Casomorphins
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What is the casomorphin?
A class of bioactive opioid peptides known as casomorphins is produced during the breakdown of casein, a primary protein in milk. The digestive or fermentation processes in the intestines release these peptides. Casomorphins are defined as any opioid released from casein during the digestive process. The human health concern pertains to casomorphins derived from beta-casein, namely the release of bovine beta-casomorphin-7 (BCM7) from cow's milk. They influence physiological processes by binding to opioid receptors, especially mu-opioid receptors (MOR). The β-casomorphins, which are generated from β-casein, are the casomorphins that have been extensively researched. The ones that fall under this category are BCM-7, BCM-5, and variations of BCM-7.
Enhanced beta-casomorphin-7 release from A1 beta-casein in cattle. (Woodford K B., 2021)
Beta casomorphin
β-casomorphins (βCMs) are a class of peptides that share a key tyrosine residue that determines their opioid activity; their chains range from 4 to 11 amino acids long. The BCM-7 heptapeptide, which was the first to be extracted and thereafter recognized most frequently, has a sequence that matches that of the parent protein's fragments 60–66. Pepsin, pancreatic elastase, and leucine aminopeptidase sequentially proteolytically degrade β-casein A1 and B (but not A2) in simulated gastrointestinal circumstances in vitro, yielding β-CM-7. The variation in results is caused by a change in the β-casein gene (SNP67) and the substitution of histidine for proline in the A1 region of the protein. Casein micelles' physical characteristics may be affected by the conformational differences in the expressed protein secondary structure caused by this amino acid substitution. The A2 variant's proline–isoleucine peptide link is also more enzymatically resistant than the A1 variant's histidine–isoleucine peptide bond. Thus, β-CM-7 is released as a result of the easier hydrolysis of A1 β-casein. In vitro digestion of A1A1 and A1A2 milk β-casein with simulated gastrointestinal digestion (SGID) verified the release of β-CM-7. Despite this, it's worth mentioning that β-casein A2 could potentially produce very minute amounts of β-casomorphin-7. Recent confirmation of β-casomorphin-7 release from A1 and A2 milk β-caseins was made by β-CMs in hydrolyzed A2 milk. It was also shown in the jejunum of healthy humans who consumed bovine milk or casein that β-casomorphin-7 was present in vivo. Even though the authors didn't say which variation of the parental protein it was, they calculated that there was enough γ-casomorphin-7 to make it biologically active.
The sequence of amino acids that make up beta-casomorphin-7 (bBCM7) is as follows: tyrosine, proline, phenylalaline, proline, glycine, proline, and Isoleucine. The first two amino acids are crucial, and the third and fourth amino acids both ensure that it will have an opioid nature. And because those three proline amino acids are so close together, bBCM7 can't be internally cleaved at the C-terminus by peptidases anymore. Consequently, dipeptidyl peptidase-4 (DPP4) is an essential enzyme for the proper N-terminal degradation of bBCM7. Even though bBCM5 is a stronger opioid than bBCM7 in vitro, shorter-chain casomorphins are not very useful in vivo due to their resistance to enzymatic degradation at the C-terminus.
The amino acid composition of the β-casomorphins formed in cow's milk. (Cieślińska A., et al., 2022)
The influence of β-casomorphins. (Daniloski D., et al., 2021)
β-Casomorphin function
Regulate lipid metabolism and blood glucose
Ischemic heart disease (IHD) is the predominant coronary ailment in humans, and the association between A1 β-casein milk consumption and IHD has been rigorously examined. Data from WHO, FAO, and evidence-based clinical research indicate that the consumption of A1 milk is a risk factor for IHD. The experimental results indicate that β-casomorphin-7 provides a protective effect against rodent diabetic cardiomyopathy by inhibiting hyperglycemia and oxidative stress. Moreover, in vitro studies utilizing cardiac preparations revealed the cardiostimulatory effect of BCM; the dietary inclusion of β-casomorphin-7, coupled with a lifestyle characterized by smoking, alcohol consumption, and saturated fat intake, may elevate the risk of heart disease. Epidemiological evidence indicates that the consumption of milk containing A1 beta-casein is linked to the onset of hypercholesterolaemia and atherosclerosis. Research including animal studies, which compared cholesterol levels in groups fed A1 and A2 milk, indicated that cholesterol levels were elevated in the A1 milk-fed animals compared to those consuming A2 milk. The deposits on the aortic surface occupied a greater area, whereas the administration of A1 beta-casein to rabbits markedly augmented the percentage of the aorta's surface area affected by fatty streaks and lesions in the aortic arch. Conversely, broiler hens administered casomorphin exhibited elevated levels of very-low-density lipoprotein, increased plasma triglycerides, and significant overall body weight with substantial abdominal fat deposits, indicating its direct influence on fat metabolism. The data indicated that the meticulously regulated laboratory environment was devoid of various confounding influences. Casomorphin has a direct correlation with the beginning of atherosclerosis based on these data.
Induce inflammatory response
The effect on allergy signaling was investigated because β-casomorphin controls cellular metabolism. Researchers discovered that the MUC5AC glands in the gut produced more mucus after consuming milk, which can release β-casomorphin when digested. The use of human breast milk in another investigation showed that the amount of IL-8 secreted in Caco-2 cells was three times higher in reported allergic milk hydrolysate. Conversely, infant formulas that were tested showed that exposure to hydrolysate of allergy milk disrupted bacterial adhesion. Additionally, these formulas had an impact on the intestinal barrier status and caused tight junction dysfunction in the intestinal epithelium due to calcium ions. There is evidence that direct injection of casomorphin into the systemic circulation alters the immunological response, according to reports that surfaced twenty years ago. These volunteers showed that β-casomorphin-7 directly released histamine in a dose-dependent manner. The observed histamine release opens the door to the potential therapeutic use of these peptide classes, which include the ability to stimulate gastric acid production in mammalian cells, dilate capillaries, and contract smooth muscles. An intriguing discovery detailing genome-wide changes in global DNA methylation patterns was noticed in the quest to explain the cause for the modularity in cells and tissues generated by β-casomorphin. Results from a genome-wide microarray expression assay in cultured human neuroblastoma cells and a next-generation sequencing method for the analysis of methyl-CpG binding domain (MBD) protein-enriched genome sequencing (MBD-seq) suggested that epigenetic mechanisms are altered in casomorphin and related milk allergies.
Promote neurite growth
β-casomorphin-5 is involved in the neurite cell line of neuro-2a mouse neuroblastoma cells. It occurs via activation of μ-opioid receptors; such activation is specific, and it does not involve activation by casomorphins of any length. β-casomorphin-5 to 9 have the minimum length of occupying the μ-opioid receptor and gives affinity in stimulating the neurite outgrowth. β-casomorphin-3,4 and [D-Pro2]-β-casomorphin-5 do not possess such affinities to activate such receptors, although they are known to possess opioid activities. Besides, the presence of Tyrosine at the N-terminal of the peptide is necessary for the best fit into the binding site of the opioid receptors. Neurite outgrowth does not involve the elevation of cAMP in response to β-casomorphin stimulation of G protein-coupled mu-receptors but by the activation of the PI3K/MAPK signaling pathway. β-casomorphin-5 to 9 can be neurotransmitters and neuromodulators, influence neural development, and are dose-dependent. The release of β-casomorphin from milk digestion would enter the systemic circulation in newborn animals and may pass through the blood–brain barrier to play a role in the growth of neurons among the newborn during suckling.
Enhance innate immunity
Mucus production contributes to the development of innate immunity, which protects the epithelial-cell linings of the respiratory and gastrointestinal systems from infections. According to new research, β-casomorphin-7 helps activate the mu-receptor on neurons, which in turn triggers the release of mucin, which protects organs. To the contrary, β-casomorphin-7 enhances mucin gene expression in rat intestinal mucin-producing cells (rMuc2 and rMuc3); conversely, animals deficient in mucin genes are more likely to be directly attacked by bacteria, which can lead to inflammation and cancer infection. The fact that the mu-receptor antagonist naloxone reversed the mucus discharge caused by β-casomorphin-7, indicating that β-casomorphin activates the opiate receptor, is strong evidence of this. As a result, peptides generated from milk products, particularly β-casomorphin-7, can enhance mucus output and boost innate immunity; however, they can also enter the respiratory tract and upregulate the MUC5AC gene expression, leading to asthma and sinusitis in individuals with weakened immune systems. Although β-casomorphin-7 use is associated with a rise in mucus secretion, which might exacerbate asthma symptoms, this same secretion would normally defend the body from potential infectious viral assaults.
Regulate T-cell proliferation
It is known that immunopeptides made from β-casein can activate or repress different immune cells, therefore modulating immunological functions. Lymphocyte proliferation is either suppressed or stimulated by β-casomorphin-7, depending on the concentration. When T-lymphocytes were treated with BCM peptides, they suppressed the growth of such cells at a lower dosage of around 10 mM. They were encouraged to multiply when these peptides were exposed to a significantly higher quantity during incubation. The administration of the µ-receptor antagonist naloxone can reverse the impact of β-casomorphin-7 doses that suppress the proliferation of human colonic lamina propria lymphocytes. The high-affinity binding of β-casomorphins to μ-opiate receptors may explain the formation of T-lymphocytes in cell-mediated immunity. The solution to the paradoxical immunomodulatory functions of β-casomorphin peptides was found in sheep colostrum, where the proline-rich peptides demonstrated resistant and differentiating actions on T-lymphocytes that were both distinct and conflicting with one another. β-casomorphin-7 has shown comparable effects on T-cell proliferation in vitro and is rich in proline at alternative locations.
Schematic illustration of overall pharmacological actions of β-casomorphin (BCM). (Thiruvengadam M., et al., 2021)
Transport and bioactivity β-casomorphins. (Daniloski D., et al., 2021)
Isolation and production of Casomorphin
In 1979, β-casomorphin 7 was initially extracted by Henschen's group from commercial β-casein using enzymatic digestion. To determine the peptide's composition, scientists utilized high-performance liquid chromatography (HPLC), paper chromatography, and gel-filtration chromatography. They found that the heptapeptide is hydrophobic because its N-terminal tyrosine is primarily occupied by prolines at alternate places (YPFPGPI). The heptapeptide lost its opioid efficacy due to structural changes that occurred during acid hydrolysis, acetylation, and enzymatic digestion, according to tests conducted on this peptide. The heptapeptide produced from β-casein following enzymatic digestion is called β-casomorphin 7, and the carboxypeptidase Y comparable digest that follows is called β-casomorphin -5, because it is similar to that one and has morphine-like action. The structure and properties of β-casomorphin-7 are identical to those of morphiceptin-enkephalin, which is generated from β-lipotropin, and this is because β-casomorphin-7 has been sequenced from β-casein.
In 2015, Haq's laboratory conducted research on the release of BCMs from the milk of Karan Fries crossbred cattle. The crossbred cattle were initially tested using PCR-RFLP for the presence of the A1 and A2 β-casein alleles. The animals that were homozygous for A1A1 β-casein, heterozygous for A1A2 β-casein, and homozygous for A2A2 β-casein were genotyped and their milk was collected thereafter. Analysis was performed at analytical RP-HPLC after SGID with pepsin, trypsin, and chymotrypsin. Assays using analytical RP-HPLC were calibrated against commercially produced BCM-5 and BCM-7. Retention durations of 24 and 28 minutes, respectively, were shown by the two chemically produced standard peptides BCM-5 and BCM-7. To show peaks at this retention durations, the hydrolysates were tested on analytical RP-HPLC. The peptide fractions were freeze-lyophilized after being collected at the intervals indicated by the retention durations on the preparative HPLC. Neither BCM-5 nor BCM-7 were detected in any of the peaks following MS-MS analysis. Nevertheless, fourteen amino acid residues of a precursor peptide sequence were successfully isolated. A seven-amino acid peptide derived from A1A1 and A1A2 milk was evaluated using competitive ELISA and an isolated organ bath after additional digestion of this fraction with the precursor sequence using elastase and leucine aminopeptidase. There was no evidence of BCM-5 in any of the milk varieties tested. When tested on rat ileum, the opioid activity of the produced peptides ranged from 0.534 to 0.595 μM for the homozygous A1 version and from 0.410-0.420 μM for the heterozygous variant, confirming the existence of BCM-7.
Release of BCM-7/5 in b-casein hydrolysates. (Haq M R U., et al., 2015)
Casomorphin uses
Regulate insulin secretion
The administration of β-casomorphin-7 in canines has demonstrated that it primarily inhibits insulin secretion, resulting in a fast decline in respiration akin to the effects of morphine on the central nervous system. Studies indicate that insulin secretion is activated at low doses of 1 nmol/kg; however, insulin secretion levels remain unchanged when the concentration of β-casomorphins employed for stimulation is elevated to 100 nmol/kg. This is not relevant to the activation of β-casomorphin-4; nonetheless, the tetrapeptide must be administered solely in its original structure, excluding any structural analogs that could impede insulin production. The use of the opiate receptor antagonist, naloxone, would entirely suppress all insulin secretion. The same was observed when β-casomorphin-5 was utilized to assess somatostatin release in dogs, indicating that the consumption of a diet containing β-casomorphin-5 stimulates somatostatin levels and contributes to the regulation of postprandial endocrine function in the intestine.
As the sleep inducers
Physiological tension and anxiety have been demonstrated to cause β-casomorphins, utilized as sleep inducers, to reduce blood pressure in neonatal rats experiencing insomnia, hence maintaining their calmness and relaxation. Following an intraperitoneal injection of β-casomorphins in 7-day-old rats, an analgesic impact was observed on the neurological system, leading to significant alterations in sleep patterns and inducing tranquility. The ratio of quiet state (characterized by the absence of body movement, save for infrequent startles) to total recording duration increased, whereas the percentage of movement during sleep diminished, illustrating the behavioral effects of BCMs and alterations in sleep patterns in male rats.
Reduce oxidative stress
BCM-7 mitigates oxidative stress in streptozotocin-induced diabetic rats. In control rats, levels of superoxide dismutase (SOD) and catalase were elevated; however, the group administered BCM-7 post-diabetes onset exhibited diminished oxidative stress, characterized by reduced NF-kB and iNOS gene expression, alongside an increase in antioxidant enzyme activity, including catalase and SOD. Consequently, it is proposed that BCM-7 may confer protection against oxidative stress in diabetes patients by obstructing the NF-KB-iNOS-NO signaling pathway in the pancreas. An increase in glutathione peroxidase activity was also noted, whereas plasma insulin levels fell, maintaining glucose levels to safeguard against hyperglycemia in diabetic rats.
β-Casomorphin-7 influence on human body systems. (Cieślińska A., et al., 2022)
References
- Cieślińska A., et al., Does a little difference make a big difference? Bovine β-casein A1 and A2 variants and human health—an update, International Journal of Molecular Sciences, 2022, 23(24): 15637.
- Woodford K B. Casomorphins and gliadorphins have diverse systemic effects spanning gut, brain and internal organs, International journal of environmental research and public health, 2021, 18(15): 7911.
- Daniloski D., et al., Bovine β-Casomorphins: Friends or Foes? A comprehensive assessment of evidence from in vitro and ex vivo studies, Trends in Food Science & Technology, 2021, 116: 681-700.
- Thiruvengadam M., et al., β-Casomorphin: A complete health perspective, Food Chemistry, 2021, 337: 127765.
- Ul Haq M R., et al., Structure and Production of Casomorphins, Opioid Food Peptides: Significant Exorphins from Food Sources, 2020: 21-38.
- Haq M R U., et al., Release of β-casomorphin-7/5 during simulated gastrointestinal digestion of milk β-casein variants from Indian crossbred cattle (Karan Fries), Food chemistry, 2015, 168: 70-79.
