Bioactives in milk
It has been long acknowledged that the bioactives in colostrum, the first fluid a mother makes soon after giving birth, are vitally important for the vulnerable newborn, to protect the undeveloped immune system against infections and stimulate growth and development, especially of the gastrointestinal tract. Colostrum milked from cows just after they have calved has been shown to provide the bioactive protectants found in mother’s milk and is marketed as a supplement to boost immune and digestive systems.
Milk also contains bioactives shown to have important benefits, much of which are attributed to antibodies. Milk proteins found in the whey component of milk have been found to have antimicrobial and immunomodulatory properties as well as having important roles in iron absorption and calcium transport. As scientific research has progressed more bioactive proteins have been discovered in milk as well as a vast array of bioactive protein fragments or peptides that are encrypted in major milk proteins. These bioactives are released by enzymatic digestion, either naturally, in the gastrointestinal tract, or by commercial processing.
Caseins are the main protein component in cows' milk and are the major source of amino acids for the new born. Three major subgroups of casein have been identified; alpha casein, kappa casein and beta casein; which are all present and bound together in micellular structures in milk. It is thought that steps utilised in milk processing, such as pasteurisation and homogenisation, may affect the casein micelle size and structure thus affecting the digestion and subsequent yield of bioactives. Additionally, the renneting process involved in the manufacture of hard cheese is thought to modify the proteins, effectively inactivating the bioactivity of the resultant digested fragments.
The peptides derived from casein digestion or processing display a range of biological effects; Bioactive peptides, released by enzymatic digestion of caseins include the Casokinins, which have antihypertensive properties, and Casoplatelins which have antithrombotic properties. A variety of peptides derived from beta caseins have been identified which have a range of biological activity including antihypertensive effects, the modulation of amino acid absorption in the gut and the modulation of various immune responses. More details of the bioactives from milk can be found in a review published in 2000, titled "Effects of milk‐derived bioactives: an overview", which presents a summary of peptides encoded in and yielded by milk proteins [1].
A range of milk proteins have also been found to yield opioid peptides which demonstrate sedative and analgesic effects, these include the casein derived Casomorphins. Further information can be found in a review published in 2000 [2] which describes in detail a range of peptides with opioid function that derive from all the casein subgroups, as well as from the whey fraction proteins alactalbumin, b‐lactoglobulin and serum albumin. Of particular interest are the opioid peptides derived from beta caseins, the beta casomorphins, which have particularly strong opioid properties.
Bioactives from beta casein variants
The difference in structure between the major beta (ß) casein variants, A1 and A2 results in a differential yield of bioactive peptides being produced during their digestion.
The difference between ß‐caseins A1 and A2 is in a single amino acid in a strand of 209. Beta casein A1 has Histidine while A2 has Proline in position 67 of the strand. This difference is also displayed between minor variants, which on the basis of this amino acid substitution and known digestion of beta casein variants A1 and A2, may be classified as 'A1 like' or 'A2 like'. These variants include: A3, D and E which, like A2, contain a Proline at this position; and B, C and F which, like A1, contain a Histidine at amino acid residue 67.
The natural mutation that gave rise to this difference is a result of a single nucleotide polymorphism at codon 67 of the ß‐casein gene; CCT (A2, Proline) ‐ CAT (A1, Histidine). It is has been demonstrated that this one amino acid difference affects the proteolytic processing of the beta casein in the mammalian gut. In vitro studies ,[3] have shown that the bioactive peptide beta casomorphin 7 (BCM‐7) is yielded by the successive gastrointestinal proteolytic digestion of beta casein A1, but not A2, resulting in differential levels of bioactives produced by the two major types of beta casein variants.
Derived from residues 60‐66 of A1 (and related variants), BCM‐7 is produced by A1's successive proteolytic digestion by pepsin, pancreatic elastase and leucine amino peptidase. Previous studies support the theory that this occurs in vivo. [4] [5] Theoretically this difference in amino acid sequence suggests a conformational difference in the secondary structure (folding of the amino acid chain) of the expressed protein, which subsequently may have ramifications on the physical properties of the respective casein micelles, further affecting digestion and properties of the caseins.
Beta Casomorphin (BCM-7)
Beta casein A1 has been shown to yield the protein fragment, or peptide, termed 'beta casomorphin 7' (BCM‐7). First reported in 1979 [6] as a casein derived opioid, BCM‐7 can subsequently be metabolised to beta casomorphin 5 (BCM‐5). These peptides have the 3rd highest and highest affinity, or binding, to opiate receptors of those reviewed [1].
BCM‐7 has been shown to be significantly more resilient to metabolic processing [7] than other studied food derived exomorphins [i]. Thus, by removing beta casein A1 from milk and thereby eliminating the yield of BCM‐7 the risk presented by opioid peptide yield from milk may be significantly reduced.
Both public [8] and private reports to a2 Corporation from consumers known to be prone to exomorphin triggered symptoms of autistic spectral disorders have detailed that when they consume a2 Milk™ (exclude A1) they find a noticeable, and in many cases, significant decrease in symptoms. Additionally, further reports that individuals exhibiting other undesirable effects following consumption of standard but not a2 Milk™ can be hypothesised to be casomorphin mediated.
These reports, coupled with published research, lead a2 Corporation to believe that the exclusion of beta casein A1 from milk products may render exomorphin yields physiologically insignificant.
BCM‐7, or further truncated forms such as BCM‐4 and BCM‐5, have been shown to have a number of effects both in vitro and in vivo, a selection of which are discussed at the end of this section. It is of note that the BCMs are established opioids, effectors of the μ‐opioid receptor which is expressed in a broad range of tissues, and have other noted effects on components of the vascular and immune systems.
It must be noted that a significant number of studies have been performed on casomorphins 4, 5, & 6. These are truncated forms of BCM‐7, and have very similar properties with regard to opioid like activity. This is supportive of the notion that the 4 NH3‐terminal residues of BCM 7 (and thus 4‐6) are important for μ‐opiate receptor binding and modulation. A comprehensive, but slightly dated, text [9] collates a number of these studies.
It is thought that biologically available BCM‐7 is truncated by exo‐peptidases (in the gut, circulation and/or tissue) thus generating BCM's 4‐6. Though one cannot state that these casomorphins are absent following the in vivo digestion of A2, it would seem plausible that the formation of BCM‐7 following A1 digestion may greatly increase the yield and thus bio‐availability of these related peptides relative to digestion of A2.
In general beta casomorphins are very stable with regard to enzymatic degradation, prone only to rapid degradation by dipeptidyl‐peptidase IV (DPP IV). DPP IV is a cell‐surface protease belonging to the prolyloligopeptidase family. It selectively removes the N‐terminal dipeptide from peptides with proline or alanine in the second position.
DPP IV is expressed on a specific set of T lymphocytes, where it is up‐regulated after activation. It is also expressed in a variety of tissues, primarily on endothelial and epithelial cells. A soluble form is present in plasma and other body fluids. It plays a role in glucose homeostasis through proteolytic inactivation of the incretins. DPP IV inhibitors improve glucose tolerance and pancreatic islet cell function in animal models of type 2 diabetes and in diabetic patients.
The role of DPP IV within the immune system is a combination of its exopeptidase activity and its interactions with different molecules. [10] Two points of note to keep in mind when considering BCM‐7 are studies that indicate that BCM‐7 has a long half life relative to other casomorphins, and the knowledge that BCM‐7 and the truncated BCM‐5 have the strongest relative affinities to opioid receptors [11]. A study on rats, published in the Journal of Dairy Science concluded that beta casomorphin is not likely to be a focus of addiction.[12]
Human beta-casomorphins
As by definition previously stated, human breast milk beta casein by virtue of amino acid 67 can be classified as A2, thus the yielding of BCM‐7 is not favoured during digestion.
Other casomorphins of varying chain lengths can nonetheless be released and it is reported that high concentrations of beta‐casomorphin‐like peptides are found in the cerebrospinal fluid and plasma of women with postpartum psychosis. Micro‐purification of human beta‐casomorphin‐8 from the milk of a woman with postpartum psychosis is further suggestive of a link [13].
Human beta‐casomorphin‐8 (BC8), BC‐immunoreactivity (BCIR) was detected in rostro‐caudally increasing levels in nineteen microscopically distinct and functionally relevant areas of mesencephalon, pons cerebri, and medulla oblongata of eight infants [14]. Data in the literature, together with those of this study indicate that beta‐casomorphins could be transported by specific mechanisms from the blood into the brain stem and that they could play a role in the central regulation of various physiological phenomena.
Regardless of comparative physiological function between human and bovine derived casomorphins, it is important to note that human beta‐casomorphin‐5 (Tyr‐Pro‐Phe‐Val‐Glu), owing to its amino acid composition is about ten times less potent than bovine beta‐casomorphin‐5 (Tyr‐Pro‐Phe‐Pro‐ Gly) [15]. Furthermore it was noted in this study monitoring the behaviour of rats, that approximately ten times more naloxone was required to antagonize the beta‐casomorphin‐5 effect than that of morphine.
Physiological characteristics of casomorphins
It should be stated that currently there presents two primary physiological effects of BCM-7: opiatelike activity, which has been widely reported; and the oxidation of LDL or peroxidation of a lipid component of LDL [16], regarded as a determining step in the development of heart disease.
The former activity may be more readily apparent with respect to physiological response and may serve to facilitate the transport of macromolecules, however it is believed that the latter observed activity of BCM-7 has a much stronger bearing on the development of arterial lesions and plaques. Absorption may not be necessary to elicit all biological effects. This is suggested by a study reporting that BCM-4 modifies the peristaltic reflex by reducing the efficacy of the reflex response and modulating the timing of the reflex pathway via opiate receptors.
This shows that casomorphins could have a local effect on the gut wall with no need for systemic absorption. [17] The theoretical scope for biological and physiological responses to exomorphins is increased dramatically if they are absorbed into circulation and transported to tissues and organs. This occurrence of such phenomena due to the ingestion and subsequent digestion of beta caseins is supported by a number of reports.
Precursors to BCM-7 have been detected in the plasma of new born cows following milk intake [18] and in new born, but not, adult dogs [19]. Additional to this, larger peptides derived from caseins have been shown to be absorbed and detected in human plasma [20]. This demonstrates the ability of the BCM-7 to be transmitted into circulation following digestion, notably in newborn or young mammals. Studies on the (non BCM specific) trans-‐epithelial transport of oligopeptides suggest absorption to be at least partially mediated by brush-‐border peptidases [21].
Further to this, studies have implicated BCM-7 to be transported across the blood-‐brain barrier (BBB) by limiting transport systems [22] in a carrier-‐facilitated [23] manner. An early study had suggested that fragments corresponding to BCMs may cross the breast parenchyma-‐blood barrier into plasma and subsequently penetrate the blood-‐brain barrier to reach the central nervous system [24]. Indeed, in a recent study which puts forward putative biological/metabolic pathways in which (bovine milk derived) casomorphins have been implicated as being related to apnea in sudden infant death syndrome [25].
Expected immediate responses to casomorphin-‐7 release have been reported in papers concerning the slowing down of down the gastrointestinal transit time (GITT) in rats [26] and dogs [27].Furthermore, a report concerning a diarrhoea patient provided with calcium caseinate as sole nutrient reported a complete remission of the condition within ten days, speculating that an inhibition of intestinal motility mediated by beta-‐casomorphins released after hydrolysis of casein in the intestinal tract may be the mechanism involved in this effect [28]. It is worth noting that a linkage has been drawn between consumption of milk and anal fissure in infants when contrasted with those that are breast fed [29] Opiate attributed effects of BCM-7 and truncated forms include studies that show:
A number of other effects have also been reported to be imparted by BCM-7 specifically, or by related truncated BCMs, some which are also shown to be opiate receptor linked:
These findings suggest that beta-‐casomorphin-‐7 can be regarded as a noncytotoxic, direct histamine releaser in humans. [37]
BCM-5 has been reported to cause concentration dependent in vitro histamine release from peripheral leukocytes of healthy adult volunteers.
(in vitro) BCM-7 affects the human mucosal immune response as represented by depression of lamina propria lymphocyte (LPL) proliferation. The anti-‐proliferative effect of BCM was reversed by the opiate receptor antagonist, naloxone. [38]
In studies on human polymorphonuclear leukocytes (PMNs), Tyrosyl -‐ prolyl -‐phenylalanyl -‐ proline (ßCM-‐4) treatment inhibited oxygen-‐free radical production and 5-‐HETE production. ßCM-‐4 seemed to act on the enzymatic activities of endogenous peroxidases that reduced hydroperoxyeicosatetraenoic acid to 5-‐HETE. [39]
Casomorphins shown to inhibit both aggregation of ADP-‐treated platelets and binding of fibrinogen to ADP-‐treated platelets'. [40]
Shown to affect immune action by stimulating human peripheral blood lymphocytes proliferation at high concentrations and suppressing proliferation at low concentrations. [41]
BCM-7 plays a role in the regulation of insulin formation in dependence of the glucose concentration as well as insulin secretion from the islets of Langerhans. [42]
In non lactating dairy cows, injection of beta-‐casomorphins 4, 5 & 7 significantly lowered the responses of serum insulin to both abomasal and intravenous glucose infusions (P<0.05). [43]
References:
[20] Wonder Milk - Today Tonight, Channel 7 Australia. Helen Wellings, July 2003.
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