ArticlePDF AvailableLiterature Review

Milk Intolerance, Beta-Casein and Lactose

Nutrients

September 2015
7(9):7285-7297

DOI:10.3390/nu7095339

License
CC BY 4.0

Authors:

Sebely Pal

Curtin University

Keith Woodford

Lincoln University New Zealand

Sonja Kukuljan

Deakin University

Suleen S Ho

Curtin University

True lactose intolerance (symptoms stemming from lactose malabsorption) is less common than is widely perceived, and should be viewed as just one potential cause of cows' milk intolerance. There is increasing evidence that A1 beta-casein, a protein produced by a major proportion of European-origin cattle but not purebred Asian or African cattle, is also associated with cows' milk intolerance. In humans, digestion of bovine A1 beta-casein, but not the alternative A2 beta-casein, releases beta-casomorphin-7, which activates μ-opioid receptors expressed throughout the gastrointestinal tract and body. Studies in rodents show that milk containing A1 beta-casein significantly increases gastrointestinal transit time, production of dipeptidyl peptidase-4 and the inflammatory marker myeloperoxidase compared with milk containing A2 beta-casein. Co-administration of the opioid receptor antagonist naloxone blocks the myeloperoxidase and gastrointestinal motility effects, indicating opioid signaling pathway involvement. In humans, a double-blind, randomized cross-over study showed that participants consuming A1 beta-casein type cows' milk experienced statistically significantly higher Bristol stool values compared with those receiving A2 beta-casein milk. Additionally, a statistically significant positive association between abdominal pain and stool consistency was observed when participants consumed the A1 but not the A2 diet. Further studies of the role of A1 beta-casein in milk intolerance are needed.

Release of beta-casomorphin-7. Adapted from Woodford [12] (reproduced with Figure 1. Release of beta-casomorphin-7. Adapted from Woodford [12] (reproduced with permission of the publisher). permission of the publisher). The two major A1 and A2 beta-casein variants can be considered as beta-casein “families”, which include The two at least major 10 A1 sub-variants. and A2 beta-casein Those within variants the A1 can family be considered are B, C, D, as F beta-casein and G. Those “families”, within the which A2 include family are at least A3, E, 10 H1, sub-variants. H2 and I [13]. Those Whether within the the A1 different family tertiary are B, C, structures D, F and of G. the Those sub-variants within the within A2 family the A1 are family A3, have E, H1, any H2 effect and I on [13]. the Whether release of the BCM-7 different is unproven. tertiary structures However, of the there sub-variants is some evidence within the that A1 the family B sub-variant have any may effect result on in the a particularly release of BCM-7 high release is unproven. of BCM-7 However, [7]. there is some evidence that A1 the beta-casein B sub-variant has may only result been found in a particularly in cattle of high European release origin. of BCM-7 Purebred [7]. Asian and African cattle A1 beta-casein has only been found in cattle of European origin. Purebred Asian and African cattle produce milk containing only the A2 beta-casein type, although some cattle presenting phenotypically produce milk containing only the A2 beta-casein type, although some cattle presenting phenotypically as Asian or African cattle may produce A1 beta-casein as a consequence of crossbred ancestry. The as Asian or African cattle may produce A1 beta-casein as a consequence of crossbred ancestry. relative prevalence of A1 and A2 beta-casein in cattle is breed-dependent, with Northern European The relative prevalence of A1 and A2 beta-casein in cattle is breed-dependent, with Northern European breeds generally having higher levels of A1 beta-casein than Southern European breeds. Guernsey and breeds generally having higher levels of A1 beta-casein than Southern European breeds. Guernsey and Fleckvieh breeds are generally considered to have a particularly high A2 allele frequency. However, Fleckvieh breeds are generally considered to have a particularly high A2 allele frequency. However, within any specific herd, basing the estimation of allele frequency on breed category is not reliable. In within any specific herd, basing the estimation of allele frequency on breed category is not reliable. In the herds in many Western countries, the ratio of A1:A2 is approximately 1:1 [10]. Herd testing for the herds in many Western countries, the ratio of A1:A2 is approximately 1:1 [10]. Herd testing for beta-casein alleles can be undertaken using DNA analysis, which is available commercially in some beta-casein alleles can be undertaken using DNA analysis, which is available commercially in some countries. Converting a specific herd by selective breeding to eliminate all A1 beta-casein from the milk countries. Converting a specific herd by selective breeding to eliminate all A1 beta-casein from the milk can be achieved within 4 years using intensive methods of animal selection that incorporate the use of can be achieved within 4 years using intensive methods of animal selection that incorporate the use of sex-selected semen, but more typically this will take 5–8 years or longer [14]. sex-selected semen, but more typically this will take 5–8 years or longer [14]. The in vivo release of BCM-7 from each liter of bovine milk will depend on the protein content of the milk The in vivo release of BCM-7 from each liter of bovine milk will depend on the protein content of the (which is in turn affected by the breed, animal feeding and component standardization procedures during milk (which is in turn affected by the breed, animal feeding and component standardization procedures milk processing), the proportion of A1 and A2 beta-casein, and possibly the specific gastrointestinal during milk processing), the proportion of A1 and A2 beta-casein, and possibly the specific conditions of the individual. There is now clear evidence that BCM-7 is released not only from milk but also gastrointestinal conditions of the individual. There is now clear evidence that BCM-7 is released not from yoghurt and cheese, and in all likelihood any milk product [6,7]. There is also evidence that there only from milk but also from yoghurt and cheese, and in all likelihood any milk product [6,7]. There is is modest release of BCM-7 in the cheese- and yoghurt-making processes, but that during the latter, also evidence that there is modest release of BCM-7 in the cheese- and yoghurt-making processes, but that certain bacteria present in yoghurt may hydrolyze BCM-7 [15,16]. Whether such bacteria consumed in during the latter, certain bacteria present in yoghurt may hydrolyze BCM-7 [15,16]. Whether such bacteria yoghurt also have a similar influence within the human gastrointestinal tract is unknown. consumed in yoghurt also have a similar influence within the human gastrointestinal tract is unknown.

…

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Nutrients 2015,7, 7285-7297; doi:10.3390/nu7095339 OPEN ACCESS

nutrients

ISSN 2072-6643

www.mdpi.com/journal/nutrients

Review

Milk Intolerance, Beta-Casein and Lactose

Sebely Pal 1,*, Keith Woodford 2, Sonja Kukuljan 3and Suleen Ho 1

1School of Public Health, Curtin Health Innovation Research Institute, Curtin University,

GPO Box U1987, Perth WA 6845, Australia; E-Mail: Suleen.Ho@curtin.edu.au

2Agricultural Management Group, Lincoln University, PO Box 85084, Lincoln 7647, Christchurch,

New Zealand; E-Mail: keith.woodford@lincoln.ac.nz

3The a2 Milk Company (Australia) Pty Ltd, PO Box 180, Kew East, Victoria 3102, Australia;

E-Mail: sonja.kukuljan@a2Milk.com

*Author to whom correspondence should be addressed; E-Mail: s.pal@curtin.edu.au;

Tel.: +61-8-9266-4755; Fax: +61-8-9266-2958.

Received: 2 July 2015 / Accepted: 21 August 2015 / Published: 31 August 2015

Abstract: True lactose intolerance (symptoms stemming from lactose malabsorption) is less

common than is widely perceived, and should be viewed as just one potential cause of cows’

milk intolerance. There is increasing evidence that A1 beta-casein, a protein produced by a

major proportion of European-origin cattle but not purebred Asian or African cattle, is also

associated with cows’ milk intolerance. In humans, digestion of bovine A1 beta-casein, but

not the alternative A2 beta-casein, releases beta-casomorphin-7, which activates µ-opioid

receptors expressed throughout the gastrointestinal tract and body. Studies in rodents

show that milk containing A1 beta-casein signiﬁcantly increases gastrointestinal transit

time, production of dipeptidyl peptidase-4 and the inﬂammatory marker myeloperoxidase

compared with milk containing A2 beta-casein. Co-administration of the opioid receptor

antagonist naloxone blocks the myeloperoxidase and gastrointestinal motility effects,

indicating opioid signaling pathway involvement. In humans, a double-blind, randomized

cross-over study showed that participants consuming A1 beta-casein type cows’ milk

experienced statistically signiﬁcantly higher Bristol stool values compared with those

receiving A2 beta-casein milk. Additionally, a statistically signiﬁcant positive association

between abdominal pain and stool consistency was observed when participants consumed

the A1 but not the A2 diet. Further studies of the role of A1 beta-casein in milk intolerance

are needed.

Nutrients 2015,77286

Keywords: milk consumption; lactose; beta-casein; lactose intolerance

1. Introduction

There is a widespread assumption within both general society and among healthcare professionals that

the dominant cause of milk intolerance is insufﬁcient lactase enzyme activity. However, the evidence,

as summarized in the 2010 National Institutes of Health consensus statement on lactose intolerance and

health, is that “many who self-report lactose intolerance show no evidence of lactose malabsorption.

Thus, the cause of their gastrointestinal symptoms is unlikely to be related to lactose” [1]. Providing

an alternative mechanism, there is now an increasing body of evidence that bovine beta-casomorphin-7

(BCM-7), derived from A1 beta-casein, is also an important contributor to milk intolerance syndrome. It

is that evidence that we discuss here, including the potential for interactions between lactose intolerance

and A1 beta-casein intolerance.

2. Literature Search and Selection of Studies for Review

The objective of this review was to assess the evidence that bovine BCM-7, which is derived from

A1 beta-casein, contributes to milk intolerance syndrome.

In vitro and in vivo animal studies and human clinical studies reporting outcomes relevant to the

formation of BCM-7 or other beta-casomorphins in the gastrointestinal system, or other outcomes

relevant to the formation of these peptides, were included in this review. Studies involving milk, milk

products and beta-casein were also considered. For in vivo animal and human clinical studies, only

studies that assessed outcomes following oral administration were included. Relevant outcome measures

included the release of beta-casomorphins in actual or simulated gastrointestinal digestion of milk, milk

products or beta-casein; opioid agonist activity following digestion of milk, milk products or beta-casein

including differences in gastrointestinal transit time; and variations in other biomarkers relevant to the

gastrointestinal system following consumption of milk, milk products or beta-casein.

Literature searches were undertaken using Medline/PubMed on 20 October 2014 using the

following search terms: Casomorphin; Beta-casomorphin; Beta-casomorphin-7; Beta-casomorphine;

Beta-casomorphine-7; A1_beta casein OR A2_beta casein; b-cm 7 OR bcm7 OR bcm-7; beta-casein

AND A1 OR A2; and A2 AND Milk. The authors’ existing EndNote X5 reference management

software library was also used to identify any additional papers not captured by the literature searches.

Studies published since October 2014 were added manually. Data were extracted manually. Studies

were assessed manually for bias, based on the information provided in each publication. We focused on

studies relevant to the stated aim of the current review.

3. Beta-Caseins and BCM-7

Beta-casein proteins make up approximately 30% of the total protein of cows’ milk [2] and may

be present as one of two major genetic variants: A1 and A2 [3]. A2 beta-casein is recognized as the

original beta-casein variant because it existed before a proline67 to histidine67 point mutation caused the

appearance of A1 beta-casein in some European herds some 5000–10,000 years ago [4]. Once milk

Nutrients 2015,77287

or milk products are consumed, the action of digestive enzymes in the gut on A1 beta-casein releases

the bioactive opioid peptide BCM-7 [5–9]. In contrast, A2 beta-casein releases much less and probably

minimal amounts of BCM-7 under normal gut conditions (Figure 1) [10]. However, it is notable that

under speciﬁc in vitro conditions relating to pH and enzyme combinations not found in the human gut,

A2 beta-casein can also release some BCM-7 [11].

Nutrients 2015, 7 3

milk products are consumed, the action of digestive enzymes in the gut on A1 beta-casein releases the

bioactive opioid peptide BCM-7 [5–9]. In contrast, A2 beta-casein releases much less and probably

minimal amounts of BCM-7 under normal gut conditions (Figure 1) [10]. However, it is notable that

under specific in vitro conditions relating to pH and enzyme combinations not found in the human gut,

A2 beta-casein can also release some BCM-7 [11].

Figure 1. Release of beta-casomorphin-7. Adapted from Woodford [12] (reproduced with

permission of the publisher).

The two major A1 and A2 beta-casein variants can be considered as beta-casein “families”, which

include at least 10 sub-variants. Those within the A1 family are B, C, D, F and G. Those within the A2

family are A3, E, H1, H2 and I [13]. Whether the different tertiary structures of the sub-variants within

the A1 family have any effect on the release of BCM-7 is unproven. However, there is some evidence

that the B sub-variant may result in a particularly high release of BCM-7 [7].

A1 beta-casein has only been found in cattle of European origin. Purebred Asian and African cattle

produce milk containing only the A2 beta-casein type, although some cattle presenting phenotypically

as Asian or African cattle may produce A1 beta-casein as a consequence of crossbred ancestry.

The relative prevalence of A1 and A2 beta-casein in cattle is breed-dependent, with Northern European

breeds generally having higher levels of A1 beta-casein than Southern European breeds. Guernsey and

Fleckvieh breeds are generally considered to have a particularly high A2 allele frequency. However,

within any specific herd, basing the estimation of allele frequency on breed category is not reliable. In

the herds in many Western countries, the ratio of A1:A2 is approximately 1:1 [10]. Herd testing for

beta-casein alleles can be undertaken using DNA analysis, which is available commercially in some

countries. Converting a specific herd by selective breeding to eliminate all A1 beta-casein from the milk

can be achieved within 4 years using intensive methods of animal selection that incorporate the use of

sex-selected semen, but more typically this will take 5–8 years or longer [14].

The in vivo release of BCM-7 from each liter of bovine milk will depend on the protein content of the

milk (which is in turn affected by the breed, animal feeding and component standardization procedures

during milk processing), the proportion of A1 and A2 beta-casein, and possibly the specific

gastrointestinal conditions of the individual. There is now clear evidence that BCM-7 is released not

only from milk but also from yoghurt and cheese, and in all likelihood any milk product [6,7]. There is

also evidence that there is modest release of BCM-7 in the cheese- and yoghurt-making processes, but that

during the latter, certain bacteria present in yoghurt may hydrolyze BCM-7 [15,16]. Whether such bacteria

consumed in yoghurt also have a similar influence within the human gastrointestinal tract is unknown.

Figure 1. Release of beta-casomorphin-7. Adapted from Woodford [12] (reproduced with

permission of the publisher).

The two major A1 and A2 beta-casein variants can be considered as beta-casein “families”, which

include at least 10 sub-variants. Those within the A1 family are B, C, D, F and G. Those within the A2

family are A3, E, H1, H2 and I [13]. Whether the different tertiary structures of the sub-variants within

the A1 family have any effect on the release of BCM-7 is unproven. However, there is some evidence

that the B sub-variant may result in a particularly high release of BCM-7 [7].

A1 beta-casein has only been found in cattle of European origin. Purebred Asian and African cattle

produce milk containing only the A2 beta-casein type, although some cattle presenting phenotypically

as Asian or African cattle may produce A1 beta-casein as a consequence of crossbred ancestry. The

relative prevalence of A1 and A2 beta-casein in cattle is breed-dependent, with Northern European

breeds generally having higher levels of A1 beta-casein than Southern European breeds. Guernsey and

Fleckvieh breeds are generally considered to have a particularly high A2 allele frequency. However,

within any speciﬁc herd, basing the estimation of allele frequency on breed category is not reliable. In

the herds in many Western countries, the ratio of A1:A2 is approximately 1:1 [10]. Herd testing for

beta-casein alleles can be undertaken using DNA analysis, which is available commercially in some

countries. Converting a speciﬁc herd by selective breeding to eliminate all A1 beta-casein from the milk

can be achieved within 4 years using intensive methods of animal selection that incorporate the use of

sex-selected semen, but more typically this will take 5–8 years or longer [14].

The in vivo release of BCM-7 from each liter of bovine milk will depend on the protein content of the milk

(which is in turn affected by the breed, animal feeding and component standardization procedures during

milk processing), the proportion of A1 and A2 beta-casein, and possibly the speciﬁc gastrointestinal

conditions of the individual. There is now clear evidence that BCM-7 is released not only from milk but also

from yoghurt and cheese, and in all likelihood any milk product [6,7]. There is also evidence that there

is modest release of BCM-7 in the cheese- and yoghurt-making processes, but that during the latter,

certain bacteria present in yoghurt may hydrolyze BCM-7 [15,16]. Whether such bacteria consumed in

yoghurt also have a similar inﬂuence within the human gastrointestinal tract is unknown.

Nutrients 2015,77288

In human milk, beta-casein is of the A2 type, with a proline at the equivalent position on the

beta-casein protein chain [17]. Human BCM-7 has a different amino acid sequence to bovine BCM-7,

with homology in ﬁve of seven amino acids (differing amino acids at positions four and ﬁve) [17,18],

and considerably weaker opioid activity [19,20]. Wada and Lonnerdal [18] examined non-digested

and in vitro-digested human milk, and reported the presence of human BCM-9 (which has a proline

at position eight), but not human BCM-7 or BCM-5 (i.e., BCM-5 is the truncated form of BCM-7).

However, Jarmolowska et al. reported the presence of both human BCM-5 and BCM-7 in colostrum

(averaging 5 and 3 µg/mL respectively), but at 2 months into the lactation period, the authors reported

much lower quantities [21]. It has been postulated that casomorphin functionality in neonates may relate

to maternal bonding, gastrointestinal function, mucosal development and sleep induction [21].

4. Opioid Characteristics of Casomorphins

Beta-casomorphins are µ-opioid receptor ligands [8,22–25]. The natural casomorphins of relevance

are BCM-5, BCM-7 and BCM-9. The most potent of these natural opioids is BCM-5. In theory,

BCM-5 could be released from BCM-7 within the human biological system by the human equivalent

of the enzyme carboxypeptidase Y [23], and there is some evidence supporting this [7,26].

Wasilewska et al. reported that bovine BCM-5 is present in the serum of exclusively breastfed human

babies whose mothers consumed bovine milk [26]. However, it has not been demonstrated whether the

BCM-5 was hydrolyzed from BCM-7 by the mothers and passed via their breastmilk to the infants, or

whether the mothers passed bovine BCM-7 via their breastmilk to the infants where it was subsequently

degraded to BCM-5 before or after intestinal absorption.

The second most potent natural casomorphin is BCM-7 and it is the major focus of this review. Bovine

BCM-7 has been identiﬁed in human jejunal contents following milk-protein feeding at levels consistent

with pharmacological effects, with 4 mg BCM-7 released from 30 g of casein after 2 h of digestion, with

further release thereafter [5]. It has also been identiﬁed in the blood of human infants [27,28] and urine

of children [29].

The third natural casomorphin of interest is BCM-9. This peptide is released from the A2 type variant

of beta-casein [5,18], but it is unlikely to be a peptide of importance in relation to A1 beta-casein because

of the histidine at position 67 on A1 beta-casein. In this regard, it is notable that Boutrou et al. [5] report

in their supplementary data considerable quantities of BCM-9 with a proline at position 67 (therefore,

by deﬁnition, derived from A2 beta-casein), whereas there was almost no BCM-9 with a histidine at this

position (i.e., from A1 beta-casein). This provides supporting evidence that BCM-9 with a histidine at

position 67 is readily broken down at the histidine cleavage point to BCM-7 within the gastrointestinal

system, whereas BCM-9 with a proline at position 67 is cleavage resistant.

BCM-9 does exhibit opioid properties, but with a binding afﬁnity to µ-opioid receptors approximately

one quarter that of BCM-7 [8]. These ﬁndings are consistent with those of Barnett et al. who conducted

a study in rats [30]. They found that, while A1 beta-casein exhibited a range of gastrointestinal effects

that were blocked by the µ-opioid receptor antagonist naloxone, no such effects occurred with A2

beta-casein following naloxone administration [30]. Of interest, BCM-9 has been identiﬁed as having

antihypertensive properties [31].

Nutrients 2015,77289

5. Beta-Caseins, Beta-Casomorphins and Delayed Intestinal Transit

µ-Opioid receptors are expressed widely in humans, including in the gastrointestinal tract [32].

µ-Opioid receptor activation is known to affect the mechanics of intestinal propulsion [33] and to play

an important role physiologically in controlling gastrointestinal function, including regulating motility,

mucus production and hormone production [34]. Gastrointestinal µ-opioid receptor activation occurs

on both enteric neurons and directly on epithelial cells [35,36]. µ-Opioid receptor agonists are known

to delay gastrointestinal transit time in humans, in a naloxone-reversible manner. For example, the

opioid codeine has been shown in humans to signiﬁcantly delay small intestinal and consequently overall

colonic transit time [37]. Additionally, in humans consuming high-ﬁber diets, Stephen et al. showed

that administration of a sufﬁcient codeine dose to double gastrointestinal transit time results in a major

controlling inﬂuence over the colonic microﬂora, and thereby colonic function, with codeine signiﬁcantly

decreasing both total stool mass and bacterial mass [38]. They concluded that “differences in bowel habit

and microbial cell metabolism between individuals on similar diets are largely attributable to differences

in mean transit time” [38]. This study also demonstrated that the quantity of codeine needed to double

transit time varied considerably between individuals, which reinforces the importance of population

sub-groups when considering intolerance issues.

Several other studies provide direct evidence that casein and/or its derivatives decrease gastrointestinal

motility, in part by reducing the frequency and amplitude of intestinal contractions [39–43]. In the canine

small intestine, a comparison of casein and soy protein on various small intestinal motility measures

(e.g., force and contraction frequency) showed that casein reduced these parameters signiﬁcantly and that

pretreatment with naloxone blocked this effect [40], suggesting a role for exogenous opioids. Similarly,

casein was also shown to delay gastrointestinal transit time in rats compared with whey protein, with

naloxone partially or completely reversing these casein effects, again indicating that the opioid activity

of casein delays transit time [43]. In rat pups fed either intact casein powder or extensively hydrolyzed

casein, small intestinal transit time was delayed [41], and the effect of the intact casein on delaying transit

time was prevented with naloxone administration. These results suggest that peptides with opioid activity

are released during digestion of intact casein, which can cause gastrointestinal transit time delays. This

effect was not evident following rat pup feeding with extensively hydrolyzed casein.

A recent animal study investigating the effects of A1 versus A2 beta-casein on gastrointestinal transit

has shown that A1 beta-casein delays gastrointestinal transit time relative to A2 beta-casein feeding [30].

Using Wistar rats fed A1 or A2 beta-casein type milk-based diets for 36 or 84 h, Barnett et al. showed

that the A1 beta-casein diets delayed gastrointestinal transit time compared with the A2 beta-casein

diets [30]. Co-administration of naloxone blocked the effects of the A1 diet on transit time, but had no

effects in rats fed the A2 diet. The results indicate that the A1 diet has direct effects on gastrointestinal

function by slowing transit time, and provides further support for a role for opioid signaling pathways in

the effects of A1 beta-casein.

6. Inﬂammatory and Immune Responses to Casomorphins in the Gastrointestinal System

There is wide-ranging evidence for both inﬂammatory and immune responses to casomorphins

within the gastrointestinal system. However, the overall implications of these responses are not

Nutrients 2015,77290

fully understood. It has been shown in both rats [30] and mice [44] that A1 beta-casein is

associated with increased levels of the inﬂammatory marker myeloperoxidase (MPO) in the colon.

This effect is eliminated by administration of naloxone, indicating that it is an opioid-dependent

response. Interestingly, intestinal inﬂammation enhances the potency of µ-opioid receptor agonists

in inhibiting gastrointestinal transit, and increases the expression of µ-opioid receptors in the mouse

intestine [45]. It has also been shown in rats that A1 beta-casein stimulates the production of the enzyme

dipeptidyl peptidase 4 (DPP4) in the jejunum [30]. However, this effect is not attenuated by naloxone

administration, indicating a non-opioid effect of A1 beta-casein on DPP4. The full implications of

this are not understood, but it is notable that DPP4 degrades the gut incretin hormones rapidly [46].

In humans, incretin hormones modulate insulin and glucose metabolism [47] and affect antroduodenal

motility [48]. DPP4 inhibitors are now widely used in the management of type 2 diabetes mellitus.

BCM-7 is also known to increase mucin production within the gastrointestinal system via an opioid

pathway [34,49]. Gastrointestinal mucus provides a protective barrier between the epithelium and the

lumen; however, excessive production has the potential to disrupt gastrointestinal function and interfere

with commensal bacteria. It has been shown in two in vitro studies that BCM-7 alters lymphocyte

proliferation, also via an opioid-dependent pathway [50,51]. The full physiological relevance of the

immunomodulatory effects of BCM-7 in animals and humans requires further investigation.

More recently, Ul Haq et al. examined possible mechanisms underlying previously observed

proinﬂammatory effects of BCM-7 [52]. In this study, mice were administered BCM-7 or BCM-5

orally, and both peptides resulted in increased expression of inﬂammatory markers (MPO, monocyte

chemotactic protein-1 and interleukin-4). Increased levels of immunoglobulins, enhanced leukocyte

inﬁltration into intestinal villi, and increased expression of Toll-like receptors in the gut were also

observed. The authors concluded that both peptides stimulate inﬂammatory responses through the

Th2pathway. The same research group reported similar gastrointestinal immune effects in mice

fed a milk-free basal diet supplemented with A1 relative to mice fed a diet supplemented with A2

beta-casein [44]. The diet containing A1 beta-casein had proinﬂammatory effects in the gut (increased

levels of inﬂammatory markers and immunoglobulins, leukocyte inﬁltration and Toll-like receptor

expression). These effects were not observed in mice fed A2 beta-casein. Taken together, these results

highlight the potential proinﬂammatory effects of A1 beta-casein, and suggest pathways by which A1

beta-casein might contribute to a variety of clinical conditions, including gastrointestinal disorders.

7. Clinical Studies of Beta-Casein Effects in the Gastrointestinal System

Much of the human evidence for intolerance to A1 versus A2 beta-casein is observational and

anecdotal, and has the potential to be inﬂuenced by the lack of a controlled environment. However, there

are two clinical studies of relevance. The ﬁrst, undertaken in Newcastle, Australia, aimed to investigate

the effect of A1 and A2 beta-caseins on constipation in young children who suffered chronically from this

condition [53]. The rationale for the trial was the considerable literature linking childhood constipation

with milk, but with the causative factor being unresolved [54,55]. The authors reported 81% resolution

of constipation during the milk-free washout period, 79% resolution during the A2 epoch and 57%

resolution with A1 beta-casein [53]. However, with only 21 children completing the trial, the results were

not statistically signiﬁcant. Accordingly, the results were reported as showing no difference between

Nutrients 2015,77291

treatments, although an alternative interpretation would have been that the trial, despite showing results

of potential clinical importance, lacked sufﬁcient statistical power and that further studies are needed. It

is also notable that both the A2 and the A1 milk were commercially sourced, and that the beta-casein

proportions were not analyzed or standardized. The A1 treatment was standard commercial milk, and

although this is sometimes referred to as “A1 milk”, at that time in Australia it would have typically

contained A1 and A2 beta-casein in approximately equal proportions. The beta-casein composition

of the “A2 milk” used in this trial is also unknown, as the so-called “A2 milk” was sourced from a

private Jersey dairy farm, and was therefore unlikely to be free of A1 beta-casein. Furthermore, the milk

treatments used in the study comprised 400 mL/day, which was apparently lower than pre-treatment

consumption for many of the participants. It is therefore possible that this low consumption may have

contributed to the resolution of constipation levels independent of a particular treatment.

The second clinical trial comparing the gastrointestinal effects of A1 versus A2 beta-casein was

conducted at Curtin University, Western Australia [56]. This trial comprised 36 participants at study

completion. Although it was initially planned to recruit those with perceived milk intolerance, all

participants had to be willing to drink 750 mL of milk per day. This led to the self-exclusion of

many potential participants who had perceived milk intolerance. Accordingly, only eight of the 36

who completed the study considered themselves milk intolerant ex ante. The trial had a blinded

cross-over design. Either A1 or A2 milk was consumed for a period of 2 weeks, followed by a 2-week

washout period. Participants then crossed over to the second treatment. Key outcomes were statistically

different Bristol Stool Scale measures (A1 milk, 3.87 versus A2 milk, 3.56, p= 0.04) with higher values

(i.e., looser and more runny stools) on A1 than A2 milk. These differences remained signiﬁcant when

participants reporting milk intolerance prior to the trial were excluded (thus, there were differences in

stool outputs in people whom reported themselves to be milk tolerant). Particularly notable was a strong

relationship between abdominal pain and increased stool looseness across all participants on the A1 diet

(r= 0.520, p= 0.001), but not on the A2 diet (r=´0.13, p= 0.43). The difference between these two

correlations (0.52 versus ´0.13) was highly signiﬁcant (p< 0.001). Similarly, while receiving A1 milk,

higher gut inﬂammation (fecal calprotectin) correlated with higher abdominal pain (r= 0.46, p= 0.005)

and higher bloating (r= 0.36, p= 0.03) scores. These relationships were absent in the same people

when they received A2 milk. Again, the difference in the correlation measures was

signiﬁcant for: (i) gut

inﬂammation and abdominal pain (A1, 0.46 vs. A2, 0.03; p= 0.02); and (ii) gut inﬂammation and

bloating (A1, 0.36 vs. A2, ´0.02; p= 0.05).

In contrast, differences in subjective measures of intolerance were not statistically signiﬁcant.

However, there were treatment differences in subjective measures of intolerance amongst the eight

participants who considered themselves milk intolerant, which are of potential clinical signiﬁcance.

The interpretation of these clinical results requires an appreciation of the body of evidence previously

discussed in this review. The expected effect of BCM-7 on gastrointestinal transit is to disrupt the

propagation of peristaltic contractions, and when considered in isolation, it might seem reasonable

to assume that this would present as constipation. However, the signiﬁcantly higher Bristol Stool

Scale values in participants receiving A1 compared with A2 beta-casein diets may instead be caused

by a combination of gastrointestinal transit delay and proinﬂammatory factors, with transit delays

potentially providing additional opportunity for fermentable oligosaccharides to undergo gas-forming

Nutrients 2015,77292

and stool-softening degradation. Prior evidence showing that intestinal inﬂammation is associated with

malabsorption of ﬂuids, nutrients and electrolytes [57,58] supports this proposition. This explanation

is also consistent with the signiﬁcant and positive association between abdominal pain and stool

consistency on the A1 diet [56]. Accordingly, it is reasonable to hypothesize that the delayed transit

effects of BCM-7 may lead to looser stools together with proinﬂammatory effects in at least some people.

The results of Ho et al. [56] are consistent with this interpretation.

It is also well understood that milk intolerances and gastrointestinal sensitivities will exhibit

differently in different individuals. Accordingly, and given the multiplicity of biological effects, it is not

unreasonable to expect that in some people, the dominant symptom from BCM-7 may be constipation,

whereas in others it might be an increased looseness of stools. The close association in the A1 epoch

between looser stools and measures of subjective discomfort, and the association between higher fecal

calprotectin values and subjective intolerance measures when participants were receiving the A1 diet in

the Ho et al. study [56] is also supportive of the hypothesis that delayed gastrointestinal transit with

associated discomfort followed by loose stools may be expected in at least some individuals.

8. The Potential for BCM-7 and Lactose Interactions

The likelihood of BCM-7 and lactose interactions deserves consideration. There are various

mechanisms by which this might occur. The ﬁrst is that the inﬂammatory characteristics of BCM-7 may

affect lactase production/activity and possibly exacerbate existing hypolactasia and consequent lactose

malabsorption symptoms in susceptible individuals. The second is that colonic inﬂammation affects

the processing of malabsorbed lactose, possibly via changes in the gut microbiota that occur with gut

inﬂammation [59]. The third is that the delayed gastrointestinal transit leads to increased opportunity

for lactose fermentation (and the opportunity for fermentation of other dietary-derived oligosaccharides).

These possibilities are consistent with the current state of knowledge of gastrointestinal symptoms related

to lactose malabsorption. However, all need to be tested in clinical investigations.

9. Conclusions

Milk intolerance is a complex problem of importance both to public health and individual health.

It is clear that lactose malabsorption (and consequent symptoms) is one element of the syndrome, but it

is also evident that there are other factors at play. The potential role of A1 beta-casein is arguably the

prime candidate requiring closer scrutiny if understanding is to be advanced.

It is important to note the considerable advancements relating to A1 beta-casein and BCM-7 that have

been made since the European Food Safety Authority (2009) report on the possible health effects of

beta-casomorphins and BCM-7 [10]. At that time, the EFSA recognized that BCM-7 exerts biological

activities such as regulatory effects on gastrointestinal motility and on gastric and pancreatic secretions.

However, they concluded that a “cause and effect” relationship could not be established between the

dietary intake of BCM-7 and assessed non-communicable diseases, which included type 1 diabetes, heart

disease and autistic spectrum disorders. Their conclusion was reached partly because BCM-7 had not

been detected in human blood following milk or casein intake, and partly because there was insufﬁcient

knowledge about the levels of BCM-7 likely to originate from the digestion of milk and its products.

Additionally, the EFSA report did not speciﬁcally address intolerance issues.

Nutrients 2015,77293

Several conclusive studies have been published since the EFSA (2009) report (as discussed in the

current review), which report that the opioid peptide BCM-7 is released in pharmacologically relevant

quantities from digestion of A1 beta-casein, but not from A2 beta-casein in the human gastrointestinal

system. It is also clear that BCM-7 has a range of effects within both in vitro models and in vivo

in animal experiments. These effects include those on gastrointestinal motility, proinﬂammatory and

immunomodulatory outcomes. Most, but not necessarily all of these effects are opioid-related. Given

the complexity of the relationships, it is reasonable to expect that exhibited symptoms will vary

between individuals.

Data from human clinical trials are limited, but statistically signiﬁcant results from the recent study by

Ho et al. are consistent with prior knowledge and scientiﬁc hypotheses drawn from in vitro investigations

and animal trials [56]. It is notable that in this study, signiﬁcant differences in stool consistency were

identiﬁed in a cohort of people who had no prior awareness of milk intolerance [56], which may be

caused by proinﬂammatory factors alongside effects on gastrointestinal transit time [30,44,56]. Further

studies are required to conﬁrm these observations.

Given the speciﬁcity of A1 beta-casein to cattle of European origin, and hence also the release of

BCM-7, the current evidence also provides a contributory explanation as to why some people report

anecdotally that they can tolerate milk from mammals such as sheep [60] and goats (GenBank Accession

No. AJ011019.3) (which contain A2-like beta-casein and not A1, because they have a proline at the

homologous position on their beta-casein chains), but not cows. It is also clear that it is feasible for dairy

farmers to breed herds of bovine cows that are free of A1 beta-casein. Indeed such herds already exist

and, where available, the dairy products are supported by consumers.

Acknowledgments

Editorial assistance was provided by Sarah Williams and Helen Roberton of Edanz Group Ltd., which

was funded by The a2 Milk Company.

Author Contributions

All authors contributed to the conceptual development of this review paper. S.P., S.K. and K.W. wrote

the paper. All authors have primary responsibility for the ﬁnal content.

Conﬂicts of Interest

Sonja Kukuljan is an employee of The a2 Milk Company (Australia) Pty Ltd. Keith Woodford

previously consulted to A2 Corporation as an independent scientiﬁc adviser. The remaining authors

declare no conﬂict of interest.

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distributed under the terms and conditions of the Creative Commons Attribution license

(http://creativecommons.org/licenses/by/4.0/).

Ho Woodford Kukuljan Pal 2014 A1 vs A2 GI effects

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October 2015

Sebely Pal · Keith Woodford · Sonja Kukuljan · Suleen S Ho

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Dec 2023

The main objective of this study was to determine the beta-casein A1/A2 polymorphism status of animals in two Holstein Friesian dairy cow herds in Győr-Moson-Sopron County, Hungary. The A1/A2 status of cattle is determined by the beta-casein gene on the sixth chromosome. The analysed single nucleotide polymorphism is non-synonymous; A1 and A2 variants of bovine beta-casein differ at position 67 of the amino acid chain: A1 variant codes for histidine and A2 codes for proline, which may affect the milk protein degradation process. The analysed polymorphism leads to key conformational changes in the secondary protein structure of beta-casein. Beta-casomorphin (known as BCM7) is released only from A1-type milk and cannot be completely degraded by enzymes during digestion. DNA isolation was performed from whole blood, and a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method with agarose gel electrophoresis was applied in order to determine individual genotypes. The results from the two dairy farms demonstrate that a high proportion of cows (86.08 and 90.74 %) carry the A2 gene variant without targeted selection. At farm "A", beta-casein polymorphisms were determined in 599 cows and 148 heifers. The genotype distribution of the cows was 47.25 % heterozygous, 38.83 % homozygous A2, whereas 14.02 % of the cows carried the A1A1 genotype. In heifers, A2A2 was already present in a remarkably high frequency (91.89 %), whereas the prevalence of heterozygotes was 7.43 %, and A1A1 animals made up only 0.67 % of the analysed heifer population. In Hungary, a growing number of dairy farms are using verified A2 homozygous breeding bull semen. The introduction of homozygous A2 sperm on the farm "A" remarkably increased A2 frequency in the heifer population. In total, 324 cows were genotyped on farm "B", where the A2A2 genotype was observed in 30.55 % of the animals. The second most common genotype was A1A2, with a genotype frequency of 60.19 %, whereas A1A1 homozygotes occurred with a 9.26 % frequency. The growing popularity of A2 milk due to potential health benefits is driving Hungarian stakeholders towards the targeted selection of dairy populations; animal genotyping is an evident approach to facilitate this transition.

Comparative Analysis of Yak Milk and Holstein Cow Milk in Composition of Protein, Amino Acid and Fatty Acid

Article

Nov 2023

A2 milk consumption and its health benefits: an update

Article

Full-text available

Oct 2023

Milk is a widely consumed nutrient-rich food containing protein variants such as casein A2 and A1. A1 differs from A2 in an amino acid at position 67 (Pro67 to His67). The breakdown of β-casein yields β-casomorphins (BCM), among which BCM-7 is extensively studied for its effects on the human body. Animal studies have shown that A1 β-casein milk increases digestive transit time and enhances myeloperoxidase activity. Individuals with lactose intolerance prefer A2 milk to conventional A1 milk, as BCM-7 in A1 milk can lead to inflammation and discomfort in sensitive individuals. A2 milk, which contains A2 β-casein, is believed to be more easily digestible than A1 β-casein. Its popularity has grown owing to reports linking A1 casein to diseases such as type 1 diabetes, heart disease, and autism. A2 milk has gained popularity as an alternative to A1 milk, primarily because of its potential benefits for individuals with certain diseases. This review aims to provide an updated understanding of A2 milk consumption and its health benefits. This review aims to provide an updated understanding of A2 milk consumption and its health benefits.

Employee Dietary Initiative Improved Chronic Symptoms

Article

Full-text available

Apr 2024

With the awareness that the Standard American Diet is a critical contributor to chronic diseases, this initiative aimed to assess the effects of a 28-day dietary challenge health care improvement project on health system employee energy level, sleep quality, gastrointestinal function, ability to concentrate, and aches/pains, including the impact of adherence level, during a period of restricted intake of gluten, dairy, and sugar offered annually from 2021 to 2023. A total of 754 employees completed the pre-challenge survey; analyses included 354 employees who completed both pre-challenge and post-challenge surveys in at least 1 year of this project. Wilcoxon signed rank tests compared presurvey and postsurvey responses to self-reported energy level, sleep quality, gastrointestinal function, ability to concentrate, and aches/pains. Analysis of variance with Tukey’s honestly significant difference tests compared self-reported adherence level with change scores, with η² representing effect size. In each challenge year, the mean rank levels of energy, sleep quality, gastrointestinal function, concentration, and aches/pains improved significantly between pre-surveys and post-surveys (all P<.001). Although an association between significant positive change and diet adherence level was found for all items in at least 1 challenge year, those who mostly or completely adhered to the challenge diet restrictions reported significantly greater positive change in energy levels and gastrointestinal symptoms than those who did not or minimally adhered in all challenge years, with small to medium effect sizes. In conclusion, Essentia Health’s employee challenge appeared to improve self-reported outcomes in 5 symptom domains, with energy levels and gastrointestinal symptoms correlating most favorably to adherence to the challenge. These findings have health and cost implications, which could be confirmed by formal research in employee and other populations.

COMPARATIVE MOLECULAR INTERACTION OF THE OPIOID RECEPTOR (MOR) WITH THE BOVINE A1 & A2 TYPE β-CASEIN VARIANTS AND ASSOCIATED HEALTH (GASTROINTESTINAL) EFFECTS

Thesis

Jan 2022

Prayukta Padelkar

Milk, has always been known as a ‘perfect food’, because of presence of vital nutrients such as energy, proteins, calcium, vitamins, etc. However, studies have suggested that these vital nutrients, especially Proteins, can adversely impact on Human health. Proteins, a very diverse family of large organic compounds, involved in many important biological processes, constitutes up to 3.3% of the total milk. A major protein component of cow’s milk, present in abundance is β-casein, which consists of several genetic variants, of which, there are two most frequent primary variants, A1 and A2. Digestion of A1 β-casein yields, the peptide β-casomorphin-7, an exorphin, suggested to contribute to an increased risk of cardiovascular diseases, type I diabetes, and sudden infant death syndrome. Besides several health disorders, β-casomorphin7 (BCM7) can target opioid receptors in various systems to influence digestion, respiration, and immunity. Also, it was observed that, the consumption of A2 Milk, have beneficial effects on Human systems as compared to A1 milk. The main focus of this study was to detect, whether the milk samples bought from the local suppliers consist of A1 or A2 type of milk, to analyse the various biomarkers associated with health disorders and to study the molecular interaction between Beta casomorphins and opioid receptors through In-silico approach. The type of milk was detected using Allele-specific PCR method. Further, the amplified products, with A1 genotype were subsequently used to detect presence of BCM-7 variants in it. Also, there are several increasing evidences that suggests, BCM-7 binds strongly to µ-opioid receptors (MOR) and activates them and subsequently, increases inflammatory response, which leads to adverse impact on human health, such as on nervous and hormonal systems, psychology (via interaction with the dopaminergic system), early life development, immune system, lactation, response to environmental stimuli, mother-child bonding. So, to study the co-relation between BCM-7, its associated disorders and the changes in the inflammatory responses, Biomarkers related with these disorders, was studied, so that they can further be used in case of risk prediction and in screening and diagnosis of disease progression. Moreover, an In-silico study was done. The primary objective was to observe the molecular interaction of casomorphin protein variants, mainly with opioid receptors and check its potency. Overall, this study demonstrates that the “β- casein A1/BCM7 issue” remains an intriguing but not exhaustively explained topic in human nutrition.

Bovine milk β-casein: Structure, properties, isolation, and targeted application of isolated products

Article

Mar 2024
COMPR REV FOOD SCI F

Casein, an important protein found in bovine milk, has significant potential for application in the food, pharmaceutical, and other related industries. This review first introduces the composition, structure, and functional properties of β‐casein. It then reviews the techniques for isolating β‐casein. Chemical and enzymatic isolation methods result in inactivity of β‐casein and other components in the milk, and it is difficult to control the production conditions, limiting the utilization range of products. Physical technology not only achieves high product purity and activity but also effectively preserves the biological activity of the components. The isolated β‐casein needs to be utilized effectively and efficiently for various purity products in order to achieve optimal targeted application. Bovine β‐casein, which has a purity higher than or close to that of breast β‐casein, can be used in infant formulas. This is achieved by modifying its structure through dephosphorylation, resulting in a formula that closely mimics the composition of breast milk. Bovine β‐casein, which is lower in purity than breast β‐casein, can be maximized for the preparation of functional peptides and for use as natural carriers. The remaining byproducts can be utilized as food ingredients, emulsifiers, and carriers for encapsulating and delivering active substances. Thus, realizing the intensive processing and utilization of bovine β‐casein isolation. This review can promote the industrial production process of β‐casein, which is beneficial for the sustainable development of β‐casein as a food and material. It also provides valuable insights for the development of other active substances in milk.

Detection and Validation of A2 Milk Suitable for Consumers Having Milk Intolerance by ELISA Method

Article

Jun 2023

Mediha Esra YAYLA

Casein proteins, which make up 80% of cow's milk, consist of mainly A1 and A2 genetic types which differ by a mutation that causes conversion from proline to histidine. Histidine-containing A1 protein undergoes proteolytic degradation in the gastrointestinal system, while this is not observed during the digestion of A2 protein. A1 milk consumption causes bloating, gas, discomfort and symptoms confused with lactose intolerance. Studies showed that A1 milk consumption may cause diabetes, coronary heart disease, arteriosclerosis, sudden infant death, and is associated with autism and schizophrenia. With an increasing trend in the world, A2A2 milk (milk without A1 protein) production in the world is becoming widespread with consumer preferences; and, A2 milk takes its place on the market shelves. With the onset of this trend, the need for a new analysis on food safety became evident. It will be required to test the absence of A1 protein in milk to be labeled as A2 milk by food control laboratories. In this study, the quantitative analysis and validation of β-casein A1 and A2 proteins in cow's milk by Enzyme-Linked ImmunoSorbent Analysis (ELISA) method was investigated. The methods have detection limits of 1.8 and 0.8 ppm, and quantitation limits of 17 and 2.4 ppm for A1 and A2, respectively.

Milk protein polymorphism: Detection and diffusion of the genetic variants in Bos genus

Article

Full-text available

Jan 1999

Comparative effects of A1 versus A2 beta-casein on gastrointestinal measures: A blinded randomised cross-over pilot study

Article

Full-text available

Jul 2014
EUR J CLIN NUTR

Background/objectives: At present, there is debate about the gastrointestinal effects of A1-type beta-casein protein in cows' milk compared with the progenitor A2 type. In vitro and animal studies suggest that digestion of A1 but not A2 beta-casein affects gastrointestinal motility and inflammation through the release of beta-casomorphin-7. We aimed to evaluate differences in gastrointestinal effects in a human adult population between milk containing A1 versus A2 beta-casein. Subjects/methods: Forty-one females and males were recruited into this double-blinded, randomised 8-week cross-over study. Participants underwent a 2-week dairy washout (rice milk replaced dairy), followed by 2 weeks of milk (750 ml/day) that contained beta-casein of either A1 or A2 type before undergoing a second washout followed by a final 2 weeks of the alternative A1 or A2 type milk. Results: The A1 beta-casein milk led to significantly higher stool consistency values (Bristol Stool Scale) compared with the A2 beta-casein milk. There was also a significant positive association between abdominal pain and stool consistency on the A1 diet (r=0.520, P=0.001), but not the A2 diet (r=-0.13, P=0.43). The difference between these two correlations (0.52 versus -0.13) was highly significant (P<0.001). Furthermore, some individuals may be susceptible to A1 beta-casein, as evidenced by higher faecal calprotectin values and associated intolerance measures. Conclusions: These preliminary results suggest differences in gastrointestinal responses in some adult humans consuming milk containing beta-casein of either the A1 or the A2 beta-casein type, but require confirmation in a larger study of participants with perceived intolerance to ordinary A1 beta-casein-containing milk.

Dietary A1 β -casein affects gastrointestinal transit time, dipeptidyl peptidase-4 activity, and inflammatory status relative to A2 β -casein in Wistar rats

Article

Full-text available

Mar 2014
INT J FOOD SCI NUTR

Abstract We compared the gastrointestinal effects of milk-based diets in which the β-casein component was either the A1 or A2 type in male Wistar rats fed the experimental diets for 36 or 84 h. Gastrointestinal transit time was significantly greater in the A1 group, as measured by titanium dioxide recovery in the last 24 h of feeding. Co-administration of naloxone decreased gastrointestinal transit time in the A1 diet group but not in the A2 diet group. Colonic myeloperoxidase and jejunal dipeptidyl peptidase (DPP)-4 activities were greater in the A1 group than in the A2 group. Naloxone attenuated the increase in myeloperoxidase activity but not that in DPP-4 activity in the A1 group. Naloxone did not affect myeloperoxidase activity or DPP-4 activity in the A2 group. These results confirm that A1 β-casein consumption has direct effects on gastrointestinal function via opioid-dependent (gastrointestinal transit and myeloperoxidase activity) and opioid-independent (DPP-4 activity) pathways.

Autistic children display elevated urine levels of bovine casomorphin-7 immunoreactivity

Article

Full-text available

Jun 2014
PEPTIDES

Elevated concentrations of circulating casomorphins (CM), the exogenous opioid peptides from milk casein, may contribute to the pathogenesis of autism in children. Because several mass spectrometry studies failed to detect casomorphins in autistic children, it was questioned whether these peptides can be detected in body fluids by mass spec. Here we demonstrated, using a novel high sensitivity ELISA method, that autistic children have significantly higher levels of urine CM-7 than control children. The severity of autistic symptoms correlated with concentrations of CM-7 in the urine. Because CMs interact with opioid and serotonin receptors, the known modulators of synaptogenesis, we suggest that chronic exposure to elevated levels of bovine CMs may impair early child development, setting the stage for autistic disorders.

Genetic polymorphism of milk proteins

Article

Jan 1992

The biology of incretin hormones

Article

Jan 2006
CELL METAB

D.J. Drucker

Bioactive peptides released from in vitro digestion of human milk with or without pasteurization

Article

Jan 2015

Background Donor pasteurized human milk (HM) serves as the best alternative for breast-feeding when availability of mother's milk is limited. Pasteurization is also applied to mother's own milk for very low birth weight infants, who are vulnerable to microbial infection. Whether pasteurization affects protein digestibility and therefore modulates the profile of bioactive peptides released from HM proteins by gastrointestinal digestion, has not been examined to date.MethodsHM with and without pasteurization (62.5 ºC for 30 min) were subjected to in vitro gastrointestinal digestion, followed by peptidomic analysis to compare the formation of bioactive peptides.ResultsSome of the bioactive peptides, such as caseinophosphopeptide homologues, a possible opioid peptide (or propeptide), and an antibacterial peptide, were present in undigested HM and showed resistance to in vitro digestion, suggesting that these peptides are likely to exert their bioactivities in the gastrointestinal lumen, or be stably transported to target organs. In vitro digestion of HM released a large variety of bioactive peptides such as angiotensin I-converting enzyme-inhibitory, anti-oxidative, and immuno-modulatory peptides. Bioactive peptides were released largely in the same manner with and without pasteurization.Conclusions Provision of pasteurized HM may be as beneficial as breast-feeding in terms of milk protein-derived bioactive peptides.Pediatric Research (2015); doi:10.1038/pr.2015.10.

Release of b-casomorphin-7/5 during simulated gastrointestinal digestion of milk b-casein variants from Indian crossbred cattle (Karan Fries)

Article

Jul 2014
FOOD CHEM

Crossbred Karan Fries (KF) cows, among the best yielders of milk in India are carriers of A1 and A2 alleles. These genetic variants have been established as the source of β-casomorphins (BCMs) bioactive peptides that are implicated with various physiological and health issues. Therefore, the present study was aimed to investigate the release of BCM-7/5 from β-casein variants of KF by simulated gastrointestinal digestion (SGID) performed with proteolytic enzymes, in vitro. β-Casein variants (A1A1, A1A2 and A2A2) were isolated from milk samples of genotyped Karan Fries animals and subjected to hydrolysis by SGID using proteolytic enzymes (pepsin, trypsin, chymotrypsin and pancreatin), in vitro. Detection of BCMs were carried out in two peptide fractions (A and B) of RP-HPLC collected at retention time (RT) 24 and 28 min respectively corresponding to standard BCM-5 and BCM-7 by MS–MS and competitive ELISA. One of the RP-HPLC fractions (B) showed the presence of 14 amino acid peptide (VYPFPGPIHNSLPQ) having encrypted internal BCMs sequence while no such peptide or precursor was observed in fraction A by MS–MS analysis. Further hydrolysis of fraction B of A1A1 and A1A2 variants of β-casein with elastase and leucine aminopeptidase revealed the release of BCM-7 by competitive ELISA. The yield of BCM-7 (0.20 ± 0.02 mg/g β-casein) from A1A1 variant was observed to be almost 3.2 times more than A1A2 variant of β-casein. However, release of BCM-7/5 could not be detected from A2A2 variant of β-casein. The biological activity of released peptides on rat ileum by isolated organ bath from A1A1 (IC50 = 0.534–0.595 μM) and A1A2 (IC50 = 0.410–0.420 μM) hydrolysates further confirmed the presence of opioid peptide BCM-7.

Consumption of β-casomorphins-7/5 induce inflammatory immune response in mice gut through Th2 pathway

Article

Apr 2014

β-Casomorphins are opioid like bioactive peptides released on digestion of β-casein of milk. In the present study in vivo impact of consumption of β-casomorphins (BCM-7/5) was evaluated on immune response of murine gut. The peptides were given to mice through oral intubation at a dose of 7.5 × 10−8 mol/day/animal for 15 days. Oral administration of both peptides individually increased (p < 0.01) phenotypic expression of inflammation associated molecules (MPO, MCP-1, IL-4 and histamine), humoral immune response (IgE, IgG, IgG1/IgG2a), infiltration of leucocytes in intestinal villi and mRNA expression of toll like receptors (TLR-2 and TLR-4) in mice gut. However, no changes in sIgA, IgA+ and goblet cell numbers were recorded. These results clearly indicate that consumption of β-casein derived peptides BCM-7/5 induce inflammatory immune response in gut, most likely through Th2 pathway.

Isotope dilution liquid chromatography-tandem mass spectrometry for simultaneous identification and quantification of beta-casomorphin 5 and beta-casomorphin 7 in yoghurt

Article

Mar 2014
FOOD CHEM

A highly selective and sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous identification and quantification of beta-casomorphin 5 (BCM5) and beta-casomorphin 7 (BCM7) in yoghurt. The method used deuterium labelled BCM5-d10 and BCM7-d10 as surrogate standards for confident identification and accurate and quantification of these analytes in yoghurt. Linear responses for BCM5 and BCM7 (R(2)=0.9985 and 0.9986, respectively) was observed in the range 0.01-10ng/μL. The method limits of detection (MLDs) in yoghurt extracts were found to be 0.5 and 0.25ng/g for BCM5 and BCM7, respectively. Analyses of spiked samples were used to provide confirmation of accuracy and precision of the analytical method. Recoveries relative to the surrogate standards of these spikes were in the range of 95-106% for BCM5 and 103-109% for BCM7. Precision from analysis of spiked samples was expressed as relative standard deviation (%RSD) and values were in the range 1-16% for BCM5 and 1-6% for BCM7. Inter-day reproducibility was between 2.0-6.4% for BCM5 and between 3.2-6.1% for BCM7. The validated isotope dilution LC-MS/MS method was used to measure BCM5 and BCM7 in ten commercial and laboratory prepared samples of yoghurt and milk. Neither BCM5 nor BCM7 was detected in commercial yoghurts. However, they were observed in milk and laboratory prepared yoghurts and interestingly their levels decreased during processing. BCM5 decreased from 1.3ng/g in milk to 1.1ng/g in yoghurt made from that milk at 0day storage and <MLQ at 1 and 7days storage. BCM7 decreased from 1.9ng/g in milk to <MLQ in yoghurts immediately after processing. These preliminary results indicate that fermentation and storage reduced BCM5 and BCM7 concentration in yoghurt.

Milk Intolerance, Beta-Casein and Lactose

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