melting temperatures. For instance, it was demonstrated in a study of Huang and coauthors that fish gelatin after the TGase-induced modification showed clear increases in both gelling temperature (from 17.3 °C to 20.4 °C) and melting temperature (from 23.8 °C to 26.1 °C) (Huang et al., 2019). When bovine gelatin was crosslinked by horseradish peroxidase, its gelling and melting temperatures had an increase level of 2 °C (Lu, Han, & Zhao, 2018). Moreover, if TGase of different addition levels was used to crosslink tilapia skin gelatin, both gelling and melting temperatures also showed an increasing trend accompanying the increase of TGase addition (Sha et al., 2023). Overall, these studies proved consistently that the crosslinked gelatin had higher gelling and melting temperatures, endowing the respective gels with higher thermal resistance. Moreover, an increase in gelling and melting temperatures of the modified gelatin has potential technological and sensory implications in the processed foods; for instance, it might cause improved thermal stability in the gelatin-based desserts or meat products, or affect textural perception for the jelly products by providing a firmer or less melt-in-mouth gel structure.

3.6. Effect of laccase-induced crosslinking on surface hydrophobicity and foaming property of tilapia gelatin

Compared with unmodified gelatin, modified gelatin I and modified gelatin II showed lower surface hydrophobicity (17.25 and 21.35 versus 30.50) (Table 4). The studied laccase-induced crosslinking thus led to decreased surface hydrophobicity in the two modified products, partly as the result of the incorporation of hydrophilic −OH groups (from tea phenols) into gelatin molecules. The hydrophilic −OH groups from the conjugated tea polyphenols had ability to increase surface hydrophilicity, and thus resulted in decreased surface hydrophobicity. Additionally, both modified gelatin I and modified gelatin II showed better foaming properties than the unmodified gelatin (Table 4), because they caused 64 %–129 % increases in foaming capacity and enhanced foaming stability. Compared with modified gelatin I, modified gelatin II possessed higher crosslinking extent, and thus showed lower surface hydrophobicity and especially much enhanced foaming properties. It was evident again by the present results that higher crosslinking extent caused much changes in interfacial property of the investigated tilapia gelatin.

Table 4.

Surface hydrophobicity (SH), foaming capacity (FC), and foaming stability (FS) at different time intervals of tilapia gelatin, modified gelatin I, and modified gelatin II.

	Tilapia gelatin				Modified gelatin I				Modified gelatin II
SH	30.52 ± 0.03c				21.35 ± 0.02b				17.25 ± 0.01a
FC (%)	92 ± 11C				151 ± 10B				221 ± 14 A
FS (%)	92	60	40	10	151	110	100	90	221	180	160	150
Time interval (min)	0	10	20	30	0	10	20	30	0	10	20	30

Different lowercase or capital letters after the values as superscripts in the same row indicate that one-way ANOVA of the means differs significantly (p < 0.05).

Agyare and Damodaran found that the TGase-induced crosslinking of whey protein isolate led to much reduction in surface hydrophobicity (Agyare & Damodaran, 2010), while Saricay and coauthors also observed that the peroxidase-catalyzed cross-linking of apo-α-lactalbumin resulted in decreased surface hydrophobicity (Saricay, Wierenga, & de Vries, 2014). However, due to the generation of protein aggregates, crosslinked proteins have enhanced interfacial property, especially the critical interfacial stability. It was found that the TGase-treated gelatin had better foam stability, especially using higher TGase addition (Calvarro, Pérez-Palacios, & Ruiz Carrascal, 2016), while the TGase-crosslinked wheat gluten hydrolysate was more able to generate stable foams (Agyare, Addo, & Xiong, 2009). When both laccase and ferulic acid were applied in the crosslinking of soy protein, its foaming stability was significantly improved due to protein polymerization (Santoso, Al-Shaikhli, Ho, Rajapakse, & Le, 2025). Moreover, when polyphenols were used to crosslink gelatin under an alkaline condition, the crosslinked gelatin received better interfacial properties in emulsions, resulting in increased interfacial layer thickness, higher layer density, and better creaming stability (Wu et al., 2024). In this study, both modified gelatin I and modified gelatin II had protein aggregates as the result of the laccase-induced gelatin crosslinking, thus were more able than the unmodified gelatin to generate cohesive and viscoelastic interfacial film that held the incorporated gases in foams. In general, the cohesive and viscoelastic film is vital to the stability of foam system in preventing water drainage or lamellae thinning, thus reduce the rate of foam rupture and provide necessary foam stability. They two thus had better capacity than the unmodified gelatin in facilitating foam formation and especially providing better foam stability.

Although the present results proved the applicability of the laccase-induced modification on tilapia gelatin to alter its gelling, colloidal, and foaming properties, possible changes in other properties such as emulsifying and rheological properties, gel texture, film formation, and other functionalities are still unknown to us and thereof should be investigated. Moreover, the bioactivities of collagen, gelatin, and their hydrolyzed products (i.e. bioactive peptides) were the topic of many past studies (Gómez-Guillén, Giménez, López-Caballero, & Montero, 2011). Thus, whether this modification might cause potential changes in gelatin bioactivity is also important and thereby recommended in the future studies.

4. Conclusion

When tilapia gelatin of low Bloom value was modified with laccase and tea polyphenols, the modified products received covalent crosslinking and tea polyphenols conjugation via the proposed four key reaction steps, and thus had increased Bloom value together with altered structure and properties. Briefly, the modified products remained typical triple-helix structure in solution, and had much ordered secondary structure, reduced in vitro digestibility, decreased surface hydrophobicity, increased gelling and melting temperatures, larger particle size and zeta-potential in solution, and improved foaming activity and stability. Moreover, higher increase in Bloom value or crosslinking extent consistently yielded much property changes in the modified products. The laccase-induced gelatin crosslinking in the presence of tea polyphenols thus is a promising strategy to valorize gelatin quality, via enhancing its gelling, foaming, and colloidal properties. Moreover, future study might focus on a investigation to reveal whether this modification could impact other gelatin properties, such as film formation, and in vitro or in vivo bioactivities.

CRediT authorship contribution statement

Ya-Zhu Xiao: Data curation, Formal analysis, Methodology, Writing – original draft. Ming-Yue Zhong: Formal analysis, Methodology. Xin-Huai Zhao: Conceptualization, Funding acquisition, Project administration, Writing – review & editing. Qing-Qi Guo: Supervision, Writing – original draft. Chun-Li Song: Methodology.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

No data was used for the research described in the article.