β-casomorphin-7: a review of occurrence, identification, techno-functionality, and effects on human health.

βCM-7 identification in the human body and its probable effects have gained increased attention during the last decades. The absorption of βCM-7 into the bloodstream has numerous potential effects for human health, given its opioid-like properties and capability to interact with diverse physiological systems (Table 4 ). βCM-7 is considered as an exorphin since it is an exogenous opioid peptide, in a similar category to that of morphine 110 . With the binding of βCM-7 to the active sites of μ-opioid receptors, it is anticipated that it causes fundamental variations that stimulate signal transduction, creating various biological responses 97 . The principal prospective agonist impact of opioid βCM-7 its ability to modulate motility, causing a delay in gastrointestinal transit time and an increase in mucus production 111 . βCM-7 is believed to have the capability of crossing the intestinal barrier and entering the bloodstream 4 . Certain characteristics remain ambiguous, for instance whether βCM-7 can affect responses by binding to μ-opioid receptors outside the gastrointestinal tract, and the cause-and-effect relationships associated with the dosage limits and exposure/duration of βCM-7 intake in the body, which can regulate biological functions. Table 4 Potential health effects of βCM-7 on human health Effects Potential mechanisms and conclusions References Gastrointestinal discomfort βCM-7 can modulate gastrointestinal proinflammatory responses, leading to symptoms such as abdominal discomfort and altered gut motility. βCM-7 can interact with μ-opioid receptors in the gut, which are established to modulate gastrointestinal motility. This interaction can slow down the movement of the intestines, resulting in constipation in some individuals. In addition, the regulation of gut motility by βCM-7 can lead to a range of bowel habit alterations, involving irregular bowel movements and discomfort. 169 , 170 Coronary disease βCM-7 may contribute to fat development in arteries, increasing the risk of coronary disease. βCM-7 may affect lipid metabolism, potentially leading to altered cholesterol levels. Dyslipidemia, characterized by abnormal levels of cholesterol and triglycerides, is a significant risk factor for cardiovascular disease. βCM-7 can promote fat accumulation in the arteries, contributing to atherosclerosis development. This disorder involves the buildup of fatty deposits in the arterial walls, which can cause heart attacks and strokes. 171 Immune system modulation By binding and activating the μ-opioid receptor, βCM-7 can influence immune responses, potentially leading to immune system modulation. βCM-7 interacts with opioid receptors in the immune system, which can regulate immune responses. This interaction may affect the balance between pro-inflammatory and anti-inflammatory cytokines, possibly modifying autoimmune disease progression. βCM-7 can activate the TH-2 (T-helper cell type 2) pathway, which is associated with allergic responses and chronic inflammation. This activation can exacerbate autoimmune conditions by promoting an overactive immune response. 172 , 173 Neurological effects Activation of the μ-opioid receptor in the central nervous system by βCM-7 can affect neurological functions, potentially contributing to conditions like autism and schizophrenia. βCM-7 may be at least relatively driving upregulation of µ-opioid receptors in the motor and somatosensory cortex of the brain. In addition, βCM7 can influence the antioxidant level in neuronal stem cells. 174 , 175 Milk intolerance symptoms βCM-7 interacts with gut microbiota, which can lead to symptoms of milk intolerance, especially in individuals sensitive to A1 β-casein. Lactose intolerant individuals may experience more noticeable gastrointestinal indicators when consuming A1 milk by reason of the combined influences of βCM-7 and lactose. There is continuing research into the prospective role of βCM-7 in worsening celiac disease symptoms, although the proof is not yet convincing. 120 , 176 Gut-brain axis effects The production and degradation of βCM-7 in the jejunum can affect the gut-brain axis, influencing both gastrointestinal and neurological health. βCM-7 indirectly affects the central nervous system through stress to the intestinal microbiota. The gut–brain axis theory was expanded to the microbiota–gut–brain axis to explain the influence of microorganisms on cognitive development and functioning. 169 , 177 Respiratory issues in infants βCM-7 released upon digestion is easily absorbed from immature gastrointestinal tract and, once in the circulation, it would cross the blood-brain-barrier of infants, physiologically still permeable, as is the case for the intestinal membrane. Once in the central nervous system, βCM-7 would bind to the µ-opioid receptors located in respiratory control centers, causing apnea and, as a result, death by asphyxiation. 97 Autism spectrum disorder Interaction with opioid receptors affecting brain development. βCM-7 is anticipated to impair autism symptoms by acting both on opioid, dipeptidyl peptidase-4 and serotoninergic systems. In addition, βCM-7 would act as an antagonist of 5-HT2 serotoninergic receptor, which has the function of regulating mood, learning, memory, behavior, thermoregulation and vasoconstriction. 153 , 178 Type 1 diabetes βCM-7 may modulate immune responses, leading to the destruction of pancreatic beta cells and increasing the risk of type 1 diabetes. βCM-7 can disrupt the management of insulin transmission. By binding to opioid receptors, βCM-7 may delay the normal signaling pathways of insulin, which is fundamental for retaining the levels of blood glucose. βCM-7 can affect glucose transporter proteins, including GLUT2 and GLUT4. These transporters are important for the uptake of glucose in the cells, and their disturbance can cause reduced glucose metabolism. 117 , 179 Schizophrenia Interaction with opioid receptors by βCM-7 can affect neurotransmitter balance, potentially contributing to the development of schizophrenia. 180 Potential health effects of βCM-7 on human health βCM-7 can modulate gastrointestinal proinflammatory responses, leading to symptoms such as abdominal discomfort and altered gut motility. βCM-7 can interact with μ-opioid receptors in the gut, which are established to modulate gastrointestinal motility. This interaction can slow down the movement of the intestines, resulting in constipation in some individuals. In addition, the regulation of gut motility by βCM-7 can lead to a range of bowel habit alterations, involving irregular bowel movements and discomfort. βCM-7 may contribute to fat development in arteries, increasing the risk of coronary disease. βCM-7 may affect lipid metabolism, potentially leading to altered cholesterol levels. Dyslipidemia, characterized by abnormal levels of cholesterol and triglycerides, is a significant risk factor for cardiovascular disease. βCM-7 can promote fat accumulation in the arteries, contributing to atherosclerosis development. This disorder involves the buildup of fatty deposits in the arterial walls, which can cause heart attacks and strokes. By binding and activating the μ-opioid receptor, βCM-7 can influence immune responses, potentially leading to immune system modulation. βCM-7 interacts with opioid receptors in the immune system, which can regulate immune responses. This interaction may affect the balance between pro-inflammatory and anti-inflammatory cytokines, possibly modifying autoimmune disease progression. βCM-7 can activate the TH-2 (T-helper cell type 2) pathway, which is associated with allergic responses and chronic inflammation. This activation can exacerbate autoimmune conditions by promoting an overactive immune response. Activation of the μ-opioid receptor in the central nervous system by βCM-7 can affect neurological functions, potentially contributing to conditions like autism and schizophrenia. βCM-7 may be at least relatively driving upregulation of µ-opioid receptors in the motor and somatosensory cortex of the brain. In addition, βCM7 can influence the antioxidant level in neuronal stem cells. βCM-7 interacts with gut microbiota, which can lead to symptoms of milk intolerance, especially in individuals sensitive to A1 β-casein. Lactose intolerant individuals may experience more noticeable gastrointestinal indicators when consuming A1 milk by reason of the combined influences of βCM-7 and lactose. There is continuing research into the prospective role of βCM-7 in worsening celiac disease symptoms, although the proof is not yet convincing. The production and degradation of βCM-7 in the jejunum can affect the gut-brain axis, influencing both gastrointestinal and neurological health. βCM-7 indirectly affects the central nervous system through stress to the intestinal microbiota. The gut–brain axis theory was expanded to the microbiota–gut–brain axis to explain the influence of microorganisms on cognitive development and functioning. βCM-7 may modulate immune responses, leading to the destruction of pancreatic beta cells and increasing the risk of type 1 diabetes. βCM-7 can disrupt the management of insulin transmission. By binding to opioid receptors, βCM-7 may delay the normal signaling pathways of insulin, which is fundamental for retaining the levels of blood glucose. βCM-7 can affect glucose transporter proteins, including GLUT2 and GLUT4. These transporters are important for the uptake of glucose in the cells, and their disturbance can cause reduced glucose metabolism. The prospective physiological impacts of βCM-7 on both humans and animals have been extensively investigated. Nevertheless, most of the studies focused on the inflammatory gastrointestinal responses and the adjustment of responses related to opioid receptors in the immune and nervous systems 112 . It is worth noting that globally marketed milk contains both A1 and A2 β-casein variants, and the health benefits of milk apply to the entire global population. Human βCM-7 has been detected in human milk, indicating that βCM-7 is present before β-casein digestion in the infant’s gut. Other βCMs have also been identified in the blood of pregnant and lactating women 113 , but not in non-pregnant women or men. This has paved the way to propose that these peptides might have a role in fetal and maternal health 65 , 114 . Notably, bovine and human βCM-7 vary by two amino acids at the four and five positions of the peptide chain (Fig. 4 ). These structural differences affect the opioid activity of βCM-7 115 , with bovine milk βCMs demonstrated to be at least 10 times more influential. The following subsections summarize findings from diverse sources, including animal models, in vitro studies, and epidemiological correlations. These do not establish causality and should be interpreted with caution. Several digestive discomfort concerns, such as bloating, flatulence, and abdominal distension are commonly associated with cow milk consumption, probably because of lactose intolerance 116 . µ-Opioid peptides receptors are widely distributed in the gastrointestinal tract. βCM-7 bioactive peptides released upon A1 β-casein digestion bind to µ-opioid receptors and cause intestinal inflammation, disturbing colonic microbiota, and eventually influencing the stool makeup 117 . An animal model study revealed that the consumption of A1 milk delayed the gastrointestinal transit time compared to A2 milk 118 . In lactose-intolerant individuals, lactose malabsorption and digestive comfort with lactose-containing milk were enhanced with milk exclusively comprising A2 β-casein 119 . In addition, it has been reported that βCM-7 might influence the gut microbiota, possibly disrupting the balance of beneficial microbiome 120 . A1 milk consumption can cause higher prevalence of heart diseases in the long term. Several studies have linked the increased risk of heart disease with A1 milk intake 121 , 122 . It has been reported that A1 β-casein intake advocated fat deposition in the blood vessels 13 . In addition, consumption of β-casein has been shown to have a role in the development of atherosclerosis or hypercholesterolemia in several animal studies including rabbits, rodents, monkeys, and pigs 17 . A1 β-casein milk-fed rabbits displayed higher levels of cholesterol and higher percent surface area of the artery obscured by fatty streaks compared to A2 β-casein-fed group 123 . A1 β-casein-derived βCM-7 has been shown to promote human low-density lipoproteins oxidation, which is associated with increased heart disease risk 124 , 125 . Conversely, consumption of A2 β-casein can protect against ischemic heart disease as the levels of high-density lipoproteins and low-density lipoproteins cholesterol were lower in A2 diets compared to the A1 diets. The effect of A1 milk-derived βCM-7 on the low-density lipoprotein’s oxidation or peroxidation of lipid components of low-density lipoproteins, is considered a critical step in the advancement of heart diseases 126 . No substantial differences were detected in the blood parameters of individuals who consumed A1 or A2 β-casein milk 127 . Furthermore, the protective effect of βCM-7 on cardiomyopathy in rats was reported 128 . In vitro hydrolysates of A1 and B variants increased 5-fold angiotensin-converting enzyme inhibition as compared to the hydrolysates of A2 and I variants (3-fold) 129 . This suggests that A1 milk has more hypotensive effects than A2 milk, and accordingly it could possibly be more beneficial for patients with heart failure, ischemic heart disease, and reduced contractility of the left ventricle. The proposed mechanisms include the oxidation of low-density lipoproteins and pro‑atherogenic effects facilitated by βCM‑7; these are derived from in vitro and animal models. Population‑level analysis and small human studies are inconsistent and confounded; current human data do not exhibit a causal relationship. It has been reported that A1 milk intake is considered as a risk factor for type-1 diabetes 130 . Hypothesized pathways include immune modulation and increased intestinal permeability causing autoimmunity; evidence is primarily from animal models and in vitro research. Type-1 diabetes linked to milk exposure in general and A1 β-casein particularly have been supported by animal studies. Type-1 diabetes is generally identified in children, and is described by the deficiency of insulin in the body 131 . Children consuming A1 milk during childhood are highly exposed to the risk of type-1 diabetes 132 . Animal studies revealed no differences between the consumption of A1 or A2 β-casein milk and type-1 diabetes 133 , while adverse consequences of A1 β-casein on type-1 diabetes were reported in other studies. Global level studies demonstrated that type-1 diabetes incidence is greater in individuals ingesting A1 β-casein 132 . In addition, Chia et al. 134 reported that early life exposure to A1 β-casein through the diet could modify the development to diabetes in non-obese diabetic mice. Case-control studies have shown an increase in antibodies against cow β-casein in individuals with type-1 diabetes. This immune reactivity has been linked to superior intestinal permeability and cross-reactivity with pancreatic islet cells 135 . However, other researchers and organizations, such as the European Food Safety Authority, did not find appropriate evidence to verify a direct contributory connection between A1 β-casein and type-1 diabetes. The consensus in the scientific community is that further well-designed studies are required to fully explain the relationship between type-1 diabetes and A1 β-casein. Until that time, the proof remains not clear. Ecological correlations and case-control outcomes are heterogeneous and do not confirm causality; more robust prospective trials are needed. A1 β-casein consumption has been associated with inflammatory responses, leading to metabolic suppression and lymphatic congestion 136 . It has been reported that more inflammatory substances accompanying heart diseases, asthma, and eczema were detected in A1 β-casein-fed mice as compared to A2 β-casein-fed mice. A1 milk has been shown to worsen eczema, acne, asthma upper, allergies, and respiratory infections. In addition, tonsillitis, bronchitis, and ear infections are driven by A1 β-casein. Because of its immune-disruptive and inflammatory effects, A1 β-casein tends to cause endometriosis, a disorder where the uterine lining cells proliferate outside the uterus. Consequently, A1 milk consumption might cause endometriosis and other reproductive obstacles 137 , 138 . The inflammation-inducing effects of βCM-7 were studied in an animal model. βCM-7 has been shown to induce inflammatory responses in mice intestines, probably through the TH-2 (CD + 4) pathway 137 . Besides boosting immune responses, oral supplementation of βCM-7 had improved several cytokines and inflammatory mediators like histamine and interleukin-4 in mice. A2/A2 milk-fed mice displayed immunomodulatory effects by modifying the levels of certain proteins like CD4/CD19 in B and T cells 139 . The study reported that diets enriched in A2 milk could alleviate the adverse impacts of growth-related immune variations, such as responses to vaccination and infections exposure. Through signals from T helper cells, βCM-7 existence in the intestine might initiate undesired immune responses. The specifics of the interactions associated with βCM-7 are still lacking, and this potential interaction is recommended to be considered when studying casein peptides-caused inflammation. The proposed link between βCM-7 and sudden infant death syndrome is highly speculative and based on limited animal studies and postmortem observations; no causal evidence exists. Sudden infant death syndrome, a most frequent cause of infant death, happens in infants aged between 1 month and 1 year 43 . Enzymatic degradation of casein after milk consumption releases small peptides with have higher levels of proline and higher stability against proteolysis. Owing to the undeveloped central nervous system of the infant, βCM can pass the blood-brain-barrier after absorption from the gastrointestinal tract. Hence, opioid peptides derived from the digested milk might depress the brain stem respiratory centers because of vagal nerve development and abnormal respiratory control in infants. This has been reported to be an influencing factor in apnea and sudden infant death. Further research is recommended to fully understand this process at different scales 140 . Casein-derived substances have been detected in the brain stem of infants experiencing sudden infant death syndrome, and βCMs have consequently been generally suggested as a potential causing factor 141 . Nevertheless, whether βCM-7 is explicitly responsible is uncertain. Compared to healthy infants, higher levels of βCM-7 in the blood and lower activities of active serum dipeptidyl-peptidase-4 were detected in infants with an apnea episode 142 . In humans, dipeptidyl-peptidase-4 is the only enzyme that can hydrolyze βCM-7 to X-Pro dipeptide at the N-terminus side. The high levels of βCM-7 cause the body to increase dipeptidyl-peptidase-4 activity in healthy children. Definitely, the privileges of human milk consumption are undebatable compared to baby formulas 143 . However, further studies are needed to mitigate the drawbacks of formulas feeding, specifically from the βCM-7 viewpoint. It has been reported βCM-7 injection into the blood of young rats and rabbits might cause apnoea 144 . Another study reported that after A1 bovine milk digestion by the mother, βCM-7 can transfer to the human milk and lead to life-threatening actions in infants 145 . Human studies are needed to understand the potential transmission of βCM-7 from the mother to the infant, specifically for mothers consuming higher levels of bovine milk 146 . Further research is essential before any clinical relevance can be inferred. Most studies linking βCM-7 to autism or neurological disorders are observational, dated, or based on animal models; causality remains unproven. βCM‑7 is a μ‑opioid receptor agonist and may cross the blood–brain barrier; proposed effects include modulation of neurotransmission and gut–brain axis signaling. These mechanistic pathways are biologically plausible but remain unverified in human clinical trials. It is assumed that βCM-7 can cross the blood-brain-barrier. In addition, the binding of pharmacological opioids to transporting proteins might protect them from the hydrolytic activity of blood peptidases and then could become a substrate for the carrier peptide transport system-1 in the blood-brain-barrier 97 , 147 . Since βCM-7 is a μ-opioid receptor agonist when it crosses over the blood-brain-barrier, this could initiate the corresponding receptors of the central nervous system, a fundamental factor of internal messaging systems that comprise enkephalins and endorphins. This activation would result in modified neural development or neurological syndromes 148 . Nevertheless, opioid receptors are not limited to the central nervous system, they also exist in the endocrine and immune system, peripheral nervous system, and even bone cells. Therefore, the prevalent presence of opioid receptors throughout the human body permits opioids to exert their functions from pain relief to modulation of bone metabolism. A1 β-casein intake may be associated with several mental, neurodevelopmental, and neurological syndromes. βCM-7 has been shown to be able to accumulate in autism- and schizophrenia-related sections of rat’s brain 149 . Furthermore, opioid peptides functions in human autism etiology have been investigated by Reichelt and Knivsberg 150 , while Sokolov et al. 151 reported that prolonged exposure to higher levels of bovine βCM may hinder early child development, potentially leading to autistic syndromes based on laboratory and clinical observations. Significantly higher levels of βCM-7 in the blood 145 and urine 152 of individuals with autism, schizophrenia, and women experiencing postpartum psychosis have been established previously, while in vitro tests revealed that βCM-7 may impact the expression of its receptor gene in autistic infants 153 . Even though activation of opioid receptor is considered a fundamental mechanism, βCM-7 has also been demonstrated to act as an antagonist to 5-HT2 serotonin receptors 151 , and in this manner, increasing its biological activities. Human evidence is restricted to small observational studies and indirect biomarkers; findings are insufficient and inconsistent to assume causality. Evidence remains speculative and requires confirmation through well-designed human trials. Beyond potential adverse consequences, βCM-7 and similar peptides derived from casein display various beneficial bioactivities. Studies have revealed antioxidant properties 120 , which may help alleviate the oxidative stress in various biological systems. In addition, βCM-7 and related peptides have shown ACE-inhibitory activities 154 , implying functions in cardiovascular health and blood pressure regulation. Immunomodulatory effects have also been reported 155 , including immune function enhancement and cytokine responses modulation in particular models. These findings underscore the dual nature of βCM-7 as both a functional constituent with physiological benefits and a bioactive peptide with potential health risks. Nevertheless, most evidence for these positive outcomes initiates from in vitro assays or animal studies, and human clinical validation remains limited. To clarify their practical implication in health and nutrition, future research should aim to verify these benefits in well-designed trials. The physiological effects of βCM-7 have been investigated; however, the strength of evidence varies significantly. Human clinical trials remain limited and frequently include small cohorts, decreasing generalizability. For example, trials comparing A1 and A2 milk usually report gastrointestinal consequences but lack long-term follow-up, making it a challenge to validate chronic health effects. On the other hand, animal studies provide mechanistic visions into cardiovascular and inflammatory responses; nevertheless, controlled experimental conditions and species variations limit direct extrapolation to humans. In vitro studies, though valuable for understanding the peptide-receptor interactions, they cannot replicate the complexity of in vivo digestion and absorption. In general, the evidence linking βCM-7 to conditions, such as autism, type 1 diabetes, and cardiovascular diseases is questionable. Associations reported in ecological experiments may be confounded by genetic and dietary factors. In addition, numerous studies do not explain the variability in the distribution of β-casein genotype or processing impacts on peptide release. Applying a framework such as grading of recommendations, assessment, development, and evaluation indicates that current evidence is of low to moderate quality, with high risk of bias and inconsistency among studies. To explain causality and dose-response relationships, future research should spotlight well-designed randomized controlled trials and population-based studies.

There is a substantial number of bioactive peptides released after dairy products protein digestion, which possess antioxidant, antihypertensive antimicrobial, and immunomodulatory activities 95 . Overall, bovine milk proteins-derived bioactive peptides have several health benefits to the human body. One of the major challenges associated with this assertion is to confirm their effectiveness and capability for in vivo bioavailability since their action could be affected by the gastrointestinal digestion and the ability to reach the targeted organs 96 . Numerous studies have focused on confirming and consolidating this suggestion, and as progressive investigations have been accomplished, it is conceivable to comprehend that the structural integrity of certain peptides is not compromised at gut level, in addition to their transportation through the epithelium, vindicated by structural changes, characteristics of hydrophobicity, molecular weight, size, and peptides charge 95 . All βCMs released by β-casein proteolysis comprise 4–11 amino acids, and the first three amino acids in the peptide chain remain the same (tyrosine-proline-phenylalanine), starting at position 60 from the sequence of the 209 β-casein amino acids (Table 1 ). This indicates that the primary difference among these peptides, categorized as βCMs, lies in the number of amino acids, which is determined by the further cleavage at position 3, eventually leading to the formation of different βCM types 81 . In human, the digestion of casein begins in the stomach, where digestive enzymes break down peptides of different lengths. This process continues in the duodenum and ultimately moves to the jejunal digestion, where further hydrolysis and the absorption of smaller peptides take place 97 . For protein to be absorbed in the intestine, it is required that the proteins should be hydrolyzed into amino acids or small peptides, such as di- and tripeptides 98 . Physiologically, the breakdown of dipeptides into free amino acids is facilitated by dipeptidyl-peptidase-4 99 , a cell surface enzyme found in several cell types, including endothelial cells and the brush border in the intestinal mucosa, and is present in several strains of intestinal microbiota, existing in a soluble form in the bloodstream 100 , 101 . For the A1 β-casein milk, whether homozygous (A1/A1) or heterozygous (A1/A2), a single amino acid substitution at His 67 permits proteases to break it down. This cleavage occurs in the small intestines through the activities of pepsin and leucine aminopeptidase, causing the release of tyrosine residue at the amino terminal by cutting the peptide bond between Val 44 and Tyr 60. In the meantime, pancreatin cleaves peptide bonds at the carboxyl terminal end by targeting the Ile 66-His 67 site of the A1 β-casein. This process results in releasing numerous amino acids, including βCM-7 (Fig. 4 ). Furthermore, dipeptidyl-peptidase-4 is recognized as the primary enzyme responsible for βCM-7 cleavage, as it specifically removes the dipeptide that contains proline (Pro 61) in the N-terminal peptides 4 . Fig. 4 Release of βCM-7 from A1 and A2 β-casein variants during enzymatic hydrolysis digestion in the small intestine. βCM-7 activates opioid receptors through the gut and body. Adapted from refs. 117 , 182 , 183 . Adapted from refs. 117 , 182 , 183 . βCMs have been stated to have a higher stability against the enzymatic degradation by proteinases and peptidases and also have a microbial aminopeptidases resistance because of the high proline structure 102 . The stability and degradation of βCM-7 were evaluated by using human gastrointestinal juice and porcine jejunal brush border membrane peptidases 103 . HPLC-ESI-MS was applied to monitor βCM-7 changes throughout the process of digestion. Whole βCM-7 was analyzed by using RP-HPLC. The study revealed that βCM-7 was partially digested by the digestive enzymes. Besides the identification of 3 proteolytic fragments f(60–65) YPFPGP, (f(62–66) FPGPI, and f(61–66) PFPGPI), the complete peptide molecule f(60–66) YPFPGPI was detected as well. The study also revealed that 42% of the initial peptide was broken down after 2 h of brush border membrane digestion, and 79% degradation was shown after 4 h. However, a small quantity (5%) was still noticeable after 24 h of gastrointestinal and brush border membrane digestion. In general, βCMs are good substrates for respective enzymes only. One of these enzymes is dipeptidyl-peptidase-4 (CD26), which is a cell-surface protease of the prolyl oligopeptides group. This enzyme is found in immune system cells, epithelial cells, and also exists in a soluble form in the extracellular fluids and blood 104 , 105 . Plasma dipeptidyl-peptidase-4 has been reported to hydrolyze βCM-5 into a mixture of YP (a dipeptide containing tyrosine and proline), FP (a dipeptide composed of phenylalanine and proline), FPG (a tripeptide made up of phenylalanine, proline, and glycine), and glycine 106 , while Osborne et al. 107 . revealed that βCM-7 prompt hydrolysis by the intestinal epithelium model with the release of 3 metabolites: GPI (a tripeptide made up of glycine, proline, and isoleucine), YP (a dipeptide consisting of proline and tyrosine), and FPGPI (a pentapeptide composed of phenylalanine, proline, glycine, proline, and isoleucine). Bioavailability of βCMs is mostly related to the most important factor - enzymatic resistance. It has been reported that both βCM-5 and βCM-7 can cross the abdominal epithelial cell monolayer 108 . In the presence of the active dipeptidyl-peptidase-4, both peptides were found to have a low permeability rate, while the absorption of βCMs increased by inactivation of this enzyme even 10 times in the case of βCM-7. Both brush border hydrolase activity and food ingredients can determine intact βCM-7 transport 109 . In addition, increased levels of calcium and glucose in the culture media have been shown to improve the effectiveness of peptide transport. This firmly implies that instability to intestine barrier, in addition to the high-sugar modern diets, could advocate the penetration of foods-derived opioid peptide. This also specifies a likely justification for βCM-7 deficiency in the urine and blood, upon subsequent casein consumption in healthy adults with ordinary functionality of the intestine barrier 107 .

Several genetic variants of β-casein, including A1, A2, A3, B, C, D, E, F, H1, H2, I, and G have been documented within cattle populations 8 , 41 . These variants originate from missense DNA mutations resulting in amino acid changes (Fig. 3 ). A1 and A2 variants are mostly detected, with B appearing less regularly, and A3, C, and I are classified as rare. The E variant is entirely detected in the Italian Piedmontese breeds, indicating a significant genetic indicator. In addition, the F variant has been detected in the Emilia Romagna area of Northern Italy, even though at very low incidences 8 . The structure of β-casein could be affected by the genetics and breed of cattle. Each copy of the β-casein allele in the dairy animals causes the formation of the comparable type of β-casein, such as A1/A1, A1/A2 or A2/A2. A1 type milk is produced by cows with A1/A1 or A1/A2 genotypes, whereas the A2 type milk is secreted only by cows with A2/A2 genotype 42 . In addition, A1 β-casein has the potential to produce βCM-7 throughout digestion 43 . To reduce the content of βCM-7, dairy farmers can control the composition of milk through selective breeding by selecting cow breeds that primarily produce the A2 variety of β-casein. In addition, genomic variations among individuals can also influence their susceptibility to the potential health effects associated with βCM-7, for instance gastrointestinal discomfort, metabolic disruptions, and neurological impacts. These inherited tendencies emphasize the complex interactions between genetics and the functional reactions to βCM-7, highlighting the need for tailored dietary consequences and additional investigations into its health inferences. Fig. 3 Alterations in the primary sequence of amino acids in bovine β-casein genetic variants. Adapted from ref. 181 . Alterations in the primary sequence of amino acids in bovine β-casein genetic variants. Adapted from ref. 181 . The transmutation in β-casein does not happen in purebred African and Asian cattle and is very uncommon in other mammal species involving human 42 . Asian herds, Jersey, Guernsey, goat, sheep, yaks, donkey, buffalo, camel, cattle and human milk predominantly contain the A2 variant of β-casein 44 . βCMs have been isolated from different milk sources, such as cattle, sheep, buffalo, and human but not yet isolated from camel and goat 45 . β-casein fraction is mostly found in the form of A2 in camel and goat milk 46 , and does not release βCM-7 during digestion 47 . The milk secreted by Northern European cow breeds, such as British Shorthorn, Holstein, Ayrshire, and Friesian commonly has a higher content of A1 β-casein variant. On the other hand, cow breeds from the Channel Islands and Southern France, for instance Jersey, Guernsey, Simmental, Limousine, and Charolea, in addition to African indigenous Zebu breeds, produce A2 β-casein milk 48 , 49 . It has also been reported that A1 protein variant is frequently found in the milk produced by hybridized and European cattle breeds, whereas domestic breed milks from India and other countries in Asia primarily have the A2 β-casein variant 50 . Estimating the β-casein alleles and genotypes in 657 buffaloes from 4 several Brazilian breeds revealed that all animals had the A2/A2 genotype, but not A1 allele, accordingly generating A2 type beneficial buffalo milk 51 . Similar findings conducted on 9 various Indian buffalo breeds have also been reported 52 , 53 . It has been reported that A2 β-casein allele occurrences in Guzera and Gir Brazilian breeds were 97 and 98%, respectively. Rangel et al. 54 reported 0.93 and 0.96 values for A2/A2 genotype incidences, respectively. In another study, A1/A1, A1/A2, and A2/A2 genotypes in Indian Frieswal crosses represented 17.5, 51.5, and 31%, respectively. The occurrence of the A2 β-casein allele accounted for 56.8% 55 . In Indian Vrindavani crosses, genotype occurrences of A1/A1, A1/A2, and A2/A2 were 12.3, 48.1, and 39.6%, respectively. The frequencies of A1 and A2 β-casein alleles represented 0.364 and 0.636 56 . In addition, Kumar et al. 55 reported that the high A2/A2 genotype frequency of Indian domestic cattle breeds applied in the crossbreeding program was responsible for the prevalence of the A2 allele despite years of crossbreeding. Because of the low levels of A1 β-casein of the milk produced by local Turkey breeds, the formation of βCM-7 did not happen and A2 category original healthful milk was attained 46 . The frequencies of A1/A2 genotype have been shown to be not species-specific but are specific to the geography and region. For instance, A1 variant occurrence in Holstein Friesen cows in Northern Europe and North America was more that 90%, whereas German Holstein Friesen cows displayed higher occurrence (97%) of the A2 variant. In other countries, the prevalence of A1 incidence in Holstein Friesen breeds varied between 40 and 65% 57 . The frequencies of A2 β-casein (60%), A1 β-casein (30%) in Italian Holstein Friesian cattle breeds were defined as A1/A1 (9.88%), A1/A2 (35.79%), and A2/A2 (36.96%) 8 . In addition, the frequencies of A1 and A2 alleles in Czech Holsteins respectively represented 0.45 and 0.55, while genotype frequencies were reported as A1/A1 (0.20%), A1/A2 (0.51%), and A2/A2 (0.29%) 58 , β-casein A1 and A2 alleles respectively represented 0.396 and 0.604 in Serbian Holstein-Friesian breeds, and the genotype incidences were determined as A1/A1 (12.26%), A1/A2 (54.72%) and A2/A2 (33.02%) 59 . A1/A2 β-casein alleles in Chinese Holstein cattle accounted for 0.432 and 0.459, respectively, and the genotype incidences were reported as A2/A2 (0.226%), A1/A2 (0.353%), and A1/A1 (0.203%) 60 . In another study, β-casein A1/A1, A1/A2, and A2/A2 alleles in Holstein Friesen Indian cow breeds were reported to be 0.216, 0.451 and 0.333%, respectively, and the genotype incidences were determined as A2 (0.559%) and A1 (0.441%) 61 . Furthermore, Gholami, et al. 62 reported that the frequencies of A1 and A2 β-casein alleles in Iranian Holstein Friesen were 0.50%, while Dinc, et al. 63 revealed that frequencies in Turkish Holstein Friesen accounted for 0.485 and 0.456% for A1 and A2, respectively. The levels of βCM-5 and βCM-7 in 30 human milk samples were evaluated in colostrum and after 1 and 4 months postnatal 28 . The study revealed that the colostrum displayed 8 to 9-times higher levels of βCM-7 compared to the milk taken after 1 and 4 months postnatal, respectively. The incidence of βCM precursors in the plasma and cerebrospinal fluid of women throughout pregnancy period and after postnatal was established. No immunoreactive βCMs was found in the plasma of non-pregnant women and men 64 . Also, it has been reported that the plasma levels of immunoreactive βCM-8 increased throughout pregnancy and continued to rise during the first week after delivery 64 . Similarly, significantly higher amounts of both βCM-5 (5.03 µg/mL) and βCM-7 (3.10 µg/mL) were found in human colostrum than in mature milk (0.58 and 0.33 µg/mL, respectively). In addition, there were no significant differences in the levels of βCMs in the milk obtained after 2 months of lactation and that collected after 4 months of lactation 65 . βCM-7 levels in A1/A1, A1/A2, and A2/A2 Holstein Friesian cows were evaluated in the first lactation 20 . Milk samples were collected after 30, 100, and 200 days of lactation, and then hydrolyzed by using different enzymes. The study showed that the milk of A1 variant cows hydrolyzed by a mixture of pepsin, trypsin, and elastase displayed higher βCM-7 values as compared to the raw milk. The study also revealed that the A2 variant milk had lower βCM-7 values throughout the experiment periods. It has been suggested that the incidence of βCM-7 in the milk secreted by A2 variant cows is undoubtedly attributed to the acid hydrolysis throughout the digestion of β-casein with pepsin. The same research also reported that βCM-7 values in the different variants of A1/A1, A1/A2, and A2/A2 increased after high temperature processing of the raw milk. In another study, raw milk samples from different breeds, ages, and lactation periods were analyzed for βCM-7 content 37 . The study revealed that there were substantial variations in the content of βCM-7 among milk samples, and the differences were attributed to the effects of breed, age, and lactation period of the cattle. With the progress in Holstein Friesian cows’ age, the levels of βCM-7 in milk declined. The Jersey breeds milk displayed significantly higher levels of βCM-7 in the first lactation animals compared to that in the milk obtained from second lactation animals. In addition, the number of peptides was also affected by the lactation stage, with higher numbers in the first week of lactation and reduced numbers in later lactation stages. Heat treatment is a crucial step in the dairy industry from a quality and safety perspective. In addition to inhibiting the growth of both pathogenic and spoilage microorganisms, heat treatment can also modify the conformation of milk proteins that constructively influences the viscosity and texture characteristics of dairy products 66 , 67 . In yogurt manufacture, secondary heating along with pasteurization are usually completed at 90–95 °C for 5 min 68 , leading to whey proteins denaturation, and accelerating the interactions between κ-casein and β-lactoglobulin 69 . The formed milk complex affects the proteolysis throughout the process of fermentation 70 . Also, the thermal processing causes the degradation of large proteins, producing lower molecular weight peptides 71 . Peptides formation from casein was not only caused by enzymatic action, but also occurred during the proteins backbone fragmentation throughout thermal treatment 72 . The same study detected five peptides not originally found in raw milk after it was heated for 30 min at 120 °C. Then, matrix-assisted laser desorption/ionization time of flight-mass spectrometry (MALDI-TOF-MS) was applied to identify the casein-originated peptides at mass-to-charge ( m / z ) values of 974.4, 2218.7, 3730.1, 4297.8, and 4436.8. None of these values corresponded those of βCMs, demonstrating that simply heating the raw milk didn’t release βCM. Thermal treatment of milk under these conditions inactivates the enzymes and is unlikely to significantly change the pH of milk, consequently peptides formation by acid hydrolysis and enzymes can be ruled out. Conversely, during heat processing of milk, the Maillard reaction might release substances that can break down the peptide bonds of milk proteins, yielding new peptides 73 . The development of new peptides throughout thermal processing depends on the thermal strength of β-casein peptide bonds 74 . βCM-7 release and stability in thermal-treated milk have been reported 20 , 27 . The content of βCM-7 in commercially pasteurized, ultra-high temperature (UHT), and in-bottle-sterilized milk was evaluated. It was shown that these products were free from βCM-7, although the products have A1 and B β-casein variants, which comprise βCM-7 sequence. Likewise, the content of βCM-7 was determined by using ELISA in several milk species including pure A1, A2, and a mixture of A1 and A2 β-casein variants after pasteurization (95 °C/20 min) and sterilization (117 °C/5 min). The content of βCM-7 tended to decline after milk heating not because of the degradation of βCM-7 peptide bonds, but due to the reactions between amino acids residues of βCM-7 and lactose, which decreased the binding affinity of the modified βCM-7 to the antibody used in ELISA, leading to undervalued results for βCM-7 content 20 . Thus, heat processing might alter the β-casein and facilitate the formation of βCM-7 in dairy products 22 , 75 . Thermal processing of milk at 85 °C/30 s or 140 °C/5 s decreased βCM-7 release in both skimmed and full-fat (either homogenized or unhomogenized) samples both in A1 and A2 milk 22 . The properties of heat load were slightly different than previously detected in another study, concluding that compared with pasteurization, UHT treatments caused an increase in βCM-7 production from A1/A1 milk although this result was not detected in A2/I milk, and the formation of βCM-7 in raw milk was not evaluated 5 . Generally, thermal processing of milk seems to delay the βCM-7 formation despite the genetic origin of milk. Probably this is the consequence of proteins denaturation induced by heating, which has been explained to influence milk proteins digestion kinetics 76 . Conversely, it is not clear if the impact of reduced βCM-7 release is initiated by a reduced β-casein hydrolyzes or due to the increased hydrolyzes of β-casein and βCM-7 into lesser fragments 22 . Microbial activity can initiate the formation of βCM-7 during the manufacture of fermented dairy products, such as cheese and yogurt 77 , 78 . The degree of βCM-7 release depends on fermentation conditions and the microbial strains utilized. Fermentation is a very critical step in milk processing into yogurt and cheese. Besides pH reduction, fermentation can also cause several alterations in milk composition brought about by glycolysis, lipolysis, and proteolysis 79 . These effects are affected by starter culture type, temperature and time of incubation, in addition to ripening time. Through fermentation, starter culture strains can release various bioactive peptides from milk proteins, including βCMs released from β-casein. Certain βCMs have been isolated from cheese types. Using Lactobacillus acidophilus L10, Lacticaseibacillus casei L26, and Bifidobacterium lactis B94 to produce probiotic yogurt 80 and Streptococcus thermophilus and L. bulgaricus for traditional yogurt fermentation 27 did not result in the development of βCM-7 or other βCMs. It has been reported that a mixture of S. thermophilus and L. bulgaricus used for traditional yogurt making enriched in PepX might break down proline-rich βCMs into many smaller peptides 36 . Fermentation of yogurt also implies a decline in the pH from 6.8 to 6.3 during first stages of fermentation, and βCM-7 might be degraded by PepX if released at this stage. Accordingly, the stage during which βCM-7 is formed or degraded in yogurt made by using S. thermophilus and L. bulgaricus remains unidentified. The interaction between κ-casein and whey proteins by disulfide bonds is well recognized to increase the viscosity of yogurt made from milk heated at 85–90 °C/5 min 81 . On the other hand, the formation of inter/intra-molecular disulfide bonds during thermal treatment is not probable for this protein due to the insufficient content of cysteine in β-casein fraction 82 . Nevertheless, β-casein modification can be achieved via enzymatic covalent cross-linking by tyrosine, glutamine, and lysine residues through transglutaminase and tyrosinase addition 83 . Yoghurt texture and proteolysis could be affected by transglutaminase 84 . It has been reported that milk fortification with transglutaminase decreased the content of peptides in yogurt, with no differences in the profile of peptides from transglutaminase-treated milk and untreated milk 85 . In addition, the content of peptides in transglutaminase-treated milk, which was still active throughout fermentation, was lower than that in transglutaminase-treated milk that had been inactivated during fermentation. Inter-molecular cross-linking throughout yogurt fermentation is associated with the increase in lactic acid, which decreases the stability of casein 86 . As shown in Table 1 , βCMs contain more tyrosine residues that have the propensity to induce enzyme-catalyzed cross-linking; consequently, the production of βCMs from β-casein might be reduced by milk treatment with cross-linking enzymes. Because of its proline-rich sequence and μ-opioid receptors affinity, βCM-7 can affect gelation profile, foam stability, and water-holding capacity through modifications in the proteolysis outlines and peptides-proteins interaction throughout fermentation and processing 75 , 87 . On the contrary, angiotensin converting enzyme (ACE)-inhibitory peptides derived mainly from casein or whey sequences caused by enzymatic hydrolysis or fermentation mostly contribute physiological antihypertensive activities 88 , 89 via renin-angiotensin-aldosterone system regulation with minimal direct influence on rheology, excluding when co-generated within extensive hydrolysates that may soften the gel or increase seruming 90 . Lactoferrin, a multifunctional glycoprotein, has several immunomodulatory, iron-binding, and antimicrobial properties and can moderately influence emulsions stability through surface activity 91 , 92 , but does not typically drive the microstructure of casein gel the way small proline-rich peptides may throughout ripening process. In addition, βCM-7 is distinct in coupling techno-functional characteristics (processing-dependent peptides generation/degradation influencing the texture) with bioactivity. ACE-inhibitory peptides and lactoferrin are more specified for health functionality and safety-profiled purposes, such as infant nutrition 93 , while their direct textural roles are generally limited or matrix-dependent. The degree of βCM-7 techno-functional effects is relative, dependent on the genotype (A1/A2), heat history, protease specificity, fermentation strains, in addition to the product type (cheese vs. yogurt). Hence, generalizations should be kept away without product-definite statistics. The worldwide interest in A2 β-casein milk has driven labeling and branding approaches that emphasize reduced βCM-7 release potential as compared to A1 β-casein milk 94 . While consumer demand has extended, regulatory positions caution against causal health requests, put emphasis on the necessity for transparent, evidence-associated messaging. There are several dairy formulation schemes, for instance (1) selective breeding for A2/A2 herds to reduce the potential formation of βCM-7; (2) processing control, including thermal behavior, pH adjustment, and selection of the starter cultures to limit proline-rich peptide persistence; (3) targeted enzymatic hydrolysis to decrease intact βCM-7 while conserving the product texture; and (4) infant formulas design bearing in mind the β-casein phenotype and proteolysis control, with cautious risk-benefit assessment. Furthermore, several companies progressively address the protein phenotype and the digestive comfort consequences, but the specific disease-risk statements are improper without robust randomized controlled trials. Innovation opportunity could be attained through integrating comparative peptidomic and digestibility analysis into product development in order to navigate functionality versus bioactivity trade-offs. βCM-7 is one among numerous dairy-derived bioactive peptides, incorporating antioxidant, ACE-inhibitory, and immunomodulatory sequences, and proteins such as lactoferrin and the milk fat globule membrane constituents. βCM-7 acquires attention for opioid receptor intervention and controversial connections; however, additional bioactives have apparent human-trial support for immunity, blood pressure, or gut barrier roles. Placing βCM-7 in this reasonable outline underlines two significances: (i) standardized in vitro digestion and ex vivo/clinical bioavailability models to benchmark physiological applications; (ii) well-driven, phenotype-controlled randomized controlled trials to explain benefit-risk and translate results to industry applications. This wider viewpoint supports evidence-based product design and responsible statement in the dairy sector.

The content of βCMs and their precursors in various milk species and dairy products has been quantified using different analytical methods. The analysis of βCM-7 in both fresh and enzymatically hydrolyzed bovine milk showed that the level of βCM-7 in hydrolyzed A1 milk was 4-fold higher than A2 milk, while traces of βCM-7 were detected in the non-hydrolyzed fresh milk 19 . Furthermore, Duarte-Vázquez et al. 21 and Lambers et al. 22 detected small quantities of βCM-7 after A2 milk β-casein digestion. In another study, the amount of βCM-7 in A1/A1 β-casein variant hydrolysate was 3.2-fold higher than that in A1/A2 variant hydrolysate, while no βCM-7 of the A2/A2 variant was detected, after enzymatic digestion 16 . In addition, higher levels of βCM-7 were released from raw milk proteins as compared to heat-treated milk 22 . The structural variations between bovine β-casein A1 and A2 affect the techno-functional properties of milk by influencing protein aggregation and product formation during manufacture. A2 β-casein, has been correlated with reduced gelation and enhanced foaming, resulting in weaker gel products with a diverse network structure because of its higher content of ionic calcium. β-casein composition and structure variations can substantially affect the characteristics of milk preparations under different processing conditions 26 . The profile of opioid peptides derived from β-casein has been determined in fermented milks and various cheese types. βCM-7 has been identified in fermented milk drinks, such as kefir and natural yogurt 27 , 28 . The peptides content could be strongly affected by several factors, for instance fermentation time and storage conditions, resulting in a low peptides content 29 . Also, precursors of βCMs or βCM-9 and βCM-10 have been detected in different cheese types, such as Gouda, Blue, Brie, Limburger, and Swiss cheeses, but the degradation during the ripening process of mature Cheddar cheese resulted in no detection of these precursors 30 , 31 . The presence of βCM-7 in other cheese varieties, such as Cheddar, Gouda, Gorgonzola, and Fontina has been also reported 27 . Most of these results were qualitative. Nevertheless, according to the existing data, it appears that the content of βCM-7 in Dutch-type semi-hard cheese types that are ripened for longer periods is lower than its content in short-ripening soft cheese varieties. Further, the presence of βCM-like and morphiceptin-like activities or specifically, βCM-5 and βCM-7 in infant formulas has been reported 32 . Moreover, an infant formula was developed using A2 milk where the levels of βCM-7 were considerably lower than that in other investigated formulas involving A1 milk-based formulas 21 . The content of βCM-7 in milk and different dairy products is summarized in Table 2 . Table 2 A summary of βCM-7 content in milk and different dairy products Source βCM-7 content unit Sample and data sets Reference Unprocessed milk 1.16 mg/Kg A total of 27 commercial dairy products, including 10 cheese samples, 4 drinking milk samples (unprocessed, pasteurized, UHT and in bottle-sterilized), 2 yogurts and 4 fermented milks containing probiotics, 1 sample of skim milk powder, 1 sample of calcium caseinate, and 2 milk protein concentrates, and 7 infant formulas (4 powder and 3 liquid products) 27 Whole pasteurized milk 0.64 Whole UHT milk 0.96 Skimmed in-bottle-sterilized milk 0.60 Yogurt 0.29–1.23 Cheese Brie 2.07 Caprine 1.5 Fontina 15.22 Gorgonzola 5.41 Gouda 21.77 Infant formula powder 0.04–0.21 mg/L liquid 0.12–0.18 A2/A2 milk 0.95–3.45 µg/mg 10 Polish Holstein-Friesian cows held in one herd and milked in the same season were included 18 A1/A1 milk 10.68–12.73 Holstein Frisian cow milk 0.76–2.61 ng/g 30 samples of raw milk were collected from different breeds 37 Jersey cow milk 1.80–7.73 Commercial cow milk 14 μg/L 10 milk samples from local markets in Ireland 39 Cheese Brie 0.60 mg/Kg 12 different cheese samples were selected. All samples were collected at the Italian market and represented industrial products 157 Gouda (2.5 months) 0.59 Gorgonzola (2 months) 7.63 Cheddar (4 months) 0.27 Milk powder A1/A1 2.7 ng/mL 177 Holstein–Friesian cows were included. Cows were kept in the same herd and fed with a balanced diet. Out of 177 cows, 18 cows were selected for the analysis. Cows were in their first lactation and were milked in the same lactation season 20 A2/A2 0.90 A1/A2 0.80 Pasteurized milk A1/A1 3.3 A2/A2 3.6 A1/A2 6.1 Sterilized milk A1/A1 2.9 A2/A2 5.4 A1/A2 7.4 Cow milk heated at 73 °C/20 s; 85 °C/5 min; 121 °C/12 min β-casein A1/A1 141.45–75.95 ng/mL 8 milk samples collected from different cow breeds 5 β-casein A2/I 19.35–24.50 β-casein A1/A1 127.25–198.10 Human milk colostrum 0–26.78 µg/mL 82 samples were collected from 30 breastfeeding mothers at 3 different time points: Colostrum (3–5 days after delivery): 30 samples. Milk 1 (1 month after delivery): 30 samples. Milk 2 (4 months after delivery): 22 samples 65 2 months 0–1.5 4 months 0–2.84 A summary of βCM-7 content in milk and different dairy products Milk and dairy products are complex food matrices, in which proteins, lipids, and lactose can interfere with extraction and identification of the targeted peptides, causing inaccurate qualitative and quantitative results. For the analysis of amino acids and peptides, reversed-phase high-performance liquid chromatography (RP-HPLC) has been widely used 33 – 35 . Several analytical techniques have been applied to determine the presence and quantity of βCM-7 in milk and dairy products (Table 3 ). Atlan et al. 36 evaluated the effects of heat treatment on βCM-5 and βCM-7 after in vitro digestion of milk demonstrating A1/A1, A2/A2, and A2/I β-casein phenotypes. The milk was treated at 73 °C/20 s, 85 °C/5 min, and 121 °C/12 min, and then subjected to in vitro gastrointestinal digestion. βCM-5 and βCM-7 were identified by using the ultra-performance liquid chromatography-mass spectrometry/mass spectrometry (UPLC-MS/MS) and further confirmed by using the ultra-high-performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS). βCM-7 was not detected in milk samples having the A2/A2 β-casein variant. However, the level of βCM-7 was in the range between 127.25 and 198.10 ng/mL in heated milk with A1/A1 β-casein variant, while it was detected at much lower levels in the range between 19.35 and 24.50 ng/mL in heated milk with A2/I β-casein variant. Table 3 A summary of the analytical techniques used for the analysis of βCMs from milk and various dairy products Matrix βCM Sample preparation Analytical method Limits of detection Key findings Reference Cow milk βCM-7 β-casein was isolated and purified from milk, and βCM-7 was separated on a C18 column at 45 °C LQ-MS 0.01 fmol/μL The concentration of βCM7 was ~4 mg/g β-casein for A1, compared to 1.4 mg/g for A2 and I 38 βCM-5; βCM-7 Milk was spiked with 200 ng of βCM5/7-s10, acidification, centrifugation, filtration, dilution, and peptides recovery by using SPE cartridges LQ-MS 0.32 ng/g for βCM-5 and 0.24 ng/g for βCM-7 βCM7 was detected in 21 out of 30 raw samples, ranging from 0.76 to 8.41 ng/g milk. βCM5 was unequivocally identified in 3 raw milk samples, at 0.4–0.64 ng/g milk 37 βCM-7 Milk samples were hydrolyzed by pepsin/trypsin/elastase. Extraction with ethanol/chloroform followed by shaking, centrifugation, freeze-drying, and purification on C18 SPE cartridges ELISA - 22 cows were characterized by the A1/A1 genotype, 86 were characterized by the A2/A2 genotype and 69 cows were characterized by the A1/A2genotype 20 βCM-7 Milk samples were spiked with 200 ng of βCM-5-d10 and 200 ng of βCM-7-d10. Centrifugation and SPE cartridges were applied UHPL-HRMS - βCM7 was released after gastrointestinal digestion of heated milk with β-casein A1/A1 phenotype, ranging from 4.94 to 7.70 ng/mg protein. βCM7 was released after gastrointestinal digestion of heated milk with β-casein A2/I phenotype, ranging from 0.71 to 0.91 ng/mg protein 5 βCM-7; βCM-9; βCM-13; βCM-21 Enzymatic digestion, chromatographic fractionation and purification HPLC-UV (280 nm) - βCM-7 was released from a genetic variant of β-casein containing a His residue at the 67th position, not from the more common Pro67 variant. Longer βCMs like βCM-9, -13, and -21 were isolated, and the enzymes responsible for their release were identified 158 Human colostrum, milk βCM-5; βCM-7 Extraction with methanol/chloroform followed by shaking, centrifugation, freeze-drying, and purification on C 18 SPE cartridges HPLC-UV (280 nm) 0.8 μg/mL βCM-5 and 0.6 μg/mL βCM-7 The content of βCM-5 and βCM-7) was significantly higher in human colostrum than mature milk. βCM-5 concentration was 5 times higher and βCM-7 was eight times higher in colostrum than in mature milk 65 Yogurt βCM-5; βCM-7 Water soluble peptides were extracted from yogurt by centrifugation and acidification. βCM-7 separation was achieved using a Kinetex C18 column Isotope dilution liquid chromatography-tandem mass spectrometry 0.5 and 0.25 ng/g for βCM-5 and βCM-7 The method had good accuracy (95-109% recovery) and precision (1-16% RSD). Neither βCM-5 nor βCM-7 were detected in commercial yogurt. βCM-5 decreased from 1.3 ng/g in milk to <MLQ in yogurt after 1 day storage. βCM-7 decreased from 1.9 ng/g in milk to <MLQ in yogurt after processing 29 βCM-9 Homogenization, removal of sugar and lactic acid on cation exchanger columns LC-MS/MS - Yogurt contained a high amount of free amino acids like His, Tyr, Thr and Lys, which have antioxidant properties 159 βCM-13 Extraction, trisodium citrate dehydrate and dithiothreitol, centrifugation, purification on SPE cartridges micro LC-TOF-MS - 50 bioactive peptides in the yogurt sample were identified. C18 cartridge showed higher extraction efficiency for bioactive peptides compared to the STX cartridge, based on the abundance ratios 160 Cheese βCM-7 Water-soluble extract was prepared, and peptides were separated by WIDEPORE XB-C18 column UPLC/HR-MS - The total level of βCM in cheese samples ranged from undetectable to 11.74 mg/kg, with Gorgonzola containing the highest amount 157 βCM-5; βCM-7 Extraction with chloroform/methanol, centrifugation, fat removal, and freeze-drying, and purification on C 18 T SPE cartridges ELISA 11.10 −3  μg/mL βCM-5 13.10 −3  μg/mL βCM-7 All cheese varieties contained both agonistic opioid peptides (βCM-5 and βCM-7) and antagonistic opioid peptides (casoxin-6, casoxin-C, and lactoferroxin A) 161 βCM-11 Water-soluble extract of cheese was prepared, ethanol was added, and finally fractionation with calcium chloride HPLC-QIT-MS operated in MS scan and MS/MS full scan - The peptide YPFTGPIPN, identified in fraction 19 of the Caprino del Piemonte cheese, had an elevated homology with βCM-11 (YPFPGPIPNSL), a multifunctional, mainly opioid, peptide isolated from intestinal chyme 162 βCM-9 Homogenization with water following by centrifugation, dialyzation, freeze-drying HPLC-GPC Fast atom bombardment-MS - 4 peptides from 8-months aged Gouda cheese 163 βCM-9; βCM-10 Extraction with deionized water, centrifugation, paper filtration, freeze-drying HPLC-TOF-MS LC-MS/MS - Quantitative determination of 49 putative taste-active metabolites in 4- and 44-week-ripened Gouda cheese 31 βCM-7 Extraction with chloroform/methanol, gel filtration Ion exchange chromatography - Based on amino acids composition and minimal mole ratios and on the N- and C-terminal amino acids, the peptide isolated from the peptide extract of Brie cheese was most probably βCM-7 164 βCM-3; βCM-4; βCM-5; βCM-6; βCM-7 Extraction with chloroform/methanol, centrifugation, fat removal, and freeze-drying HPLC-UV (220, 254, 280 nm) 2 μg/mL (βCMs) A major peak at 34.6 min, corresponding to βCM-7 in the augmented sample. Recovery of βCM in the organic extract, using HPLC calculated peak area and the βCM-7 calibration line, was 55 ± 12% 165 Fermented milk βCM-4 Extraction with chloroform/methanol, absorption/desorption on charcoal/evaporation HPLC-UV (220 nm) LC-MS MS scan (200–800  m/z ) - Chromatogram profiles showed differences in peptide elution time, concentration and number. An opioid peptide, βCM 1–4 (60–63), was detected in a peptide extract derived from the fermented milk 166 Infant formulas βCM-5; βCM-7 Milk coagulation, centrifugation, filtration, and freeze-drying HPLC-MS/MS MRM transitions ( m/z ) βCM-5 580.3 → 408.2; 286.0 βCM-7 790.3 → 381.1; 30.2 - All commercial infant formulas contained variants A1, A2 and B of β-CN and released 16-297 nmol βCM-7 per 800 L, the recommended daily intake 167 Cheese, milk, yogurt, infant formulas, dried milk βCM-5; βCM-7 Heating samples, pH adjustment, centrifugation, filtration, freeze-drying, then peptides were extracted by mixing with 0.1% trifluoroacetic acid in deionized water, centrifugation, and filtration HPLC-MS/MS MRM transitions ( m/z ) βCM-5 580.3 → 408.2; 286.0 βCM-7 790.3 → 381.1; 30.2 - βCM-7 was detected only in cheeses, except for Taleggio, Caprino, and Grana Padano, with levels ranging from 0.01-0.15 mg/kg. Infant formula digests contained βCM-7 at 0.04–0.21 mg/L 27 Milk and cheese βCM-5; βCM-7; βCM-10; βCM-11 Extraction, fat removal, purification on polymeric SPE C 18 cartridges Nanoelectrospray with ion-trap mass spectrometry - NanoMate-QIT-MS method provided good linearity ( r 2  > 0.999) and reproducibility (RSD ≤ 7%) for quantification of βCM in spiked milk and cheese samples. The method was able to detect and identify βCM-7 in a milk sample from a local supermarket 39 Enzyme modified cheese βCM-7 Centrifugation, methylene chloride and water, concentrated by Speed Vac HPLC-API-MS - Enzyme-modified cheese process simultaneously produced bioactive peptides that might provide savory, nutritional, medical, and health benefits. βCM stability may be dependent on the type of enzyme in the system 168 ELISA enzyme–linked immunosorbent assay, HPLC-API-MS high-performance liquid chromatography combined with atmospheric pressure ionization-mass spectrometry, HPLC-GPC high-performance liquid chromatography-gel permeation chromatography, HPLC-UV high-performance liquid chromatography-ultraviolet detector, LQ-MS liquid chromatography-tandem mass spectrometry, SPE solid-phase extraction, UHPL-HRMS ultra high-performance liquid chromatography-high resolution accurate mass Orbitrap™ mass spectrometry. A summary of the analytical techniques used for the analysis of βCMs from milk and various dairy products βCM-5; βCM-7 0.32 ng/g for βCM-5 and 0.24 ng/g for βCM-7 Milk samples were hydrolyzed by pepsin/trypsin/elastase. Extraction with ethanol/chloroform followed by shaking, centrifugation, freeze-drying, and purification on C18 SPE cartridges βCM-7; βCM-9; βCM-13; βCM-21 βCM-5; βCM-7 βCM-5; βCM-7 βCM-5; βCM-7 11.10 −3  μg/mL βCM-5 13.10 −3  μg/mL βCM-7 βCM-9; βCM-10 βCM-3; βCM-4; βCM-5; βCM-6; βCM-7 βCM-5; βCM-7 βCM-5; βCM-7 βCM-5; βCM-7; βCM-10; βCM-11 ELISA enzyme–linked immunosorbent assay, HPLC-API-MS high-performance liquid chromatography combined with atmospheric pressure ionization-mass spectrometry, HPLC-GPC high-performance liquid chromatography-gel permeation chromatography, HPLC-UV high-performance liquid chromatography-ultraviolet detector, LQ-MS liquid chromatography-tandem mass spectrometry, SPE solid-phase extraction, UHPL-HRMS ultra high-performance liquid chromatography-high resolution accurate mass Orbitrap™ mass spectrometry. βCMs peptides including βCM-5 and βCM-7 in raw cow milk were determined using LC-MS/MS 37 . The levels of βCM-5 ranged from 0.40 to 0.64 ng/g milk, while βCM-7 was in the range between 0.76 and 8.41 ng/g milk. In another study, LC-ESI/MS was applied to identify βCM-7 profile in skimmed Danish Holstein cow milk 38 . Four milligrams of βCM-7 per one gram of β-casein were detected after 120 min duodenal digestion of the A1 β-casein variant as compared to 1.4 mg from A2 and I β-casein variants. In addition, LC-MS/MS was also used to quantify βCM-5 and βCM-7 in yogurt 29 . The study showed that βCM-7 decreased from 1.9 ng/g in milk to less than the method limits of detection in yogurt immediately after fermentation. In another study, ELISA was used to study the content of βCM-7 as affected by milk pasteurization and sterilization 20 . The results showed that raw milk processing at high temperatures slightly affected the variations of βCM-7 originating from various β-casein genotypes. Juan-García et al. 39 reported that quadrupole ion-trap mass spectrometry (QIT-MS)-ESI-MS enabled interpreting the major fragmentation pathways for βCMs and structures for the major fragment ions that were perceived in the MS. In a recent study, Caira et al. 40 identified the profile of βCM-5, βCM-6, and βCM-7 in various bovine milk blue cheeses, and described the mechanisms involved in the release of these peptides, considering their source from cheese with A1 and A2 β-casein variants. The nanoLC-ESI-Q-Orbitrap-MS/MS and developed computational devices were employed to analyze the peptidomes profile of Stilton, Gorgonzola, Bleu d’Auvergne, and Bergader blue cheeses made from milk comprising both A1 and A2 β-casein variants. This methodology established the incidence of βCM-7 in all cheese types, even though at different levels. The study revealed that all cheese types having A1 and A2 β-casein variants displayed proteolysis, leading to an obvious release of βCM-7 due to the intensive action of several Penicillium roqueforti exopeptidases. Gorgonzola cheese displayed different βCM-7 levels, which might be attributed to the variations in milk composition, microbial populations, or cheese-making conditions, which could affect the proteolytic formation of βCM-7 throughout ripening. Remarkably, substantial levels of A1 and A2 β-casein derived βCM-8 precursors, specifically (60 Tyr-Pro-Phe-Pro-Gly-Pro-Ile-His 67) and (60 Tyr-Pro-Phe-Pro-Gly-Pro-Ile-His 67) were detected among the different types of cheese samples. This underscores the dynamic nature of cheese ripening, during which precursors of βCM-8 might release bioactive βCMs, whereas βCM-5, βCM-6, and βCM-7 undergo further proteolysis.

βCM-7 is a bioactive peptide released by the A1 form of β-casein fraction during digestion or cheese making (fermentation), but not by A2 form. The amino acids sequence of βCM-7 is Tyr 60 -Pro 61 -Phe 62 -Pro 63 -Gly 64 -Pro 65 -Ile 66 . βCM-7 is a known μ-opioid receptor agonist that may affect the gastrointestinal functioning directly and may also exert influences elsewhere in the body, such as on the neurological, cardiovascular, and endocrine systems. In this review, we have aimed to highlight the incidence, composition, and identification procedures of βCM-7, as well as the techno-functional properties of βCM-7. The content of βCM-7 in milk and dairy products could be affected by several factors, such as genetics, animal breed, lactation number and period, processing methods, and cross-linking. The potential health effects of βCM-7 are complicated, including both beneficial and adverse effects. While some studies suggested its responsibility for supporting gut health and immune responses regulation, others raise concerns about its association with disorders, such as diabetes, cardiovascular diseases, and autism. The complexity of βCM-7 impacts on human health highlights the necessity for further research to clarify its mechanisms of action and long-term implications. As the dairy industry continues to progress, understanding the distinctions of βCM-7 will be critical for the development of safer and more beneficial dairy products. Upcoming studies should be targeted to specify clearer understandings into the balance between βCM-7 health benefits and risks, eventually enabling better dietary recommendations and public health guidelines. Future research should spotlight randomized controlled trials to explain whether βCM-7 exerts clinically relevant outcomes on human health, as current evidence is largely associated.

Current evidence proves that A1 and A2 β-casein vary by a single amino acid substitution at the position 67 (His vs Pro), which affects peptides release throughout the process of digestion. βCM-7 is a heptapeptide (Tyr-Pro-Phe-Pro-Gly-Pro-Ile) mainly released from the A1 β-casein, and its presence in milk and different dairy products has been validated using robust analytical methods, such as the liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), ELISA, and the HPLC. Furthermore, established techno-functional impacts include variations in foaming and gelation characteristics between A1 and A2 milk, supported by experimental data. On the other hand, relationships between βCM-7 and health consequences, such as type-1 diabetes, autism, and cardiovascular diseases remain controversial and hypothetical. These connections are principally based on mechanistic probability, for instance opioid receptor binding and gut-brain axis signaling, and the epidemiological associations, which are not consistent and often confounded. Evidence from animal models and small experimental studies cannot verify causality, and findings should be interpreted with risk avoidance. In addition, well-designed randomized controlled trials should be highlighted to explain whether these associations have clinical significance.

In order to ensure reproducibility and transparency, a comprehensive literature search was carried out according to the PRISMA 2020 guidelines. The databases searched involved PubMed, Web of Science, Scopus, Google Scholar, etc. to improve systematic coverage. In addition, the search was conducted by using patterns of keywords and Boolean operators, such as (“β-casomorphin-7” or “βCM-7”) and (“A1 β-casein” or “milk peptides”) and (“techno-functional properties” or “health effects”). The search screened publications up to 2025. Furthermore, reference lists of appropriate articles were checked for additional sources. The studies were incorporated in this review if they met specific criteria, such as (1) focused on βCM-7 occurrence, identification, techno-functional characteristics, and/or health influences in milk and different dairy products; (2) reported animal studies, human clinical trials, or in vitro experiments related to βCM-7; (3) published in English and in peer-reviewed journals; and (4) applied analytical approaches such as high-performance liquid chromatography/mass spectrometry (HPLC-MS), enzyme–linked immunosorbent assay (ELISA), or comparable practices. The exclusion criteria were studies that are not definitely addressing βCM-7; non-peer-reviewed sources, such as conference abstracts; publications in languages other than English; and studies lacking adequate quantitative data or methodological detail. All the reclaimed records were introduced into reference management software, and the duplicate studies were removed. In addition, the titles and abstracts were independently screened. Then, full-text articles were evaluated for eligibility according to predefined measures. Inconsistencies were resolved through discussion. Figure 2 shows the PRISMA flow diagram that explains the identification, screening, eligibility, and inclusion levels. The diagram reports the records number at each stage and the exclusion reasons. Fig. 2 Research flowchart describing articles selection process using the PRISMA approach for systematic reviews and meta-analyses. Research flowchart describing articles selection process using the PRISMA approach for systematic reviews and meta-analyses. To enhance the methodological rigor, human clinical trials were distinguished from animal and in vitro studies. Evidence quality was graded using the GRADE approach, taking into consideration factors, such as study design, bias risk, consistency, and directness. In addition, high-quality evidence, for example randomized controlled trials were given higher weight in the discussion, whereas limitations of lower-quality evidence were obviously noted. The following data were extracted for each included study; (1) study type (human, animal, in vitro), (2) sample size and population characteristics, (3) analytical methods used for βCM-7 detection, (4) key findings on the techno-functional characteristics and health effects, and (5) quality rating. In addition, the data were narratively synthesized because of the heterogeneity in the study designs and outcomes. The findings were grouped by thematic relevance and evidence type where possible.

Milk is considered as an exceptional nutritional resource, offering substantial amounts of proteins, fats, and carbohydrates (lactose), along with essential micronutrients like minerals and vitamins, making it a valuable component in human dietary practices 1 . Mature bovine milk protein is categorized into two main groups: casein and whey proteins at an approximate ratio of 80:20 2 . Casein proteins can be further classified into four fractions, specifically α s1 -, α s2 -, β-, and κ-casein, in a ratio of 4:1:4:1, respectively 3 . In bovine milk, β-casein represents ~40% of the total caseins and one third of the total protein content. β-casein can be further subdivided based on the variations in its amino acids profile, which are determined by the CSN2 gene located on chromosome 6 4 . According to the amino acid substitution arising from the cleavage site in the polypeptide chain, 12–15 genetic variants of β-casein exist, with variants A1 (His 67) and A2 (Pro 67) predominant in modern cattle, which incidence depends on the genetics of cow 2 , 5 . The difference between these variants is a mutation in the amino acid polypeptide chain at the position 67 6 , 7 . β-casein variants can be separated into two of the most prevailing families, specifically A1 family, including A1, B, and F variants; and A2 family, involving A2, A3, and I variants 6 . A single nucleotide polymorphism in modern European cattle several millennia ago led to the prevalence of the A1 β-casein variant (155.2 g/mol), despite all cattle initially originating from the A2 β-casein variant (115.1 g/mol), considered the oldest β-casein proteoform 8 . Limited information exists regarding the structural and functional disparities between β-casein proteoforms, beyond the β-casein polypeptide chain composition. The distinctions between A1 and A2 β-casein genetic variants have sparked discussions about Holstein-Friesian cattle genetic selection 9 , the impact of these variants on the physicochemical properties of milk and dairy products 10 , milk yield and composition 11 , and market dynamics 12 . β-casomorphin-7 (βCM-7), a bioactive opioid peptide, has gained considerable relevance in the field of health and nutrition sciences. The digestion of β-casein by digestive enzymes, such as pepsin and pancreatic proteases is the main factor to release this peptide. Among the two predominant variants of β-casein (A1 and A2), βCM-7 is mainly released from the A1 variant, which is found in the milk of specific cow breeds, for instance Friesian and Holstein. On the other hand, the A2 variant, being present in some other breeds, such as Jersey and Guernsey, does not release βCM-7 after digestion 13 . βCMs are peptides containing 4–11 amino acids (Table 1 ), all of them start with tyrosine residue, which plays a critical role in their opioid activity 14 . The chemical structure of the different βCM types is shown in Fig. 1 . βCM-7 heptapeptide was the first extracted, and most frequently identified, and its sequence is associated with the section 60–66 of the parent protein 15 . The amino acids sequence of βCM-7 is as follow: Tyr-Pro-Phe-Pro-Gly-Pro-Ile. It has been reported that after in vitro simulated digestion, βCM-7 is released by continuous proteolytic digestion of A1 β-casein due to the action of pepsin, pancreatic elastase, and leucine aminopeptidase 16 . The difference between A1 and A2 β-caseins arose from a single nucleotide polymorphism of β-casein genes and replacement of proline by histidine in A1 β-casein. The substitution of this amino acid caused conformational changes in the expressed secondary structure of protein, thereby affecting the physical characteristics of the particular casein micelles 17 . The peptide bond found between histidine and isoleucine in A1 β-casein has a lower enzymatic resistance as compared to that found between proline and isoleucine in the A2 β-casein. Accordingly, the A1 variant is more easily hydrolyzed, leading to βCM-7 release 18 . In vitro simulated digestion has confirmed the release of βCM-7 from A1/A1 β-casein and A1/A2 β-casein 19 . Conversely, Cieślińska et al. 20 and Duarte-Vázquez et al. 21 revealed that small quantities of βCM-7 could also be released from A2 β-casein. βCM-7 release from both A1 and A2 β-caseins has been also reported by Lambers et al. 22 , while Haq et al. 16 , didn’t detect βCMs in hydrolyzed A2 milk. Furthermore, other in vivo studies have also reported the presence of βCM-7 in the jejunum of healthy individuals who consumed bovine milk or casein 23 . The parental protein variant was not indicated in the study; however, it was predictable that the amount of βCM-7 was adequate to prompt its biological activities. Fig. 1 Chemical structures of different βCM types. Data collected from PubChem database; https://pubchem.ncbi.nlm.nih.gov . Table 1 A list of β-casomorphins, including their amino acids sequence, molecular formula, and molecular weight. Data adapted from Kim et al. 156 β-casomorphins Amino acid sequence Molecular formula Molecular weight (g/mol) β-casomorphin 3 Tyr-Pro-Phe C 23 H 27 N 3 O 5 425.48 β-casomorphin 3-amide Tyr-Pro-Phe C 23 H 28 N 4 O 4 424.5 β-casomorphin 4 Tyr-Pro-Phe-Pro C 28 H 34 N 4 O 6 522.61 β-casomorphin 4-amide Tyr-Pro-Phe-Pro C 28 H 35 N 5 O 5 521.62 β-casomorphin 5 Tyr-Pro-Phe-Pro-Gly C 30 H 37 N 5 O 7 579.60 β-casomorphin 6 Tyr-Pro-Phe-Val-Gly-Pro C 38 H 49 N 6 O 10 749.8 β-casomorphin 7 Tyr-Pro-Phe-Pro-Gly-Pro-Ile C 41 H 55 N 7 O 9 789.93 β-casomorphin 8 Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro C 46 H 62 N 8 O 10 887.0 β-casomorphin 9 Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro-Asn C 50 H 67 N 9 O 13 1036.18 β-casomorphin 10 Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro-Asn-Ser C 55 H 72 N 10 O 13 1185.36 β-casomorphin 11 Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro-Asn-Ser-Leu C 60 H 79 N 11 O 14 1334.54 Amino acids abbreviation: Tyr tyrosine, Pro proline, Phe phenylalanine, Gly glycine, Ile isoleucine, Asn asparagine, Ser serine, Leu leucine. Chemical structures of different βCM types. Data collected from PubChem database; https://pubchem.ncbi.nlm.nih.gov . A list of β-casomorphins, including their amino acids sequence, molecular formula, and molecular weight. Data adapted from Kim et al. 156 Amino acids abbreviation: Tyr tyrosine, Pro proline, Phe phenylalanine, Gly glycine, Ile isoleucine, Asn asparagine, Ser serine, Leu leucine. The possible health consequences of βCM-7 have been a topic of contention. The interest in βCM-7 is attributed to its prospective physiological attributes that are facilitated by the interactions between βCM-7 and the μ-opioid receptors widely distributed in the human body. The endogenous opioid system, which is composed of these receptors, plays a fundamental role in the regulation of reward, pain, and numerous physiological activities 24 . Due to the ability of βCM-7 to interact with these receptors, several studies investigated its effects on immune functions, abdominal health, and neurological disorders. It has been suggested that βCM-7 might be associated with gastrointestinal discomfort, particularly in individuals with gastrointestinal concerns like lactose intolerance 25 . βCM-7 can cross the blood-brain-barrier and impact the functions of the central nervous system, presenting alarms about its potential task in particular neurological diseases, such as schizophrenia and autism. Despite the increasing research efforts, the potential health effects of βCM-7 remain debatable. It is crucial to note that most reported associations between βCM-7 and human disorders are correlational, not causal, and command validation through well-designed clinical trials. Some studies claimed that the effects of βCM-7 are insignificant and only applicable in certain inhabitants, whereas others emphasized the necessity for further comprehensive analyses to entirely understand its effect. The variability in βCM-7 release from different β-casein variants makes this issue more complicated. In this context, the present review aims to provide an insightful assessment of the current state of knowledge of βCM-7, embracing/encompassing its biochemical characteristics, mechanisms of action, identification, and analytical methods. In addition, the factors affecting the content of βCM-7 and its potential effects on human health are also highlighted.