Metal-Free Production of Natural Blue Colorants: Novel Insights from Anthocyanin-Protein Interactions

Academic Background

With increasing consumer awareness of health and natural food ingredients, the demand for natural colorants has been on the rise. However, natural blue colorants are scarce, and their production poses significant challenges. Currently, the food industry primarily relies on synthetic blue colorants, which may pose potential health risks. Therefore, the development of safe and stable natural blue colorants has become a critical research direction in food science. Anthocyanins (Acns), commonly found in plants, can exhibit a range of colors from orange-red to blue-purple. However, in vitro environments, the blue stability of anthocyanins is poor, particularly under neutral or alkaline conditions, where the blue color tends to fade.

Traditionally, anthocyanins form stable blue colorants through complexation with metal ions, especially acylated anthocyanins, which exhibit excellent blue stability. However, metal complexation methods are costly and may pose risks of metal ingestion. Therefore, finding a metal-free method for producing natural blue colorants is a current research focus. This study proposes a novel metal-free strategy to generate stable blue colorants through the interaction of anthocyanins with proteins and delves into the underlying mechanisms.

Source of the Paper

This paper is authored by Wenxin Wang, Peiqing Yang, Fuqing Gao, Yongtao Wang, Zhenzhen Xu, and Xiaojun Liao, affiliated with the College of Food Science and Nutritional Engineering at China Agricultural University and the Institute of Quality Standard & Testing Technology for Agro-products at the Chinese Academy of Agricultural Sciences. The paper was published in 2025 in the Journal of Advanced Research, with the DOI 10.1016/j.jare.2024.02.018.

Research Process

1. Experimental Materials and Methods

• Experimental Materials: The study used six common non-acylated 3-glucoside anthocyanins (cyanidin-3-glucoside, delphinidin-3-glucoside, petunidin-3-glucoside, malvidin-3-glucoside, pelargonidin-3-glucoside, and peonidin-3-glucoside), with human serum albumin (HSA), bovine serum albumin (BSA), and lysozyme (Lys) as model proteins.
• Sample Preparation: Anthocyanin solutions were prepared under acidic conditions, and protein solutions were dissolved in neutral buffers. The interactions between anthocyanins and proteins were studied using visible absorption spectroscopy, fluorescence quenching, and molecular simulations.
• Visible Absorption Spectroscopy: The absorption spectra of anthocyanin-protein mixtures were recorded using a UV-Vis spectrophotometer to analyze changes in maximum absorption wavelength (λmax) and absorbance.
• Fluorescence Quenching Assay: Fluorescence intensity changes upon protein-anthocyanin binding were measured using a fluorescence spectrophotometer to calculate binding constants (Kd).
• Molecular Docking and Dynamics Simulations: Molecular docking was performed using AutoDock Vina, and molecular dynamics simulations were conducted using GROMACS software to analyze the binding modes and stability of anthocyanins with proteins.

2. Experimental Results

• Interaction of Anthocyanins with HSA: The study found that the absorption spectra of anthocyanins exhibited significant bathochromic shifts upon binding to HSA, particularly for anthocyanins with two or more hydroxyl or methoxyl substituents on the B-ring (e.g., cyanidin-3-glucoside, delphinidin-3-glucoside, and petunidin-3-glucoside). These anthocyanins showed a λmax exceeding 600 nm, displaying blue hues.
• Binding Mechanisms: Through molecular simulations and thermodynamic parameter analysis, it was found that the binding of anthocyanins to HSA was primarily driven by enthalpy changes (ΔH), especially due to hydrogen bonding between hydroxyl groups on the B-ring and polar amino acid residues in HSA. In contrast, the binding of malvidin-3-glucoside to HSA was mainly driven by entropy changes (ΔS), with methoxyl substituents enhancing hydrophobic interactions with non-polar amino acid residues in HSA.
• Selectivity of Proteins: The study revealed that different proteins selectively enhanced the blue coloration of anthocyanins, with BSA and Lys showing significant blue-enhancing effects on different anthocyanins. Notably, malvidin-3-glucoside exhibited a λmax of 642 nm upon binding to BSA, displaying a vivid blue hue.

3. Color Stability

• Color Loss Mode: The study found that HSA and BSA significantly improved the color stability of anthocyanins. Over 12 hours, the binding of anthocyanins to proteins notably slowed down the fading of color, particularly when the protein concentration reached a certain threshold, resulting in optimal color stability.

Research Conclusions

This study is the first to demonstrate that widely distributed non-acylated 3-glucoside anthocyanins can generate diverse blue hues through interactions with specific proteins without relying on traditional metal complexation methods. The study reveals the critical role of hydroxyl or methoxyl substituents on the B-ring of anthocyanins in blue color generation. Specifically, cyanidin-3-glucoside, delphinidin-3-glucoside, and petunidin-3-glucoside generate blue colors through interactions with HSA or Lys, primarily driven by enthalpy changes, while malvidin-3-glucoside achieves blue coloration through interactions with HSA or BSA, mainly driven by entropy changes. This discovery offers a simple, effective, and metal-free strategy for producing natural blue colorants.

Research Highlights

• Metal-Free Blue Colorant Production Method: The study proposes a novel metal-free strategy to generate stable blue colorants through anthocyanin-protein interactions, overcoming the limitations of traditional metal complexation methods.
• Optimization of Anthocyanin Structures: The study highlights the critical role of hydroxyl or methoxyl substituents on the B-ring of anthocyanins in blue color generation, providing theoretical guidance for the future development of novel natural blue colorants.
• Selectivity of Proteins: The study reveals that different proteins exhibit significant selectivity in enhancing the blue coloration of anthocyanins, particularly with BSA and Lys, which result in vivid blue hues.

Research Significance

This study not only theoretically elucidates the mechanism of anthocyanin-protein interactions but also provides new insights for the development of novel natural blue colorants in the food, cosmetic, and other industries. Future research can further optimize the blue stability of anthocyanin-protein systems and explore their potential in practical applications.