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

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 ß-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 ß-casein variants of KF by simulated gastrointestinal digestion (SGID) performed with proteolytic enzymes, in vitro. ß-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 28min 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 ß-casein with elastase and leucine aminopeptidase revealed the release of BCM-7 by competitive ELISA. The yield of BCM-7 (0.20±0.02mg/g ß-casein) from A1A1 variant was observed to be almost 3.2 times more than A1A2 variant of ß-casein. However, release of BCM-7/5 could not be detected from A2A2 variant of ß-casein. The biological activity of released peptides on rat ileum by isolated organ bath from A1A1 (IC50=0.534-0.595µM) and A1A2 (IC50=0.410-0.420µM) hydrolysates further confirmed the presence of opioid peptide BCM-7.

Comparative evaluation of cow ß-casein variants (A1/A2) consumption on Th2-mediated inflammatory response in mouse gut.

PURPOSE: Recently, apprehension has been raised regarding "A1/A2 hypothesis" suggesting relationship between consumption of A1 "like" variants of cow ß-casein and various physiological disorders. The information available is based on either the human epidemiological data of milk consumption or in vitro trials on cell lines with ß-casomorphin peptides. The direct scientific evidence establishing the link between consumption of A1/A2 "like" milk and health is scanty. Thus, under present investigation, in vivo trials in mice were undertaken to study the effect of feeding three genetic variants (A1A1, A1A2 and A2A2) of cow ß-casein milk on gastrointestinal immune system as it is the first and foremost site of immunological interactions. METHODS: Animals were divided into four groups for feeding with basal diet (control) and ß-casein isolated from milk of genotyped (A1A1, A1A2 and A2A2) dairy animals, respectively. Gut immune response was analyzed by spectrophotometric assessment of MPO activity, quantitative sandwich ELISA of inflammatory cytokines (MCP-1 and IL-4), antibodies (total IgE, IgG, sIgA, IgG1 and IgG2a) and qRT-PCR of mRNA expression for toll-like receptors (TLR-2 and TLR-4). Histological enumeration of goblet cells, total leukocytes and IgA(+) cells was also carried out. RESULTS: It was observed that consumption of A1 "like" variants (A1A1 and A1A2) significantly increased (p < 0.01) the levels of MPO, MCP-1, IL-4, total IgE, IgG, IgG1, IgG2a and leukocyte infiltration in intestine. TLR-2 and TLR-4 mRNA expression was also up-regulated (p < 0.01) on administration of A1 "like" variants. However, no changes in sIgA, IgA(+) and goblet cell numbers were recorded on consumption of any of the ß-casein variants. CONCLUSION: It is reasonable to conclude that consumption of A1 "like" variants of ß-casein induced inflammatory response in gut by activating Th2 pathway as compared to A2A2 variants. The present study thus supports the purported deleterious impacts of consumption of A1 "like" variants of ß-casein and suggests possible aggravation of inflammatory response for etiology of various health disorders.