Identification of the human β-casein C-terminal fragments that specifically bind to purified antibodies to bovine β-lactoglobulin (original) (raw)
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Immunoregulatory peptides in bovine milk
Bovine milk is known to contain a number of peptide fractions that can affect immune function. The vast majority of immunoregulatory peptides that have been characterised are hydrolysate derivatives of major milk proteins. Recent research has also indicated that the metabolic activity of probiotic lactic acid bacteria can generate de novo immunoregulatory peptides from milk, via enzymatic degradation of parent milk protein molecules. In contrast, relatively little is known of endogenous, preformed immunoregulatory peptides in milk that may be relevant to modulating human health. The natural in vivo role of preformed and enzymatically derived peptides is likely to be one of regulation of the neonatal (bovine) gastrointestinal tract immune system, in order to modulate immune function with respect to the development of immunocompetence and avoidance of undesirable immunological responses (e.g. tolerance, and hypersensitivity to nutrients). There is scope for the further characterisation of both the origin and function of milk-derived immunoregulatory peptides, so that their potential to influence human health can be fully appraised. This review highlights our current knowledge of milk-derived immunoregulatory peptides, and outlines areas that are of relevance for further research.
Excretion of Dietary Cow's Milk Derived Peptides Into Breast Milk
Frontiers in Nutrition, 2019
Nanoflow-HPLC-tandem mass spectrometry (MS/MS) was used to analyze the peptide fraction of breast milk samples collected from a single non-atopic donor on different days (10 samples) after receiving an oral load of cow's milk (by drinking 200 mL of bovine milk). In addition, breast milk was sampled from the same lactating mother over a 6-h period at five time points after drinking cow's milk. We aimed to trace the intra-individual variability and to define a time profile of the excretion of dietary peptides into breast milk. Overall, 21 peptides exclusively originating from both bovine caseins and whey proteins with no match within the human milk proteome were identified in the breast milk samples. These peptides were missing in the breast milk obtained from the mother after a prolonged milk-and dairy-free diet (three samples). The time course of cow's milk-derived β-Lg f(125-135) and β-casein f(81-92) in breast milk was determined from the MS ion intensity of the peptide signals. No intact cow's milk gene products were detected by HPLC-MS/MS analysis and Western blotting with anti-β-Lg antibody, but dot-blot analysis confirmed the occurrence of β-Lg fragments in the enriched peptide fraction of breast milk. These data suggest shifting the analytical perspective for the detection of dietary food allergens in breast milk from intact proteins to digested peptide fragments. The possible sensitization and elicitation potential or the tolerogenic properties of such low amounts of dietary peptides for the breastfed newborns remain to be explored.
Analytical and Bioanalytical Chemistry, 2011
The aim of this work was the determination of peptides, which can function as markers for identification of milk allergens in food samples. Emphasis was placed on two casein proteins (α-and β-casein) and two whey proteins (α-lactalbumin and β-lactoglobulin). In silico tryptic digestion provided preliminary information about the expected peptides. After tryptic digestion of four milk allergens, the analytical data obtained by combination of reversed-phase high performance liquid chromatography and quadrupole tandem mass spectrometry (LC-MS/MS) led to the identification of 26 peptides. Seven of these peptides were synthesized and used for calibration of the LC-MS/MS system. Species specificity of the selected peptides was sought by BLAST search. Among the selected peptides, only LIVTQTMK from β-lactoglobulin (m/z 467.6, charge 2+) was found to be cow milk specific and could function as a marker. Two other peptides, FFVAPF-PEVFGK from α-casein (m/z 693.3, charge 2+) and GPFPIIV from β-casein (m/z 742.5, charge 1+), occur in water buffalo milk too. The other four peptides appear in the milk of other species also and can be used as markers for ruminant species milk. Using these seven peptides, a multianalyte MS-based method was developed. For the establishment of the method, it was applied at first to different dairy samples, and then to chocolate and blank samples, and the peptides could be determined down to 1 ng/mL in food samples. At the end, spiked samples were measured, where the target peptides could be detected with a high recovery (over 50%).
2011
The present review describes selected peptides derived from bovine milk proteins and demonstrates their effect on the human immune system. Apart from their obvious nutritive value milk proteins and products of their degradation (peptides) have multiple biological functions. Bovine milk, fermented milk drinks and cheeses are the most abundant sources of biologically active peptides. One of the primary functions of milk is to protect the health of a newborn organism by the virtue of the fact that milk contains many proteins, which exhibit bacteriostatic and bactericidal properties in their intact form. Ingestion of bovine milk by humans causes that bioactive peptides are evoked from delivered proteins during the course of digestion. They possess not only immunomodulatory, but also antibacterial, antiviral and antifungal properties.
International Dairy Journal, 2006
The use of hydrolysed formulas for feeding babies is efficient for preventing allergic diseases. Several hypoallergenic formulas (HF) based on hydrolysed cows’ milk proteins have been commercialised. However, some children fed such formulas suffer allergic reactions. β-lactoglobulin (βLG) is the main allergen present in cows’ milk. In addition to the whole protein, βLG fragments have also shown allergenic character. Detection
Food and Bioproducts Processing, 2014
Protein A mimetic peptide ligands have several benefits over conventional Protein A/G ligands, namely that they are small in size, have low production costs, are stable over a wide range of pH values and can withstand cleaning by harsh sanitization agents such as sodium hydroxide. In this paper, a hexamer peptide (HWRGWV) affinity matrix was used for the isolation of bovine immunoglobulins from various dairy streams (skim milk, acid whey and colostrum). Bound immunoglobulins were recovered in elution buffer (0.2 M sodium acetate buffer, pH 4.0) fractions with a purity of >85% in a single step. The peptide resin has achieved a maximum equilibrium adsorption capacity of 23±0.58 mg.mL-1 of resin for bovine IgG and had a dynamic binding capacity of 11.8±0.03 mg.mL-1 at residence time of 2 min. These results suggest that the hexamer peptide chromatography could potentially be used for the selective purification of bovine immunoglobulins from dairy streams. This method has promise as an alternative to conventional Protein A/G chromatography for direct capture of immunoglobulins from streams containing relatively high immunoglobulin concentrations such as colostrum, transgenic or hyper-immune milk.
Isolation and purification of beta-lactoglobulin from cow milk
Veterinary World, 2015
The present study was undertaken to standardize a convenient method for isolation and purification of β-lactoglobulin (β-lg) from cow milk keeping its antigenicity intact, so that the purified β-lg can be used for detection of cow milk protein intolerance (CMPI). Materials and Methods: Raw milk was collected from Gir breed of cattle reared in Haringhata Farm, West Bengal. Milk was then converted to skimmed milk by removing fat globules and casein protein was removed by acidification to pH 4.6 by adding 3 M HCl. β-lg was isolated by gel filtration chromatography using Sephacryl S-200 from the supernatant whey protein fraction. Further, β-lg was purified by anion-exchange chromatography in diethylaminoethyl-sepharose. Molecular weight of the purified cattle β-lg was determined by 15 percent one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was analyzed by gel documentation system using standard molecular weight marker. Results: The molecular weight of the purified cattle β-lg was detected as 17.44 kDa. The isolated β-lg was almost in pure form as the molecular weight of purified β-lg monomer is 18kDa. Conclusion: The study revealed a simple and suitable method for isolation of β-lg from whey protein in pure form which may be used for detection of CMPI.
Simple Purification Method for Beta-Lactoglobulin from Buffalo Milk
Advances in Animal and Veterinary Sciences, 2014
It has been observed that 82 percent of milk-allergic patients are sensitive to betalactoglobulin (β-lg), a major milk protein that accounts for approximately 10 to 15 percent of total milk proteins. The modification in β-lg is considered a promising venture to mitigate milk allergies. With the aim of standardizing a convenient method for isolation and purification of β-lg from buffalo milk, the present study was designed to keep its antigenicity intact, so that, the purified β-lg can be used to detect buffalo milk protein intolerance. Raw milk was collected from Murrah breed of buffalo from Haringhata farm (West Bengal) and converted to skimmed milk by removing fat globules. Casein protein was removed by acidification to pH 4.6 by adding 3 M HCl. The β-lg was isolated by gel filtration chromatography using Sephacryl S-200 from supernatant whey protein fraction. Further, βlg was purified by anion-exchange chromatography using DEAE-Sepharose. Molecular weight of the purified buffalo β-lg was 18.05kDa as assessed by the gel documentation system using standard molecular weight marker (range 14.3 to 97.4 kDa) in 15 percent onedimensional SDS-PAGE. The isolated β-lg was almost in pure form as the molecular weight of purified β-lg monomer is 18kDa. The study revealed a simple and suitable method for isolation of β-lg from whey protein in pure form that might be exploited for any basic and applied studies related to buffalo milk protein intolerance.