Milk protein is a high-quality protein characterized by a rich amino acid profile and excellent nutritional properties, and it has been widely applied in food, health food and pharmaceutical industries. Protein fragments isolated from milk protein, namely sequences containing 2 to 20 amino acid residues that exert beneficial effects on human physiological functions, are defined as bioactive peptides [1-2]. With the advancement of research, bioactive peptides derived from milk protein have attracted extensive attention. Released from milk protein via enzymatic hydrolysis, fermentation and other processes, these peptides possess multiple biological functions including antioxidant, antibacterial, immunomodulatory and antihypertensive activities, demonstrating great application potential. Therefore, the extraction technology and bioactivities of milk protein peptides have become a hot research topic.

1 Functions of Milk Protein-derived Bioactive Peptides

The health-promoting properties of milk-derived bioactive peptides have been validated by numerous studies. According to international consensus [27], such protein fragments promote health by regulating metabolic and physiological processes. Their functional characteristics cover diverse bioactivities such as antioxidation, blood pressure reduction, antibacterial action, antithrombotic effect, cholesterol regulation and immunomodulation [28]. These ingredients can be used to develop functional foods, medical foods and dietary supplements, holding remarkable potential for health intervention. Notably, bioactive peptides generated by enzymatic hydrolysis of different milk proteins exhibit characteristic functional differences, with obvious heterogeneity in action mechanisms and activity intensity [29-30] (Figure 2).

Antihypertensive Activity

Cardiovascular diseases are mainly triggered by hypertension, overweight, smoking, insulin resistance (diabetes) and insufficient physical exercise [31]. Angiotensin-converting enzyme (ACE) serves as a key blood pressure regulator, as it catalyzes the conversion of angiotensin I into angiotensin II, a hormone that constricts blood vessels and thereby elevates blood pressure. ACE inhibitors are commonly used for hypertension treatment by suppressing this conversion process to lower blood pressure [32]. Many synthetic ACE-inhibitory peptides have been developed into anti-hypertensive pharmaceuticals; however, their application is associated with adverse side effects including cough, cutaneous allergic reactions, hypotension and renal impairment [33]. It has been discovered that bovine milk protein can produce ACE-inhibitory peptides, whose potential to inhibit ACE activity is under investigation [34]. Two critical ACE-inhibitory tripeptides, Ile-Pro-Pro and Val-Pro-Pro, with ACE inhibitory effects, were isolated from casein fermented by Lactobacillus helveticus and Saccharomyces cerevisiae [35] (Table 2). Rubak et al. [36] reported that Lactobacillus kefiri JK17 and Lactobacillus kefiri YK4 produced multiple ACE-inhibitory peptides including FSDIPNPIGSE, MPFPKYPVEPF and KALPMHIR from fermented milk. Another study [37] demonstrated that lactoferrin fermented by Kluyveromyces marxianus yielded antihypertensive peptides such as DPYKLRP, which reduced the systolic blood pressure of spontaneously hypertensive rats by 27 mmHg.

Antioxidant Activity

Oxidative stress is another inducing factor for cardiovascular diseases. It is identified by the generation of reactive oxygen species, including hydroxyl radicals, free radicals, superoxide molecules and non-radical peroxides such as hydrogen peroxide [52]. Macromolecules such as RNA, DNA, proteins and lipids are primary targets attacked by reactive oxygen species, which can induce a variety of human disorders including atherosclerosis, necrosis, cell apoptosis and carcinogenesis [53]. It has been reported that intake of dietary antioxidants can mitigate oxidative stress by boosting antioxidant defense mechanisms. One study [17] showed that a hexapeptide named YFYPEL, generated from pepsin-hydrolyzed bovine αs1-casein, acts as a superoxide radical scavenger. In research on hydrolysates of β-lactoglobulin treated with Corolase PP, the peptide sequence WYSLAMAASDI was released and exhibited radical scavenging activity [40]. Another study [41] found that when camel milk proteins were hydrolyzed with different enzymes, trypsin hydrolysis produced three peptides with antioxidant activity (TPDNIDIWLGGIAEPQVKR, RLDGQGRPRVWLGR and VAYSDDGENWTEYRDQGAVEGK). In addition, studies on peptides YFPQL, YQEPVLGPVR and LLY derived from buffalo casein [42] proved their potent radical scavenging capacity. The antioxidant efficiency of bioactive peptides arises from the properties of specific amino acids, which enhance the affinity of peptides for hydrophobic targets.

Antithrombotic Activity

Thrombosis refers to a pathological condition caused by elevated platelet activity or defective fibrinolysis in veins or arteries. Myocardial infarction and stroke are primarily induced by arterial thrombosis, which is aggravated during atherosclerosis. Hence, antithrombotic drugs are required to treat these disorders [54]. One study [44] illustrated the similarity between the RGDS sequence derived from human lactoferrin and short bovine milk peptide (KRDS sequence), both of which possess antithrombotic activity. Both KRDS and RGDS block platelet aggregation by inhibiting the binding of fibrinogen to glycoprotein receptor αIIbβ3. According to a study [55], the amino acid sequence MAIPPKKNQDK (κ-casein f106–116) from casein can prevent blood coagulation, bind to targets and further inhibit the connection between fibrinogen and platelets. Similarly, research [17] indicated that two bioactive peptides (amino acid sequences 163–171 and 165–171) from goat milk κ-casein and casein glycomacropeptide (CGMP) displayed strong antithrombotic activity during tryptic hydrolysis. Moreover, a study [46] showed that three peptides (QVTSTEV, KDQDK and TAQVTSTE) released during the enzymatic hydrolysis of goat milk CGMP completely inhibited platelet aggregation.

Immunomodulatory and Antibacterial Activity

Milk protein peptides such as hydrolysates of α-lactalbumin and β-lactoglobulin have shown prominent immunomodulatory effects in adaptive and innate immune responses, manifested as lymphocyte activation and proliferation, cytokine regulation, antibody production, enhanced phagocytosis of macrophages and stimulated immunoglobulin synthesis [56]. In terms of antibacterial properties, whey lactoferrin (including lactoferricin and its derived peptides) and other milk peptides have been verified to exert antibacterial activity against Gram-positive bacteria, Gram-negative bacteria (e.g., Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa), yeasts and fungi [57]. The activity of antimicrobial peptides (usually composed of 12–50 amino acids) is affected by total amino acid number, disulfide bond positions, charge distribution and sequence composition. The key structural features for the interaction between antimicrobial peptides and microorganisms include amphipathic structure (hydrophilic and hydrophobic amino acids at two ends of peptide sequence) and positive charge, which determine how antimicrobial peptides bind to microbes. For instance, peptide sequence YQEPVLGPVRGPFPI obtained from bovine β-casein and RPKHPIKHQGLPQEVLNENLLRF from bovine milk isracidin showed antibacterial activity against E. coli DH5α; while TKLTEEEKNRLNFLKKISQRYQKFALPQYLK derived from bovine αs2-casein f151–181 was active against both Bacillus subtilis and E. coli [46]. In addition, Wada et al. [48] found that peptide QELLLNPTHQIYPVTQPLAPVHNPISV released from human milk β-casein f185–211 also possessed antibacterial activity. Other studies [49,54] demonstrated that peptide KCRRWQWRMKKLGA from casein exhibited antibacterial effects against Staphylococcus aureus and E. coli. Whey lactoferrin can be converted into lactoferricin, which plays a vital role in the body’s immune system [58]. For example, lactoferrin combined with triple therapy (rabeprazole, clarithromycin and tinidazole) can effectively eradicate Helicobacter pylori [59]. Research [60] also indicated that bovine lactoferrin combined with erythromycin or ampicillin completely inhibited the invasiveness of group A streptococci and improved the survival rate of epithelial cells undergoing apoptosis, and this effect was validated by in vivo tests. Furthermore, supplementation with vitamin D and whey protein for 13 weeks significantly relieved chronic low-grade inflammation in elderly patients with sarcopenia [61]. β-lactoglobulin and α-lactalbumin in whey protein can also enhance the immune response of human neutrophils [62]. Based on these findings, milk proteins show potential against pathogens and other immune-related diseases via immune regulation.

Cholesterol-lowering Activity

Cholesterol is an essential component required for the synthesis of vitamin D, bile acids and hormones. However, excessive cholesterol in blood deposits in arteries to form plaques, resulting in arteriosclerosis. This reduces oxygen delivery to the heart and further promotes the progression of cardiovascular diseases [48,63]. Mice fed cholesterol-free and cholesterol-rich diets respectively revealed a significant correlation between milk protein intake and decreased total cholesterol levels. Two key bioactive peptides recovered from β-lactoglobulin, β-lactorphin and lactostatin, help reduce cholesterol [64]. Tryptic hydrolysate of the β-lactoglobulin peptide sequence Ile-Ile-Ala-Glu-Lys exhibits cholesterol-lowering activity [51]. Samtiya et al. [10] proved that bovine milk-derived bioactive peptides TDVEN, VLPVPQ, LQPE and VAPFPE exerted antihypercholesterolemic effects in vitro by reducing the solubility of cholesterol micelles. Besides, according to Nielsen et al. [65], four peptides isolated from β-lactoglobulin help lower cholesterol levels by inhibiting cholesterol breakdown in the digestive system. Cholecystokinin, an appetite suppressant released from milk protein, can be used for body weight maintenance and is known as an anti-appetite bioactive peptide [66].

2 Bioavailability of Whey Peptides

One of the most critical challenges in bioactive peptide research is ensuring the bioavailability of these peptide sequences to achieve expected health benefits. Hydrolysis of peptide sequences by gastrointestinal proteases directly influences their biological effects. Therefore, besides verifying peptide bioactivity, it is necessary to evaluate their gastrointestinal tolerance and whether they can be efficiently absorbed into the bloodstream to function in vivo [67]. Research on bioavailability is particularly critical because peptides may be further degraded into inactive fragments inside the gastrointestinal tract, impairing their final health benefits. To ensure bioactive peptides reach their action sites intact, researchers need to consider peptide stability and their changes during digestion [48]. The study by Boelsma et al. [68] showed that although some peptides displayed antihypertensive activity in in vitro assays, not all in vitro-active peptides could function effectively in vivo. For example, they found two milk protein-derived peptides with in vitro antihypertensive effects, yet only one of them effectively inhibited ACE activity and lowered blood pressure in vivo. This finding highlights the importance of peptide bioavailability: only peptides resistant to digestive enzyme degradation, efficiently absorbed and delivered to target cells can exert prominent biological effects in practical applications. Hence, future research should focus more on optimizing peptide stability and absorption to boost their potential as functional food ingredients or pharmaceuticals.

Current studies have identified bioavailable peptides derived from milk protein, which can enhance the absorption of minerals essential for human health. Such peptides are obtained via in vitro digestion. A study [69] found that whey protein isolate peptides increased iron bioavailability by 45%–100%. In addition, Wang et al. [70] investigated hydrolysates of β-lactoglobulin and α-lactalbumin complexed with iron, and evaluated mineral absorption, intracellular iron retention and iron transport across Caco-2 cells in vitro. High iron absorption was achieved from the synthesized complexes, confirming their resistance to the gastrointestinal environment. Similarly, Wang Rongchun et al. [71] determined zinc absorption when bound to peptides obtained from milk protein hydrolyzed by trypsin, pancreatin and papain, and indicated that peptide fragments chelated with zinc were resistant to enzymatic activity in the gastrointestinal tract. Studies on complexes formed by whey-derived peptides with iron and zinc represent one of the most promising research directions for alternative therapies for anemia and mineral deficiency [72].

On the other hand, iron-peptide complexes are regarded as a promising source to improve iron bioavailability. Peptides (TPEVDDE, VRTPEVDDE, DDDLTDDI, FKDLGEEH) isolated from pancreatin-hydrolyzed protein can raise iron solubility from 0% to nearly 100% at pH 7.0 and remain stable under simulated gastric digestion conditions (solubility: 50.8%–89.4%). Likewise, reports [73] stated that the iron binding sites of these peptides correspond to carboxyl groups. In addition, nanotechnology, a rapidly developing field, plays an important role in the utilization of milk protein peptides. The properties of nanomaterials may differ greatly from conventional bulk materials of the same compound. This phenomenon arises not only from the large specific surface area of nanomaterials, but also from physicochemical interactions at the nanoscale that dominate the overall performance of the system [74]. To enable bioactive peptides to function effectively in vivo, nanodispersions can serve as efficient carriers to incorporate bioactive peptides into food matrices, improving their in vivo bioavailability and efficacy [75-76].

Peptides are absorbed via transporters expressed in the intestinal tract (PepT1), which mediates the transport of small peptides, while oligopeptides undergo passive transport through the hydrophobic regions of epithelial cell membranes [77]. In both cases, the process depends on the specific properties of each peptide. For PepT1, it prefers to transport neutral, nonpolar hydrophobic peptides [78]. For the paracellular pathway, it tends to transport small, low-molecular-weight peptides that are hydrophilic, neutral or negatively charged [79]. Peptides with large molecular weight, hydrophobicity and positive charges tend to be transported via endocytosis [80]. Peptides resistant to intestinal peptidases can be transported into the blood at micromolar concentrations; therefore, they can retain bioactivity for minutes or hours when kept intact [79].

Knowles et al. [81] discovered 31 peptides resistant to gastrointestinal digestion from milk protein hydrolysates. Experiments on the C2C12 cell line identified six peptides with the highest bioavailability (TKIPA, NLPPL, PVPQ, VGIN, VAGT and KVPQ), whose capacity to protect cells from free radical damage was verified. In contrast, other research [82] indicated that although sequences in peptide fractions generated by intestinal peptidase possessed strong inhibitory capacity against dipeptidyl peptidase-IV, their activity was lower than that of precursor peptides derived from β-lactoglobulin.

Therefore, the challenge of delivering bioactive peptides lies in their hydrolysis in intestinal epithelial cells and blood plasma. In addition, their extremely low concentration upon reaching target sites may weaken bioactivity. Accordingly, it is necessary to identify or design peptides with high affinity for PepT1 or high bioavailability for functional food formulation. Moreover, cellular and molecular research should be carried out to elucidate the molecular mechanism by which bioactive peptides cross intestinal epithelium and exert bioactivity in target tissues. Further human trials are required, and effective and safe strategies combining chemical and molecular biological techniques should be explored to improve the bioavailability of whey-derived peptide fractions.

3 Applications of Milk Protein Peptides

Owing to their diverse bioactivities, whey bioactive peptides have become a research hotspot for functional food development. Current research focuses on developing commercial products by safely incorporating such ingredients. Their mechanism of action mainly lies in regulating disease-related signaling pathways, making them adjunctive interventions for multiple disorders. In existing research [81], approximately 86% of bioactive peptide-fortified foods are concentrated in dairy products, including fermented milk, cheese and yoghurt. Notably, numerous innovative products claiming specific bioactivities have emerged in the market. For instance, BioZate® manufactured by Davisco Foods International, USA, is a hydrolyzed whey protein isolate with protein purity over 80%. Studies [27] have confirmed that β-lactoglobulin-derived bioactive peptides contained in BioZate® exert antihypertensive effects. The company’s BioPureGMP™ contains 86% glycomacropeptide, an ingredient demonstrated in clinical trials to deliver multiple bioactivities including caries prevention, enhanced coagulation function, antibacterial and antiviral effects, and appetite regulation via satiety [82].

Similarly, multiple commercial products are available on the market. Vivinal® ALPHA from Borculo Domo Ingredients (Netherlands) is rich in α-lactalbumin and contains peptides with relaxing and hypnotic effects; Praventin™ from DMV International (Netherlands) is a milk protein hydrolysate mainly sourced from lactoferrin, sold as capsules for dietary supplementation and recommended for adjuvant acne treatment [27]. Other whey-based dietary supplements fortified with bioactive peptides include Dermylex™ tablets from Advitec (Canada), recommended for psoriasis treatment; Hilmar™ 8390 from Hilmar Ingredients (USA), with anti-hypertensive and anti-diabetic properties; and NOP-47™ from Glanbia Nutritionals (USA), featuring anti-inflammatory activity [83-85]. In recent years, the application of milk bioactive peptides has expanded beyond the above products to meat and sausage products. As the first step in developing functional meat products, milk protein hydrolysates have been added to pork sausage formulations. Results showed enhanced antihypertensive potential with stable effects during storage. This indicates that incorporating whey hydrolysates into pork sausages can serve as an effective approach to deliver antihypertensive peptides through animal-derived food [81].

4 Conclusion

As natural functional ingredients, milk protein peptides exhibit promising prospects in precision nutrition and health intervention. This paper systematically analyzes core preparation technologies including enzymatic hydrolysis, microbial fermentation and bioinformatics. Enzymatic hydrolysis is the preferred industrial method thanks to its high reaction efficiency and controllable products, while microbial fermentation holds unique potential for functional fortification of traditional foods. Milk protein peptides exert remarkable effects on cardiovascular health, metabolic syndrome intervention and immune homeostasis maintenance through multiple mechanisms including ACE inhibition, free radical scavenging and immune regulation. Research on their structure-activity relationships provides theoretical support for targeted peptide design. At present, this ingredient has been successfully applied in the development of antihypertensive dairy products, antibacterial dressings and anti-inflammatory dietary supplements. A variety of commercial products such as BioZate® and BioPureGMP™ have validated their health benefits.

Future research should focus on solving three core issues: 1) improving preparation efficiency via enzyme immobilization and fermentative metabolic regulation; 2) enhancing peptide bioavailability using nano-delivery systems; 3) deeply elucidating the transmembrane transport and intracellular signal transduction mechanisms of active peptides. Meanwhile, more efforts should be made to construct clinical-grade peptide libraries and conduct safety assessments, promoting the expansion of milk protein peptides from functional foods to foods for special medical purposes. Interdisciplinary innovation will accelerate breakthroughs in this field and ultimately realize full-chain translation from basic research to health products.

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