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. 2026 Sep 19;39:104478. doi: 10.1016/j.fochx.2026.104478

Novel ternary covalent complexes (OsrhLF-EGCG-Mal) based on plant-derived recombinant lactoferrin (OsrhLF) by polyphenol-carbohydrate dual-modification strategy

Xueya Li a,1, Xiaoyan Guan a,1, Yeting Wu b, Wanrong Wu a, Yingying Li a, Rui Liu a, Zixian Xia a, Yifeng Ding a, Yanchun Shao a,⁎, Youyou Lu a,c,⁎
PMCID: PMC13635526  PMID: 42835271

Abstract

Bovine lactoferrin was widely used in food industry but suffered from resource dependence, environmental sensitivity and digestive inactivation susceptibility. As a promising alternative, the characteristics of plant-derived recombinant lactoferrin (OsrhLF) remained unclear. Herein, novel OsrhLF-EGCG-Mal ternary covalent complexes were innovatively constructed via dual-modification strategy with epigallocatechin gallate (EGCG) and maltodextrin (Mal). Results confirmed that covalent conjugation altered the spatial conformation of OsrhLF and affected its antioxidant activity, EGCG and Mal exerted effect on OsrhLF against gastrointestinal digestion. Analysis of short-chain fatty acid (SCFAs) in vitro feces fermentation verified the regulatory role of the OsrhLF-EGCG-Mal. Finally, the OsrhLF-EGCG-Mal enhanced water-holding capacity of the apple matrix, a critical functional advantage - in food processing and preservation. This study not only addressed the key shortcomings of lactoferrin application in food systems but also expanded the industrial application of OsrhLF in the field of food chemistry.

Keywords: Lactoferrin, Complexes, Epigallocatechin gallate, Maltodextrin, Food chemistry

Graphical abstract

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Highlights

  • •

    Novel ternary complexes were prepared based on plant-derived recombinant lactoferrin.

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    It was prepared through dual modification strategy of polyphenols-carbohydrates.

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    The association between ternary complexes and SCFAs metabolism was established.

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    The covalent ternary complexes affected water-holding capacity on apple surfaces.

1. Introduction

Lactoferrin (LF) is a single-chain globular glycoprotein and a key active protein among milk-derived proteins. It exhibits multiple biological functions, including immunoregulatory, antibacterial, iron-binding, and antioxidant activities, and is particularly important for infant growth and development (B. Wang, Timilsena, Blanch, & Adhikari, 2017; Wei et al., 2022). However, natural LF faces supply bottlenecks: the preparation cost of human-sourced LF is high, and the production yield of bovine-sourced LF (bLF) extracted from milk is limited, making it difficult to meet the demand for large-scale applications. In view of this, multiple studies have confirmed that LF has been successfully expressed in various biological expression systems, such as microorganisms, plants, and animals, providing a highly promising technological approach to solve the problem of high cost and low yield of natural LF (García-Montoya, Cendón, Arévalo-Gallegos, & Rascón-Cruz, 2011). However, compared with the widely used and safe bLF, research on plant-derived recombinant LF (OsrhLF) is relatively scarce and requires more in-depth exploration. In addition, LF might be highly sensitive to changes in environmental conditions, including pH, ionic strength, enzyme activity, and temperature (F. Liu, McClements, Ma, & Liu, 2023). To address this limitation, recent studies have shown that covalent modification of LF with naturally occurring active ingredients in food, such as dietary polyphenols and carbohydrates,could effectively enhance the structural stability and functional activity of LF, providing a feasible solution to overcome its application bottleneck.

Polyphenols are important secondary metabolites in plants, exhibiting a broad spectrum of biological activities, including antioxidant, anti-inflammatory, antibacterial, gut microbiota-modulating, and neuroprotective effects (X. Liu et al., 2021; Q. Wang et al., 2022). Among polyphenol-rich sources, green tea undergoes minimal fermentation during processing, which helps preserve the natural components in tea, particularly catechins, which constitute approximately 30% of its dry weight. Among the various catechins, epigallocatechin gallate (EGCG) exhibits the most potent biological activity and the highest abundance, accounting for 50%–80% of the total catechin content in green tea (S. Zhang et al., 2024). The molecular structure of EGCG features multiple phenolic hydroxyl groups, which confer upon it strong antioxidant capacity, but its structure may undergo autoxidation in the presence of trace metal ions (Musial, Kuban, & Gorska, 2020). In vivo, EGCG is primarily absorbed by intestinal epithelial cells, and the metabolites generated through its interaction with the gut microbiota are crucial to human health (Lai, Baig, Wong, & Zhu, 2020). Given its various physiological activities, particularly antioxidant and anti-inflammatory properties, EGCG holds significant promise in the development of functional foods (Li et al., 2022).

Carbohydrates play a crucial role in energy metabolism and storage in the body, and possess significant nutritional and biological activities (Jiang et al., 2021; Tang et al., 2019). Maltodextrin (Mal) is a sugar polymer produced from starch or amylopectin via enzymatic hydrolysis, refining, and drying. It exhibits high solubility, low hygroscopicity, anti-caking properties, and strong thermal stability. It is widely used in the food industry and serves as an excellent microcapsule wall material, and a fat substitute to mimic the mouthfeel of fat (Akbari, Eskandari, & Davoudi, 2019). In a complex dietary system, these three components interact with each other, affecting dietary flavor and texture, and even modulating human health. Therefore, it is of great significance to rationally assemble foodborne proteins, dietary polyphenols, and carbohydrates through different methods to elucidate the interaction mechanisms among their components, which is essential for the development and application of protein-based food products. The biopolymers formed via different covalent interactions between milk-derived proteins (e.g., lactoferrin, whey protein, casein, albumin, etc.) and dietary polyphenols or carbohydrates exhibit excellent curcumin (Cur) loading, delivery, and controlled-release capabilities, which significantly enhance the physicochemical stability (e.g., photostability, acid stability, water solubility, etc.) and biological activities (e.g., anticancer and antioxidant activities) of Cur, while showing no cytotoxicity (Amiri, Mahmazi, & Danafar, 2020; Chang, Wang, Hu, & Luo, 2017; H. Peng et al., 2017; S. Peng et al., 2020; Y. Wang et al., 2021). Proteins, polyphenols, and carbohydrates can form conjugates with distinct properties depending on the type and sequence of reactions involved (F. Liu, Ma, Gao, & McClements, 2017). In the first method, protein-carbohydrate covalent complexes are synthesized first, followed by covalent modification with polyphenols. The drawback of this method is that it requires strict control of reaction conditions to prevent loss of protein activity. In the second method, synthesize protein-polyphenol covalent complexes first, followed by covalent modification with carbohydrates. In the third method, carbohydrates are first coupled with polyphenols, and the conjugates are then reacted with proteins to form ternary complexes. The latter two methods offer advantages in product purification, as free small molecules, unreacted monomers, and catalysts from the covalent reactions are typically more readily removed via dialysis or ultrafiltration. The resulting complexes often combine the beneficial properties of the components, such as the surface activity of proteins, steric stability of polysaccharides, and the antioxidant activity of polyphenols.

In existing studies, a large amount of research has focused on the development of lactoferrin conjugates, mainly using lactoferrin from various sources as protein sources. Yang, Liang, and Xu (2020) prepared a ternary complex of lactoferrin-oat-β–glucan-curcumin, mainly relying on intermolecular interactions (hydrophobic interactions and hydrogen bonding interactions) to form amorphous non-covalent complexes. Liu, McClements, and Gao (2016) prepared chlorogenic acid-lactoferrin-dextran ternary complex, and studied the application of a ternary covalent complex as emulsifier in β-carotene lotion. Wang, Du, and Chai (2025) covalently bound LF with Rosa roxburghii Tratt fruit polysaccharides (RTFP) via the Maillard reaction, the findings suggested that the novel emulsifier showed potential to stabilize emulsions. Although studies have shown that recombinant lactoferrin expressed in rice is similar to natural LF in basic functions such as iron binding and antibacterial activity, the plant expression system endows LF with a unique plant type N-glycosylation pattern, which may affect the functional properties of the product (such as hydrophobicity, antioxidant activity, etc.). Currently, research on using OsrhLF for polyphenol and polysaccharide modification and evaluating its functional properties is still rare. Therefore, this study focuses on the preparation of a novel ternary complex by combining OsrhLF with polyphenols and polysaccharides, and compares its physicochemical and functional properties with their counterparts in bLF. The digestive characteristics, gut microbiota metabolism, and fruit preservation of the complex were evaluated to fill these research gaps.

As research advances, it has been found that the covalent modification involving plant-based lactoferrin and polyphenols and carbohydrate compounds represents an effective means to break the limitations of LF applications. Therefore, this study employed bLF as a reference and incorporated plant-derived recombinant OsrhLF to synthesize ternary covalent complexes with EGCG and Mal. This study further analyzed the differences in reaction mechanisms, structure, physicochemical properties, and functional characteristics of LF-EGCG-Mal covalent complexes, and investigated the differences in nutritional characteristics and their effects on short chain fatty acids (SCFAs) metabolism, along with their impact on food water retention, using in vitro digestion models and fecal fermentation experiments. This study not only addressed the key shortcomings of lactoferrin application in food systems but also expanded the industrial application of OsrhLF in the field of food chemistry.

2. Materials and methods

2.1. Materials

bLF (95%) and DPPH were purchased from Shanghai Yuanye Biotechnology Co., Ltd., OsrhLF, ≥95% was obtained from Wuhan Healthgen Biotechnology Corp. (−)-epigallocatechin gallate (EGCG) (98%), 2, 4, 6-Tri(2-pyridyl)-s-triazine (TPTZ), Trolox (Shanghai McLean Biochemical Technology Co., Ltd.). Maltodextrin (Mal), 8-Anilino-1-naphtholenesulfonic acid (ANS) (Shanghai Yien Chemical Technology Co., Ltd.). o-Phthalaldehyde(OPA), 2,2′-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS), 5,5’-Dithiobis-(2-nitrobenzoic acid) (DTNB) (Meilun Biotechnology Co., Ltd.). Pepsin, trypsin, bile salt, Dithiotretol (DTT), BCA protein concentration assay kit (Beijing Solarbio Technology Co., Ltd.). SCFAs standard samples (chromatographic grade) were provided by Macklin (Shanghai, China).

2.2. Methods

2.2.1. Preparation method of LF-EGCG-mal ternary complexes

bLF and OsrhLF protein solutions (0.4%, w/v) were prepared with distilled water, mixed well and hydrated overnight at 4 °C. Meanwhile, EGCG stock solution was prepared (0.05%, w/v). The LF solution and EGCG stock solution were mixed in a 1:1 (v/v) ratio, the pH was adjusted to 9.0, and the mixture was exposed to air and stirred continuously at 25 °C for 24 h to allow full oxidation of the polyphenols. After reaction, the mixed solution was transferred to a dialysis bag (molecular weight cut-off 8000–14,000) and dialyzed with deionized water for 48 h. The water was changed every 6–8 h to ensure that unreacted free polyphenols were completely dialyzed. Glycosylation modification of LF-EGCG complexes was performed through a wet heat Maillard reaction. Mal (LF: Mal = 3:4, w/w) was added to the LF-EGCG solution, magnetically stirred for 2 h at room temperature, and then hydrated overnight at 4 °C. The pH of the sample was adjusted to 11.0 and it was incubated at 75 °C for 6 h. The sample was freeze-dried to obtain the LF-EGCG-Mal covalent complexes. Meanwhile, bLF and OsrhLF solutions without EGCG and Mal were used as controls.

2.2.2. Determination of free amino groups

This study used the OPA method: 1.905 g of borax and 50 mg of SDS were completely dissolved in 25 mL of deionized water, followed by the addition of 1 mL of OPA and 44 mg of DTT to make up a final volume of 50 mL as the OPA working solution. Then, 20 μL of sample solution was mixed with 200 μL of OPA working solution and incubated at 35 °C for 2 min, and the absorbance was measured at 340 nm, calculated the content of free amino groups using serine as a standard.

2.2.3. Characterization of LF-EGCG-mal ternary covalent complexes

2.2.3.1. Fluorescence (FL) spectral analysis

Fluorescence spectra were recorded using a fluorescence spectrophotometer with LF autofluorescence as a probe. The concentration of the sample solution was 0.2 mg/mL, and the excitation and emission wavelengths were set at 295 nm and 300–500 nm, respectively. The excitation and emission slit widths were 5 nm and 2.5 nm, respectively.

2.2.3.2. Potential measurement

The samples were diluted to a concentration of 0.5 mg/mL with distilled water, and the zeta potential was measured using a nanoparticle analyzer. The temperature was set to 25 °C, the equilibrium time was 1 min.

2.2.4. Antioxidant activity of LF-EGCG -mal ternary covalent complexes

Referring to the previously reported methods with slight modifications (Jiao, Chen, Fan, & Young Quek, 2019), the antioxidant capacity of ternary covalent complexes was evaluated using multiple assays. The antioxidant activity of the samples was comprehensively assessed using DPPH radical scavenging method, ABTS+ radical scavenging method, and FRAP iron reduction ability method. For the DPPH method, the absorbance was measured at 517 nm after reacting the sample with 0.04 mg/mL DPPH in anhydrous ethanol solution with the sample for 1 h. The ABTS+ rule required the preparation of a 1:1 mixture of 7 mmol/L ABTS and 2.45 mmol/L K₂S₂O₈ in the dark for 16 h, followed by dilution to 734 nm with an absorbance of 0.70 ± 0.01 and reaction with the sample for 1 h for measurement. The FRAP method used a working solution mixed with 300 mmol/L acetic acid buffer (pH 3.6), 10 mmol/L TPTZ, and 20 mmol/L FeCl₃ at a ratio of 10:1:1 to react with the sample for 1 h, and the absorbance was then measured at 593 nm.

2.2.5. Digestive properties of LF-EGCG-mal ternary covalent complexes

Based on the in vitro digestion model developed by Brodkorb et al. (Brodkorb, Egger, & Alminger, 2019) and optimized, gastric digestion fluid (SGF, containing 6.9 mmol/L KCl and other components) and intestinal digestion fluid (SIF, containing 6.8 mmol/L KCl and other components) were prepared using a 1.25 × electrolyte reserve solution. Simulated gastric phase: Mixing 5 mL of sample solution with 5 mL of simulated gastric juice containing pepsin, shake at 37 °C for 2 h. Simulated intestinal phase: Adjusted the pH of gastric digestion solution to 7.0, then 10 mL of simulated intestinal juice containing trypsin was added, and the mixture was incubated with shaking at 37 °C for 2 h. During the digestion process, took samples every 30 min for relevant measurements. The BCA method was used to determine the content of LF in the supernatant of digestive juice, in order to calculate its changes in digestibility. The EGCG content was determined using the Folin phenol method. Prepared 2% EGCG standard solution (w/v) with distilled water, diluted it to different concentrations, took 0.5 mL of EGCG standard solution with different concentrations, added 2.5 mL of 0.2 N phenol reagent, reacted at room temperature in the dark for 5 min, then added 7.5% Na2CO3 solution (w/v), mixed well and reacted at room temperature in the dark for 2 h. Aqueous solution treated in the same manner served as the blank control. The absorbance was measured at 760 nm using a UV–visible spectrophotometer to generate the EGCG standard curve. Similarly, the changes in absorbance at 760 nm before and after digestion were measured to indicate the bioavailability of EGCG.

2.2.6. In vitro fecal fermentation analysis of LF-EGCG-mal ternary covalent complexes

2.2.6.1. Collection and pretreatment of fecal samples

This study used SPF-grade BALB/c, C57BL/6, and KM mice, aged 5–6 weeks, with half male and half female. All animals were provided by the Experimental Animal Center of Huazhong Agricultural University. All procedures performed in this study were in compliance with the institutional guidelines and were approved by the Ethics Committee of Huazhong Agricultural University. The ethical approval reference number was HZAUMO-2025-0229. At the experimental endpoint, mice were euthanized via intraperitoneal injection of an overdose of sodium pentobarbital (150–200 mg/kg). Mixing 1 g of mixed feces with 0.1 mol/L sterile PBS buffer at a ratio of 1:10 (m/v) for subsequent inoculation. Referring to the method proposed by Huang et al. (Huang et al., 2024), a slight modification was made: the fecal slurry was inoculated at a concentration of 10% (v/v) into a basic culture medium (carbon free medium) as the original fecal microbiota, and 10% of the sample solution was inoculated. The control group consisted of bLF and OsrhLF sample solutions without the addition of EGCG and Mal. All samples were incubated anaerobically for 72 h. Fermentation broth samples were collected after 0 h, 24 h, 48 h, and 72 h of fermentation, and the fermentation broth was centrifuged at 8000 ×g for 10 min at 4 °C, to obtain the fermentation supernatant for further analysis.

2.2.6.2. Determination of SCFAs content

One milliliter of fermentation broth was taken, and 100 μL of 50% sulfuric acid was added, allowed to stand at room temperature for 5 min with vortex mixing once per minute, then added 1 mL of anhydrous ether and vortexed for a short time. Centrifuged at 5000 ×g for 10 min, took the upper organic phase, and filtered it through a 0.22 μm filter membrane. The concentration of SCFAs was determined by DB-FFAP polar column (30 m × 250 μm × 0.25 μm) combined with GC/MS. The measurement conditions were: injection volume of 2 μL, split ratio of 20:1, solvent delay of 3.5 min, operating temperature of 240 °C for 2 min, scanning starting point of 35.00 and scanning endpoint of 350.00. The column temperature started at 40 °C and was maintained for 3 min, then rised to 180 °C at a rate of 8 °C/min and was maintained for 3 min, and then rised to 220 °C at a rate of 20 °C/min and was maintained for 3 min. Compound identification was performed by comparing the retention time of standard samples with the corresponding MS spectra, and quantification was performed by deconvoluting the base peak ion area.

2.2.7. Application of LF-EGCG-mal ternary covalent complexes in fresh cut apples

Washing fresh apples with clean water, disinfect the surface with 75% alcohol under a sterile operating table. Removing the core with a sterile knife, cut into evenly thick (about 1 cm) slices, and discarded the irregular parts at both ends. Soaked each sample in distilled water, bLF, OsrhLF, H-bLF, H-OsrhLF, H-bLF-EGCG-Mal, and H-OsrhLF-EGCG-Mal solutions for 1 h, removed and drained the water, immediately weighed it with an electronic balance, and recorded the weight and corresponding number of each sample accurately. Samples were taken every 1 h, weighed again, and recorded as Wₜ. The weighing process should be rapid to avoid additional moisture loss during the weighing process. The change in moisture content was calculated for each treatment group at each time point. The formula for calculating the change in moisture content was: ΔW = W₀ - Wₜ, where ΔW represents the change in apple weight.

2.2.8. Data analysis

The data results were presented as “Mean ± SD” and plotted by Excel 2019 and Origin 2018 software. t-tests, one-way ANOVA, and multiple range tests were analyzed using IBM SPSS Statistics 26.0 software, with p < 0.05 indicating the level of significant difference.

3. Results and discussion

3.1. Changes in free amino content

When characterizing the formation of protein–polyphenol–carbohydrate complexes, assessing amino acid content is a very important method for analyzing the degree of compound binding (Rohn, 2014). This method is also commonly used to determine the extent of the Maillard reaction, which occurs when free amino groups of proteins and carbonyl groups of reducing sugars covalently link to form Schiff bases (Spotti et al., 2013).

From Fig.1A, it could be seen that there was no significant change in the color state of bLF and OsrhLF solutions before and after the reaction, while LF-EGCG solution turned yellow brown. Compared with this, there was no visible difference in the color state of H-LF-EGCG-Mal solution. This phenomenon might indicate the reaction between LF and EGCG. From Fig.1B, it could be seen that compared with LF, the free amino content of H-LF, LF-EGCG, and H-LF-EGCG-Mal was significantly reduced, and H-LF-EGCG-Mal was lower than LF-EGCG. It was hypothesized that DTT reduces disulfide bonds, thereby indirectly disrupting some non-covalent interactions. These results suggested that protein cross-linking might occur between bLF and OsrhLF after heating. EGCG may have formed C—N covalent bonds with the side chain amino groups of LF, and the addition of Mal further reacted with the ε-amino group of lysine residue or the α-amino group of terminal amino acid in the LF-EGCG complex, leading to a significant reduction in free amino group content. This finding suggests the possible formation of C—N covalent bonds in the system.

Fig. 1.

Fig. 1

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Changes in free amino content and putative reaction mechanism of LF-EGCG-Mal covalent complexes. (A) Solution states. (B) Free amino content. (C) Putative reaction mechanism of LF-EGCG-Mal covalent complexes.

Based on the research conducted by Liu et al. (2016), we speculated on the possible reaction mechanism between LF and EGCG, Mal (as shown in Fig.1C). We hypothesized that, in the presence of O2, increasing the pH to alkaline conditions leads to the oxidation of the phenolic hydroxyl groups in EGCG, forming quinones. The oxidized EGCG subsequently forms dimers or higher polymers. EGCG quinone reacts with the amino or sulfhydryl side chains of LF, generating covalent C—N or C—S bonds. Meanwhile, the hydroquinone may undergo rearrangement, which can be re-oxidized to bind a second protein, thereby crosslinking protein molecules and ultimately resulting in polymerization. Following the formation of the LF-EGCG covalent polymer, the free amino groups (N-terminal amino groups) of the resulting polymer can react with the active reducing end of maltodextrin to form an N-glycosylamine with the release of one water molecule. The N-glycosylamine then rearranges to form an Amadori product, which in subsequent reactions undergoes oxidation, fragmentation, dehydration, and free radical reactions, ultimately yielding the LF-EGCG-Mal copolymer. In this study, the aforementioned binding process requires further in-depth investigation to explore and validate the relevant binding mechanisms.

3.2. Basic characteristic changes

3.2.1. FL spectrum changes

Spectroscopy characterization of LF-EGCG-Mal covalent complexes were shown in Fig.2.

Fig. 2.

Fig. 2

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Spectroscopy characterization of LF-EGCG-Mal covalent complexes. (A) Fluorescence spectroscopy of H-bLF-EGCG-Mal. (B) Fluorescence spectroscopy of H-OsrhLF-EGCG-Mal.

From Fig.2, it could be seen that both bLF and OsrhLF had strong fluorescence emission peaks around 334 nm. In comparison, the fluorescence intensity of H-OsrhLF was significantly lower than that of H-bLF, possibly attributable to the instability of the H-OsrhLF solution, which rendered it prone to aggregation under external stress. Compared with H-LF, the fluorescence intensity of the H-LF-EGCG-Mal covalent complex was almost completely quenched. Based on the above experimental results, it was speculated that the amino groups of bLF and OsrhLF side chain tryptophan simultaneously participated in the covalent reaction with EGCG and Mal. The strong interactions between the three components significantly reduced the fluorescence quantum yield of LF. This change has also been reported in previous studies (Xu, Yue, Yuan, & Zhao, 2023; Z. Zhang, Holden, Wang, & Adhikari, 2023). In addition, the emission maximum (λmax) of LF shifted after heat treatment, exhibiting a red shift. It was possible that LF unfolded and the exposure of tryptophan residues reduced its environmental hydrophobicity.

3.2.2. Potential changes

As shown in Fig.3A and 3B, after heating, the net charge of LF changed from positive to negative. This change may be attributed to heat-induced structural unfolding, which exposed negatively charged residues and thus increased the overall negative charge. Overall, the absolute zeta potential value of the H-LF-EGCG-Mal complex significantly increased to approximately −30 mV, indicating that the ternary complex solution exhibited enhanced stability. The increase in net negative charge can be attributed to the covalent conjugation of EGCG and Mal to LF, as this modification consumed the positively charged amino acid residues of LF through covalent bond formation.

Fig. 3.

Fig. 3

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Zeta potential of LF-EGCG-Mal covalent complexes. (A) Zeta potential of H-bLF-EGCG-Mal. (B) Zeta potential of H-OsrhLF-EGCG-Mal.

3.3. Changes in antioxidant activity

As shown in Fig.4A, compared with LF, the DPPH scavenging activity of H-LF was reduced. Compared with H-LF, the DPPH scavenging activity of LF-EGCG and H-LF-EGCG-Mal was significantly increased, and the scavenging activity of H-LF-EGCG-Mal was slightly higher than that of LF-EGCG. The trend of ABTS radical scavenging activity and FRAP reducing ability of LF-EGCG-Mal ternary covalent complex was consistent with the DPPH scavenging activity (Fig.4B, 4C). These results indicate that the covalent synergistic effect between EGCG and Mal significantly enhanced the antioxidant capacity of LF. It can be speculated that EGCG, acting as an effective hydrogen-atom or electron donor, covalently bound with LF and introduced a large number of phenolic hydroxyl antioxidant groups, thereby effectively neutralizing various free radicals. The covalent structure formed by it could also stabilize free radicals. At the same time, the intermediate and browning products produced by Mal glycosylation also had free radical scavenging activity (Lue et al., 2010; Zhang et al., 2023). In addition, the products of the Maillard reaction could act as metal chelating agents to bind with metal ions, and might interrupt the initiation pathway of lipid peroxidation by reducing the concentration of transition metals, thereby exerting antioxidant effects (Nooshkam & Varidi, 2020). The FRAP results indicated that the LF-EGCG-Mal ternary covalent complexed facilitated the removal of metal ions. Compared to H-bLF-EGCG-Mal, H-OsrhLF-EGCG-Mal had a stronger iron reduction ability, suggesting that OsrhLF had a higher iron content.

Fig. 4.

Fig. 4

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Antioxidant activity of LF-EGCG-Mal covalent complexes. (A) DPPH scavenging activity. (B) ABTS scavenging activity. (C) FRAP iron reducing capacity.

3.4. The effect of digestion characteristics

Changes in LF digestibility and EGCG release rate in each gastrointestinal digestion period were shown in Fig.5.

Fig. 5.

Fig. 5

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Changes in LF digestibility and EGCG release rate in each gastrointestinal digestion period. (A) Gastrointestinal digestibility of bLF group. (B) Gastrointestinal digestibility of OsrhLF group. (C) Gastrointestinal release rate of EGCG in the ternary covalent complexes.

Fig.5A and 5B showed the effect of covalent modification of EGCG and Mal on the digestibility of bLF and OsrhLF. Overall, with the increase of digestion time, the digestion rates of each sample gradually increased. The gastric digestion rates of bLF and OsrhLF were 66.67% and 62.27%, respectively, and the intestinal digestion rates reached 79.83% and 88.72%, respectively, which indicated that bLF and OsrhLF had a higher degree of digestion, absorption, and utilization ability (Bhat, Morton, El-Din A. Bekhit, Kumar, & Bhat, 2021). In contrast, for bLF, the digestion rate at each stage after heat treatment was higher than before heating, whereas OsrhLF showed the opposite trend. This might be due to the disordered protein structure after heating, which was easily degraded into small peptides and amino acids under the action of digestive enzymes. OsrhLF, on the other hand, was prone to form insoluble precipitates in the solution system and inhibited the hydrolysis of digestive enzymes (Arimboor & Arumughan, 2011). During the gastrointestinal digestion stage, the LF digestion rate of the ternary covalent complexes was lower. The final digestion rates of bLF-EGCG-Mal and OsrhLF-EGCG-Mal were only 46.50% and 47.88%, respectively, indicating that EGCG and Mal had a good protective effect on LF. This might be attributed to the interaction between polysaccharide Mal and polyphenol EGCG with digestive enzymes, and LF-EGCG-Mal was a high molecular weight polymer that could reduce enzyme accessibility (W. Wang et al., 2023). In addition, in Fig.5C, EGCG was released slowly throughout the entire stage of gastrointestinal digestion. The final EGCG release rates of bLF-EGCG-Mal and OsrhLF-EGCG-Mal were 64.04% and 65.71%, respectively, with no significant difference between them. It was speculated that if LF-EGCG-Mal acted on the human body, EGCG could be absorbed and utilized in a sustained and slow manner. In other words, LF might serve as a delivery system to achieve good delivery effects for EGCG. However, further in-depth research is needed to determine the ultimate effect.

3.5. The effect of changes in short chain fatty acids

Short-chain fatty acids (SCFAs) are metabolites produced by gut microbiota fermentation of carbohydrates. They are the main energy source for intestinal epithelial cells and play an important role in protecting intestinal mucosal function. SCFAs can not only maintain the mechanical barrier of the intestinal mucosa but also strengthen the immunological barrier and participate in intestinal immune regulation. In addition, SCFAs could selectively promote the growth of beneficial bacteria, inhibit the reproduction of pathogenic bacteria, and maintain the balance of gut microbiota. Therefore, the level of SCFAs production was a key indicator for evaluating the metabolic function of the gut microbiota ecosystem. The composition of gut microbiota is diverse and complex, among which Bifidobacterium, Blautia, Gemmiger, and Collinsella could metabolize and produce SCFAs to maintain intestinal homeostasis, enhance barrier function, and have a positive effect on host metabolism (Huang et al., 2024). Substrate composition, gut microbiota composition, and transit time can all affect the amount and relative proportion of individual SCFAs. Among them, acetic acid, propionic acid, and butanoic acid were produced in large quantities in the colon and were the most abundant SCFAs, while the production of formic acid and valeric acid was relatively low. Therefore, it was of great significance to explore the metabolism and SCFA generation ability of LF-EGCG-Mal ternary covalent complexes by different host bacterial populations.

Fig.6 showed the types and contents of SCFAs produced by anaerobic fermentation of bLF, OsrhLF, H-bLF-EGCG-Mal, and H-OsrhLF-EGCG-Mal solutions at different time points (0 h, 24 h, 48 h, and 72 h).

Fig. 6.

Fig. 6

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The content changes of SCFAs in vitro fecal fermentation between different groups. (A)BLAB/c;(B)C57BL/6;(C)KM.

As shown in Fig.6A, the types and contents of SCFAs produced by BALB/c mice at different time points during in vitro fermentation were presented. During the period from 0 h to 24 h, there was only a slight increase in the content of acetic acid, propionic acid, and butanoic acid, which remained at a low level until 48 h. At 72 h, the content of various SCFAs increased significantly. Compared to LF, the production of various SCFAs in the ternary complexes was relatively low. It might be that the LF-EGCG-Mal ternary complex has a complex and stable structure, which was not easily affected and degraded by specific enzymes, releasing small molecule substances and therefore not easily utilized by intestinal microbiota. As shown in Fig.6B, in the fecal fermentation broth of C57BL/6 mice and BALB/c mice, the production of acetic acid, propionic acid, butanoic acid, and total acid showed a similar increasing trend.

As shown in Fig.6C, in the fecal fermentation system of KM mice, the early generation of SCFAs was relatively slow but showed a gradual increasing trend, especially the production of propionic acid was low from 0 h to 24 h. However, during the 48 h fermentation stage, especially in the LF-EGCG-Mal ternary complex group, the content of propionic acid and butanoic acid showed a significant increase. But after 48 h, it showed a downward trend, which was different from the changes in SCFAs content in the first two types of mice.

3.6. Application in fresh cut apples

The moisture content is an important factor affecting the quality of fresh cut apples, directly reflecting their freshness. The aim of this study was to evaluate the effect of LF-EGCG-Mal ternary complex on reducing water evaporation in fresh cut apple slices. Short-term weight loss measurements of apple slices were used to directly reflect their water loss (as shown in Fig.7).

Fig. 7.

Fig. 7

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Mass changes of LF-EGCG-Mal ternary covalent complexes in fresh cut apples. (A) Heat map of weight changes in each group within 1 h. (B) Heat map of weight changes in each group within 2 h. (C) Heat map of weight changes in each group within 3 h. (D) Heat map of weight changes in each group within 4 h. (E) Heat map of weight changes in each group within 5 h. (F) Heat map of weight changes in each group within 6 h. (G) Trends of overall weight changes in each group.

From Fig.7, it could be seen that the weight changes of apple slices in different treatment groups increased with the prolongation of time, indicating that the cells of fresh cut apples were damaged during processing, leading to increased transpiration and water loss. Comparing different treatment groups, it was found that the weight change of the distilled water treatment group was the largest after 6 h, and the water loss was the highest (Fig.7G). The OsrhLF and bLF groups showed similar effects and were lower than the distilled water group. The H-OsrhLF-EGCG-Mal treatment group showed the best effect, effectively slowing down the water loss of fresh-cut apples and better maintaining their moisture content. Compared to the LF group, the bLF-EGCG-Mal group had less water loss, suggesting that the ternary complex can form an effective barrier to retard water evaporation. It can be speculated that the film-forming property of LF, the cross-linking capacity of EGCG, and the filling effect and hydrophilicity of Mal together exert a synergistic effect. The LF–EGCG–Mal ternary complex might assemble into a dense and stable composite film on the surface of fresh-cut apple slices upon application or soaking: LF constructed a membrane skeleton to achieve physical barrier, EGCG enhanced the density of the membrane layer through cross-linking to reduce water permeation channels, while Mal filled the pores of the membrane layer and adsorbed surface free water. The underlying mechanisms still require further research in the future.

4. Conclusion

This study demonstrated that polyphenol-carbohydrate dual modification synergistically improved the physicochemical and functional properties of plant-derived recombinant lactoferrin (OsrhLF) by constructing OsrhLF-EGCG-Mal ternary covalent complexes. Importantly, this research effectively addressed the key limitations of direct lactoferrin application in food systems, thereby expanding the research and development pathway for LF-based functional food products and offering new technical support for the industrial application of OsrhLF. However, further research is still needed to resolve individual differences in the efficacy of LF-containing ternary complexes, this will help meet the personalized application needs in functional food development and fruit preservation in the future, and further promote the practical application of recombinant lactoferrin-based food chemistry complexes.

CRediT authorship contribution statement

Xueya Li: Writing – original draft, Conceptualization. Xiaoyan Guan: Writing – original draft, Conceptualization. Yeting Wu: Methodology, Data curation. Wanrong Wu: Methodology, Data curation. Yingying Li: Investigation. Rui Liu: Validation. Zixian Xia: Formal analysis. Yifeng Ding: Software. Yanchun Shao: Supervision. Youyou Lu: Writing – review & editing, Supervision, Resources.

Funding

This work was financially supported by National Key Research and Development Program of China [grant number 2024YFF1106500] and Natural Science Foundation of Wuhan [grant number 2024040801020285] and Hubei Technology Innovation Plan Program [grant number 2025BBB075].

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.

Acknowledgements

We would be grateful to the Wuhan Zhaoyang Plan Project of Wuhan Association for Science and Technology; we would be grateful for the support of Yeting Wu of Animal Sciences and Technology, College of Veterinary Medicine, Huazhong Agricultural University.

Contributor Information

Yanchun Shao, Email: yanchunshao@mail.hzau.edu.cn.

Youyou Lu, Email: lvyouyou@mail.hzau.edu.cn.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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