Elżbieta Kostyra

Polymorphism of bovine beta-casein and its potential effect on human health.

Proteins in bovine milk are a common source of bioactive peptides. The peptides are released by the digestion of caseins and whey proteins. In vitro the bioactive peptide beta-casomorphin 7 (BCM-7) is yielded by the successive gastrointestinal proteolytic digestion of bovine beta-casein variants A1 and B, but this was not seen in variant A2. In hydrolysed milk with variant A1 of beta-casein, BCM-7 level is 4-fold higher than in A2 milk. Variants A1 and A2 of beta-casein are common among many dairy cattle breeds. A1 is the most frequent in Holstein-Friesian (0.310-0.660), Ayrshire (0.432-0.720) and Red (0.710) cattle. In contrast, a high frequency of A2 is observed in Guernsey (0.880-0.970) and Jersey (0.490-0.721) cattle. BCM-7 may play a role in the aetiology of human diseases. Epidemiological evidence from New Zealand claims that consumption of beta-casein A1 is associated with higher national mortality rates from ischaemic heart disease. It seems that the populations that consume milk containing high levels of beta-casein A2 have a lower incidence of cardiovascular disease and type 1 diabetes. BCM-7 has also been suggested as a possible cause of sudden infant death syndrome. In addition, neurological disorders, such as autism and schizophrenia, seem to be associated with milk consumption and a higher level of BCM-7. Therefore, careful attention should be paid to that protein polymorphism, and deeper research is needed to verify the range and natureof its interactions with the human gastrointestinal tract and whole organism.

Transport of μ-opioid receptor agonists and antagonist peptides across Caco-2 monolayer

Milk is the source of beta-casomorphins--biologically active peptides with opioid activity--which are suspected to play various roles in the human body. The local influence of exogenous opioid peptides on gastrointestinal functions has been widely reported. After passing the gut barrier, beta-casomorphins may affect the functions of immunological system, as well as dopaminergic, serotoninergic and GABA-ergic systems in brain, regulate the opioid receptor development and elicit behavioral effects. However, possibilities and mechanisms of the intestinal transport of beta-casomorphins in human body in vivo have not been reported so far. In our research, the transepithelial transport of micro-opioid receptor agonists--human beta-casomorphin-5 and 7(BCM5, BCM7) and antagonist--lactoferroxin A (LCF A) have been investigated using Caco-2 monolayer. In order to determine the pathway of investigated peptide transport across Caco-2 monolayer, two directions of the transport (apical to basolateral and basolateral to apical) have been studied. All investigated peptides were transported across the human intestinal cell line Caco-2 and the curves of cumulative amount of transported peptides in time were linear in each case. In addition, the hydrolysis of beta-casomorphins during 60 min of experiment by dipeptidyl peptidase IV was observed. The data suggest the possibility of transport of opioid peptides derived from food across human intestinal mucosa.

Changes of β-casomorphin content in human milk during lactation

Milk is the best, complete food important for the development and nourishment of a neonate. Except for nutrients, milk contains biologically active opioid peptides derived from beta-casein, named beta-casomorphins (BCMs), which can exert effects in the gastrointestinal tract as well as in the whole body of neonates. The content of beta-casomorphins in human milk during maturation phases has not been studied so far. The aim of this study was to determine the content of beta-casomorphin-5 and -7 in human milk in different phases of lactation. A significantly higher concentration of both beta-casomorphins was found in colostrum than in mature milk. The concentration of beta-casomorphin in milk collected in the second month of lactation was similar to the level obtained in the fourth month of lactation. The content of beta-casomorphins in human milk was observed with the period of lactation. The level of opioid peptides may depend on the function of these peptides in neonates body and may be associated with the maturation process.

The influence of μ-opioid receptor agonist and antagonist peptides on peripheral blood mononuclear cells (PBMCs).

Milk is one of the main source of biologically-active peptides that may function as regulatory substances called food hormones. After passing the gut-blood barrier, the μ-opioid receptor agonist and antagonist peptides may become the new factors influencing various functions of the human organism. The aim of the conducted research was to determine the influence of μ-opioid receptor agonist peptides: human and bovine β-casomorphin-7 (h/bBCM-7) and antagonistic peptides: casoxin-6 and- D (CXN-6/D) on proliferation and cytokine secretion of human peripheral blood mononuclear cells (PBMCs). The PBMCs proliferation was measured by the use of the BrdU test, which assesses the DNA synthesis activity and the WST-1 test which assesses the activity of mitochondrial dehydrogenase enzymes. The influence of all the investigated peptides on secretion of IL-4, IL-8, IL-13 and IFN-γ was determined by the use of the ELISA tests. Incubating the cells with the peptides has not caused any changes to their enzymatic activity, which has been proved by a WST-1 test. When using a BrdU test, however, it has been observed that there appear changes to proliferation of PBMCs correlated to amounts of bromodeoxyuridine incorporated into the cellular DNA. Moreover, changes to secretion of IL-4 and IL-13 by the cells under the influence of agonists were detected, as well as changes to secretion of IFN-gamma under the influence of all the examined substances. The obtained results provide information on immunomodulatory effects of food-derived opioid peptides, which may be of clinical significance especially in the case of allergic diseases in newborns.

Milk from cows of different β-casein genotypes as a source of β-casomorphin-7.

The aim of this study was to quantify β-casomorphin-7 in raw, hydrolyzed and processed milk in different stages of the cow lactation. The obtained results lead to the following conclusion: the highest amount of β-casomorphin-7 released from the hydrolyzed and processed milk is related to the β-casein A1 allele, irrespective of a lactation period. Some traces of β-casomorphin-7 in milk from cows with the β-casein A2 variant are probably a result of the acid hydrolysis of β-casein during its digestion with pepsin. It has been shown that processing of raw milk at high temperatures affects, in a slight degree, the differences between β-casomorphins-7 originating from different β-casein genotypes. The obtained results suggest a possibility to provide a new nutritional factor for milk quality based on the content of β-casomorphin-7 liberated in vivo from milk digested by a mixture of the gastrointestinal enzymes.

Influence of candidate polymorphisms on the dipeptidyl peptidase IV and μ-opioid receptor genes expression in aspect of the β-casomorphin-7 modulation functions in autism

Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder with population prevalence of approximately 60-70 per 10,000. Data shows that both opioid system function enhancement and opiate administration can result in autistic-like symptoms. Cow milk opioid peptides, including β-casomorphin-7 (BCM7, Tyr-Pro-Phe-Pro-Gly-Pro-Ile), affect the μ-opioid receptor (MOR) and are subjected to degradation resulting from the proline dipeptidyl peptidase IV (DPPIV, EC 3.4.14.5) enzyme activity. The presence of MOR and DPPIV activity are crucial factors determining biological activity of BCM7 in the human body. Our study examined the effect of β-casomorphin-7 on the MOR and DPPIV genes expression according to specific point mutations in these genes. In addition, we investigated frequency of A118G SNP in the MOR gene and rs7608798 of the DPPIV (A/G) gene in healthy and autistic children. Our research indicated correlation in DPPIV gene expression under the influence of BCM7 and hydrolyzed milk between healthy and ASD-affected children with genotype GG (P<0.0001). We also observed increased MOR gene expression in healthy children with genotype AG at polymorphic site A118G under influence of BCM7 and hydrolyzed milk. The G allele frequency was 0.09 in MOR gene and 0.68 in the DPPIV gene. But our results suggest no association between presence of the alleles G and A at position rs7608798 in DPPIV gene nor alleles A and G at position A118G of the MOR and increased incidence of ASD. Our studies emphasize the compulsion for genetic analysis in correlation with genetic factors affecting development and enhancement of autism symptoms.

The impact of pea protein hydrolysates on bacterial physiological activity—An in vitro study

So far, food proteins have been perceived hitherto purely as a source of nutrients indispensable for maintaining life. However, latest findings strongly indicate that food proteins may release biologically active peptides in a consequence of enzymatic degradation. Such hydrolysates may be used as food components in order to beneficially influence human health. Additionally, such modified peptides may affect the balance of bacteria inhabiting human gastrointestinal tract and thus bring about health complication of the host. Although pea seeds are of significant nutritional value due to their high contents of proteins, carbohydrates and fibre, they are also responsible for health inconveniences resulting from their susceptibility to digestion and occurrence of antinutritional as well as allergic compounds. The enzymatic degradation may pass over these nutritional obstacles by liberating hydrolysates empowered not only to exert their impact on the human physiology but also on bacterial intestinal ecosystem. Therefore, the aim of this study was to evaluate the influence of pea protein hydrolysates on bacteria typical for the small intestine. Pea protein hydrolysates have proved to diversely modulate physiological activity of bacteria existing in different states. The observed detrimental effect on planktonic bacteria was abolished in the case of bacteria immobilized to the solid surfaces, confirming the protective effect of biofilms. Additionally, Lactobacilli displayed adaptive properties enabling them to utilize pea protein hydrolysates regardless their state of existence.

Content of β-casomorphins in milk of women with a history of allergy

The prevalence of food allergies increased over the past decade. Most symptoms of food allergy appear during the first 2 yr of life. The aim of this study was to determine the β‐casomorphin‐5 and ‐7 (BCMs) in colostrum and milk of 12 breast‐feeding women with a history and clinical manifestation of food allergy. The results were compared with the data obtained from a control group of healthy age‐matched breast‐feeding women. The level of BCM in women with food allergy was constant during lactation, whereas the highest level of opioid peptides was found in colostrums of healthy women with a subsequent rapid decrease in mature milk. These differences in BCMs profile between allergic and healthy breast‐feeding women suggest that BCM content in the human milk may be an indicator of allergic conditions.

Cow's-milk-induced infant apnoea with increased serum content of bovine β-casomorphin-5.

772 S uddenly occurring life-threatening events with symptoms such as apnoea, changes in skin colour (eg, blue or cyanotic), altered muscle tone (eg, floppy, stiff), coughing, choking, or gagging are called apparent life-threatening events (ALTE). About 7.4% to 10% of infants with ALTE, mostly in its recurring form, cannot be saved and they die of sudden infant death syndrome (SIDS) (1–3). A number of different and independent causes of ALTE pathogenesis have been described. Those causes include diseases of various organs and systems, infections, nonaccidental traumas, and Münchausen syndrome by proxy (1,4,5). A coexistence of digestive tract disorders is also diagnosed in >50% of infants with ALTE. Gastro-oesophageal reflux is 1 of the most often diagnosed disorders; however, its role in the genesis of infantile apnoea has not been fully explained (6). ALTE aetiology is explainable and diagnosable only in half of all of the cases (2,3). The causes of ALTE described before do not include an opioid action of the exogenous peptides released from milk b-caseins. We report a case of a breast-fed infant with recurrent apnoea episodes, which have always been preceded by his mother’s consumption of fresh cow’s milk. A biochemical examination has revealed a high level of b-casomorphin-5 (BCM-5) in the child’s serum. We speculate that it is an opioid activity that may have a depressive effect on the respiratory centre in the central nervous system and induce a phenomenon called milk apnoea. A full-term male infant from a rural family, age 7 weeks, was referred to a macroregional SIDS prevention centre to diagnose the cause of his recurrent ALTE. Apnoea had been occurring since the child was 3 weeks old and its clinical course kept getting more serious. The infant’s mother noticed that those events occurred only after her consumption of cow’s milk, particularly when she had consumed large amounts of it (up to 2 L/d). Before and during her pregnancy, the mother drank milk with no adverse effects. The boy’s ALTE occurred during breast-feeding or directly after that, and was manifested as sudden, 20-, 30-, or 40-second-long apnoea with generally lowered muscle tone. Most of the episodes required

Glucose and calcium ions may modulate the efficiency of bovine β-casomorphin-7 permeability through a monolayer of Caco-2 cells.

Milk and dairy products provide a lot of valuable nutritive elements. They are also sources of biologically active peptides, including β-casomorphins that manifest the properties of morphine. An activity of DPPIV seems to be most crucial factor decreasing the efficiency of the β-casomorphin-7 (BCM7) transport. The increase of BCM7 concentration in blood may intensify symptoms of apparent life threatening events (ALTE), autism, schizophrenia, and allergy. This study aimed at identifying the influence of several selected substances on a transport efficiency of bovine BCM7 through an intestinal monolayer in a Caco-2 cell model system. Applying the ELISA method, the permeability coefficient of BCM7 through the Caco-2 monolayer was calculated. TEER values were used to evaluate the integrity of Caco-2 cell monolayers. An increase of glucose and Ca(2+) concentrations in the culture medium was accompanied by an increase of the BCM7 transport efficiency. The lowest permeability coefficients of BCM7 were observed for the membranes with high electrical resistances. The transport was enhanced in the presence of milk infant formulas, whereas no changes were observed when using μ-opioid receptor antagonist (casoxin-6). The results may be useful in understanding the pathogenesis of inflammation and food allergy in infants.