Anthocyanidins (ACNs) are a class of water-soluble pigments containing 2-phenylbenzopyran moieties that are widely found in various tissues and organs of plants (Khoo, Azlan, Tang, & Lim, 2017). After being absorbed by the human body, they will generate many beneficial physiological health effects (Atnip, Sigurdson, Bomser, & Giusti, 2017; Luiza et al., 2022). The main site of ACN absorption is the gastrointestinal tract, of which approximately 25% is absorbed in the stomach, 10–20% in the small intestine, and 30–75% through metabolites from microbial fermentation in the colon for human health benefits. In stomach, ACNs are rapidly transported to the plasma of portal vein, abdominal aorta and heart within 30 min by organic anion-carrying bis-translocase in gastric mucosa; In small intestine, ACNs are first hydrolyzed to glycosides and glycosides by β-glucosidase, and then absorbed either by passive diffusion or by sodium-dependent glucose cotransporter proteins; Upon arrival at the colon, ACNs are first cleaved by colonic microbial esterases via cleavage of the ester bond with spontaneous ring-opening to a number of structurally simple compounds such as phenolic acids and protocatechuic acid, and then absorbed by the colon (Fernandes, de Freitas, & Mateus, 2014; Han et al., 2019; Lila, Burton, Grace, & Kalt, 2016).

Notably, ACNs are much more stable in the stomach than in the intestine, with approximately 89% of ACNs being stable in the stomach compared to only 38% after intestinal digestion (He & Giusti, 2010; McGhie & Walton, 2007). It means that ACNs have a greater chance of being absorbed in its native intact form in the stomach. Therefore, the natural high stability of ACNs in the stomach can be utilized to keep them there for as long as possible for enhancing absorption. However, as a flavonoid small molecule compound, ACNs have a short residence time in the stomach, which makes it difficult to achieve theoretically high absorption in the stomach. Existing delivery systems for ACNs rely on the stacking of multiple materials, which inevitably leads to the introduction of chemical reagents (e.g., for pH adjustment and cross-linking), thus posing an obstacle to ACNs as a daily nutraceutical supplement (Cui et al., 2022; Dong et al., 2024; Liao et al., 2021; Mao et al., 2023; Sharma, Pandita, & Bhosale, 2023; Zhou et al., 2022).

Micellar casein is a natural component of bovine milk, which is a spherical micelle of αS1-, αS2-, β-, and κ-casein self-assembled in a colloidal Ca2+-driven process (Dalgleish, 2011; Holt, Carver, Ecroyd, & Thorn, 2013). The region where colloidal Ca2+ and phosphoserine residues on casein subunits together are known as colloidal calcium phosphate (CCP). Additionally, there is serum Ca2+ (∼30% of the total) that is not involved in self-assembly (Barone, Yazdi, Lillevang, & Ahrné, 2021; De Kruif & Holt, 2003; Reiter, Reitmaier, & Kulozik, 2022; Schäfer, Hinrichs, Kohlus, Huppertz, & Atamer, 2021). In the natural state, colloidal Ca2+ maintains a constant dynamic equilibrium with serum Ca2+, which can be simplified as: Ca3(PO4)2 ↓ + 2H+⇌ 2HPO42− + 3Ca2+ (Holt, 2004; Lewis, 2011). Casein has gastric curdling properties due to the exposure of the micellar structure after hydrolysis by pepsin. As a physical processing technique, high hydrostatic pressure (HHP) breaks non-covalent bonds (Schrader, Buchheim, & Morr, 1997) as evidenced by the transformation of colloidal Ca2+ into serum Ca2+, which leads to the partial dissociation of micelles (Anema, Lowe, & Stockmann, 2005; Baier, Schmitt, & Knorr, 2015; Huppertz & De Kruif, 2007). However, HHP-induced micellar dissociation has been found to be temporary and reversible, as serum Ca2+ released from CCP gradually interacts with the submicelles again, which occurs within a few hours after depressurization (Considine, Patel, Anema, Singh, & Creamer, 2007; Huppertz, Fox, & Kelly, 2004; Knudsen & Skibsted, 2010). Furthermore, since gastric fluids contain uncertain concentrations of Ca2+ (John & Steven, 2009; Kalantzi et al., 2006; Lindahl, Ungell, Knutson, & Lennernäs, 1997), which results in unpredictable gastrointestinal digestive behavior and release behavior of ACNs.

In this study, HHP (100, 300, 500 MPa) was applied to modulate the CCP of casein micelles and HHP-induced casein was adopted for delivery of ACNs. To simulate human gastric fluid, Ca2+ levels (0.15, 2, 5 mM) in a static in vitro simulated digestion experiment (INFOGEST 2.0) were adjusted to mimic the state of gastric fluid after a meal. We wish to provide a physical processing means based on HHP to create a simple casein-anthocyanins delivery system (CN-ACNs) with Ca2+ as a structural modifier to achieve delayed digestion of the system. It is designed to prolong the gastric residence time of ACNs. We took advantage of the high stability of ACNs in the stomach, in order for ACNs to fully accomplish their beneficial effects.