Β-casein Fragment Research Articles

Adaptation of Stadier's apparatus for electrophoresis of main milk proteins

The object of research is a Stadier-type apparatus for analytical electrophoresis of proteins. In the milk proteins research, in addition, there is a need to carry out serial express analyzes of their various groups, as well as the isolation of individual homogeneous fractions. The dimensions of working chambers for analytical, express, and micro preparative electrophoresis of caseins and milk whey proteins were proposed to solve this task. For each type of electrophoresis, different chambers and formers are used without changing the design of the apparatus. The apparatus is suitable for electrophoretic systems used for the analysis of milk proteins. Analysis of casein in the anodic system of a homogeneous polyacrylamide gel in the presence of urea allows identification of the main fractions: αS1-CN-8P, αS1-CN-9P, αS2-CN-10P, αS2-CN-11P, αS2-CN-12P, αS2-CN-13P, β-CN-5P, ϰ-CN-1P and three β-casein fragments f(29-209), f(106-209) and f(108-209). Express electrophoresis in the presence of urea reveals four fractions of caseins: αS1-CN, αS2-CN, β-CN, and ϰ-CN. The analysis of whey proteins in the Davis native disc electrophoresis system allows identification of β-Lg A, β-Lg B, α-La, BSA fractions, and a group of immunoglobulin fractions. The express electrophoregram differs by a common band A and B variants of β-Lg. Due to an adequate selection of electrophoretic systems, it is possible to identify semi-quantitatively all the main fractions of milk proteins under analytical or express mode. The adapted apparatus also makes it possible to conduct micro preparative electrophoresis and obtain the main fractions of milk proteins. In this case, the yield of electrophoretically pure proteins is: β-CN-5P (23±5 %), β-Lg (A+B) (27±6 %), α-La (11±3 %), and purified groups of αS1-CN-8P+αS1-CN-9P (25±6 %), αS2-CN-(10-13P) (6±1.5 %) and ϰ-CN-1P (7±2 %). The apparatus could be used at enterprises producing dairy protein products

Impact of a real food matrix and in vitro digestion on properties and acute toxicity of polystyrene microparticles

Although it is proved that humans ingest microplastics via food, and microplastics were found in human tissues, blood and feces, there needs to be more data on the properties and health-related effects of plastic particles that interact with food and undergo digestion. This study aimed to examine the impact of a real food matrix, milk, on the behavior and gastrointestinal fate of polystyrene microparticles (PSMP). In the presence of the food matrix, the net negative ζ-potential values of PSMP (diameter size of 1.823 μm) decreased significantly due to the formation of the corona, mostly consisting of α and β-casein fragments. Protein corona profiles and morphologies of particles incubated with whole and skim milk were found to be similar, and the protein profiles were completely altered after in vitro digestion simulation. In vitro and in vivo toxicity studies showed that neither bare PSMP nor food-interacted PSMP pose acute toxicity on the Caco-2 cell line and zebrafish embryos under the chosen experimental conditions. In summary, these results may contribute to a better understanding of changes that microplastics undergo in foods. Further studies on repeated exposure or chronic toxicity are needed to fully reveal the effect of food matrix on microplastic toxicity.

Multiple phosphorylated protein selective analysis via fluorous derivatization and liquid chromatography-electrospray ionization-mass spectrometry analysis

Post-translational modification of proteins is involved in protein function and higher-order structure. Among such modification, phosphorylation is an important intracellular signal transduction pathway. Many studies on phosphorylated protein analysis using liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) have been developed. However, there are few reports on the analysis of highly phosphorylated proteins because of their handling difficulty. Hence, we developed an analytical method that converts multiple phosphate groups contained in the peptides into perfluoroalkyl groups for selective analysis using fluorous affinity. Here, tryptic digested β-casein fragment peptides [RELEELNVPGEIVE(pSer)L(pSer)(pSer)(pSer)EESITR and FQ(pSer)EEQQQTEDELQDK] were used as model phosphorylated peptides. 1H,1H,2H,2H-Perfluorooctanethiol (PFOT) and 2,2,2-trifluoroethanethiol (TFET) were used as derivatization reagents for mono-phosphorylated peptides and multi-phosphorylated peptides, respectively, to derivatize via β-elimination/Michael addition. The derivatives were analyzed by LC-ESI-MS. A fluorous LC column is typically used to selectively retain the fluorous-derivatized peptides, which are expected to be separated from contaminants and non-phosphorylated peptides. When this method was applied to β-casein, TFET- and PFOT-derivatized peptides were strongly retained in the fluorous LC column and clearly separated from non-phosphorylated peptides on the chromatogram. Therefore, the developed method enables quantification of mono- and multi-phosphorylated peptides and is suitable for application in proteomics.

Apical-to-basolateral transepithelial transport of cow's milk caseins by intestinal Caco-2 cell monolayers: MS-based quantitation of cellularly degraded α- and β-casein fragments.

Casein (CN) is the major milk protein to nourish infants but, in certain population, it causes cow's milk allergy, indicating the uptake of antigenic CN and their peptides through the intestinal epithelium. Using human intestinal Caco-2 cell monolayers, the apical-to-basal transepithelial transport of CN was investigated. Confocal microscopy using component-specific antibodies showed that αs1-CN antigens became detectable as punctate signals at the apical-side cytoplasm and reached to the cytoplasm at a tight-junction level within a few hours. Such intracellular CN signals were more remarkable than those of the other antigens, β-lactoglobulin and ovalbumin, colocalized in part with an early endosome marker protein (EEA1) and decreased in the presence of cytochalasin D or sodium azide and also at lowered temperature at 4°C. Liquid chromatography coupled with mass spectroscopy analysis of the protein fraction in the basal-side medium identified the αs1-CB fragment including the N-terminal region and the αs2-CN fragment containing the central part of polypeptide at 100-1,000 fmol per well levels. Moreover, β-CN C-terminal overlapping peptides were identified in the peptide fraction below 10 kDa of the basal medium. These results suggest that CNs are partially degraded by cellular proteases and/or peptidases and immunologically active CN fragments are transported to basal side of the cell monolayers.

Purification and identification of β-casein phosphopeptide (1-25).

The β-casein phosphopeptide 1-25 (βCPP) is involved in calcium binding, cellular transduction, and dental remineralization. The objective of this work was to improve upon the original protocol commonly used for isolation of this phosphopeptide from β-casein. This method exploits the isoelectric point of β-casein fragments to selectively precipitate βCPP. The highest βCPP extraction yield reported to date with this protocol is 14.4±0.5% of theoretically available βCPP. The present work optimizes 2 steps in this procedure, namely the length of trypsin digestion and incorporation of cold acetone precipitation, to increase the yield to 32.3±5.4%. Reverse-phase HPLC indicated high purity of the isolate, whereas mass spectrometry confirmed 2 forms of the phosphopeptide: fragments 1-25 (87%) and 2-25 (13%). The adaptation of the existing protocol represents a significant improvement in extraction yield and facilitates preparation of larger amounts of high-purity βCPP for subsequent analysis and use in functional foods and other applications.

Determination of changes in bovine plasma and milk proteins during naturally occurring Escherichia coli mastitis by comparative proteomic analysis

The aim of this study was to investigate changes in plasma and milk proteins in response to Escherichia coli infection in cows. Plasma and milk were collected from healthy cows, cows suffering from mild E. coli mastitis, and cows suffering from severe E. coli mastitis. Protein composition was examined by two-dimensional gel electrophoresis coupled with mass spectrometry. Plasma haptoglobin and α-1 acid glycoprotein demonstrated greater expression in mastitic cows compared with controls, but there were no difference between mildly and severely mastitic cows. Milk from mildly mastitic cows showed increased albumin and casein variants. Severely mastitic cows showed lower casein levels and increased anti-microbial and acute phase proteins. Milk α-1 acid glycoprotein and cathelicidins were associated with severe mastitis. A greater number of β-casein fragments that corresponded to β-casein isoforms were found in milk from mildly mastitic cows. These results suggest that caseins levels decreased and the concentrations of anti-microbial and acute phase proteins increased corresponding to the degree of E. coli mastitis. Nevertheless, further studies are needed to determine whether cathelicidin could serve as a diagnostic marker for mastitis.

Bioavailability and Kinetics of the Antihypertensive Casein-Derived Peptide HLPLP in Rats

The aim of this study was to investigate the oral bioavailability and kinetics of the milk casein-derived peptide HLPLP, which had previously demonstrated antihypertensive effect in spontaneously hypertensive rats. HLPLP disposition after single intravenous (4 mg/kg body weight) and oral (40 mg/kg body weight) doses was studied in rats. Plasma concentrations of HLPLP [β-casein fragment f(134-138)], and two derived fragments found after HLPLP administration, LPLP [β-casein fragment f(135-138)] and HLPL [β-casein fragment f(134-137)], were determined by ultrahigh performance liquid chromatography (UPLC) coupled on line to a Q-TOF instrument. For HLPLP, the elimination half-lives (T1/2β) were 7.95 min after intravenous and 11.7 min after oral administration. The volume of distribution at steady state (Vss = 30.8 L/kg) suggests a considerable uptake of HLPLP into tissues. HLPLP was converted to the peptides LPLP and HLPL. After HLPLP intravenous administration, the elimination half-lives (T1/2β) for these biotransformed peptides, LPLP and HLPL, were 8.38 and 10.9 min, respectively. After oral administration, HLPLP was rapidly absorbed with an absorption half-life (T1/2a) of 2.79 min. The oral bioavailability of HLPLP was found to be 5.18%. Our study suggested that HLPLP was rapidly absorbed and eliminated after oral administration, biotransformed into smaller fragments LPLP and HLPL, and distributed throughout the body by the circulation blood. The present pharmacokinetic information from a preclinical kinetic study in rats can also play an important role in designing future kinetic studies in humans for assessing HLPLP dose-response relationship.

The Role of Anionic Peptide Fragments in 1N4R Human Tau Protein Aggregation

Cellular protein degradation systems are necessary to avoid the accumulation of misfolded or damaged proteins. Deficiency in these systems might cause to partial degradation of misfolded proteins and generation of amyloidogenic fragments. Protein misfolding is believed to be the primary cause of neurodegenerative disorders such as Alzheimer's disease (AD). In this study, we investigate effect of two anionic peptide fragments including, an acidic fragment of human Aβ (Aβ1-11) and a phosphorylated fragment of β-Casein (Tetraphosphopeptide), on tau protein aggregation. According to our results, these peptide fragments, induced tau fibrillization in vitro. In sum, we suggest that structural and conformational characters of inducer are as important as charge distribution on anionic inducer molecules however more experiments would be need to exactly confirm this suggestion.

Characterization of a bioactive peptide with cytomodulatory effect released from casein

To obtain a bioactive peptide released from casein and investigate the cytomodulatory effect of this bioactive peptide, Lactobacillus helveticus CICC 22171, isolated from traditional Chinese dairy product, was directly used to hydrolyze casein (5 %) at 37 C for 48 h. Ultrafiltration, Sephadex column chromatography and high-speed counter-current chromatography were used to purify the peptide. After obtaining the peptide (named as β-Casobapt-ZW), a battery assays, including acridine orange/ethidium bromide (AO/EB) staining, TUNEL assay and flow cytometry analysis, were used to determine the cytomodulatory effect on Caco-2 and IEC-6 cells. The results showed that β-Casobapt-ZW could induce Caco-2 cell shrinkage, chromosome aggregation and apoptotic. The apoptotic rate could reach to 20.36 %. However, the effects were not obvious for IEC-6 cell, and the apoptotic rate was only 3.1 %. β-Casobapt-ZW showed obvious effect on inducing Caco-2 cells apoptosis and could not have significant influence on the growth of IEC-6 cells. The molecular weight of this peptide was about 1,020 Da, and the amino acid sequence was EPVLGPVRGP. It belonged to a fragment of β-casein (β-CN, f210–219).

A simple competitive enzyme-linked immunosorbent assay for the specific detection of the multiphosphorylated 1–25 β-casein fragment – ERRATUM

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A peptidomic approach to biomarker discovery for bovine mastitis

Bovine mastitis is usually caused by either Gram positive or Gram negative bacteria, reducing the quantity and quality of milk produced. This investigation using capillary electrophoresis and mass spectroscopy, studied peptides in milk from cows with clinical mastitis in comparison to milk from healthy cows to identify biomarkers for mastitis. In addition, the milk peptidome from udders infected with Gram positive Staphylococcus aureus (S. aureus) or with Gram negative Escherichia coli (E. coli), was examined to assess differential diagnosis between the causative agent. Comparison of the peptidome between healthy (n=10) and mastitic milk (n=27) identified 154 peptides for a biomarker panel which in a model for diagnosis of mastitis showed 100% sensitivity and specificity. β-casein and α(s1) casein provided the majority of peptides identified in this model. The peptidome comparison of milk from mastitis cases caused by S. aureus (n=8) or E. coli (n=11) revealed a biomarker panel of 47 peptides which discriminated between cause of infection with a sensitivity of 75% and a specificity of 100%. β-casein fragments were the most common of the peptides in this model. Peptide biomarkers of milk could be used in the diagnosis of mastitis and can discriminate between these two bacterial causes. The paper describes an innovative approach to the use of gel free proteomics to identify the peptides that are present in milk during clinical mastitis, which is a major cause of loss of production to dairy farmers worldwide. The use of capillary electrophoresis, liquid chromatography and mass spectrometry has been able to identify panels of peptides which can be used for disease diagnosis and for differential diagnosis of the causative bacteria of the infections of the mammary gland. As well as contributing to our knowledge of the pathophysiology of bovine mastitis the results could be the basis of improved detection and differential diagnosis of the disease.

A simple competitive enzyme-linked immunosorbent assay for the specific detection of the multiphosphorylated 1–25 β-casein fragment

A specific and simple competitive enzyme-linked immunosorbent assay (ELISA) was developed to determine bovine β-casein phosphopeptides (β-CPP) in casein phosphopeptides (CPP) or CPP complexes such as casein phosphopeptide amorphous calcium phosphate complexes added into dairy products. The method combines sample pretreatment designed for CPP enrichment and anti-β-CPP(f(1-25)) monoclonal antibody 1A5 (mAb 1A5). The mAb 1A5 bound specifically to the tryptic phosphopeptides from β-casein but not from αs1- or αs2-casein. Reactivity was also influenced by the extent of the phosphorylated form of serine residues. Based on the sequence-specific recognition and contribution of phosphorylated serine residues, the epitope of mAb 1A5 was found to reside within the cluster motif Ser(P)-Ser(P)-Ser(P)-Glu-Glu and the surrounding residues in β-CPP. The competitive ELISA developed here can be used as an alternative to specialised and expensive techniques such as mass spectrometry. In particular, it is suitable for the measurement of CPP or CPP complexes in dairy products, which contain closely related endogenous molecular species.

Digestive Enzymes of the Crustaceans Munida and Their Application in Cheese Manufacturing: A Review

Crustaceans Munida (fam. Galatheideae, ord. Decapodi) were fished in the Southern Adriatic Sea and their proteolytic activities were characterized and tested for potential application in cheese manufacturing. Enzymes extracted from whole crustaceans, mainly serine proteases, showed high caseinolytic and moderate clotting activities. Analysis by 2D zymography of the digestive enzymes extracted from Munida hepatopancreas, showed the presence of several isotrypsin- and isochymotrypsin-like enzymes in the range of 20–34 kDa and 4.1–5.8 pI. Moreover, specific enzymatic assays showed the presence of aminopeptidases and carboxypeptidases A and B. Overall, optimum activity was achieved at pH 7.5 and 40–45 °C. Caseinolytic activity, determined both spectrophotometrically and by SDS gel electrophoresis, indicated higher activity on β-casein than on α-casein. Miniature cheddar-type cheeses and Pecorino-type cheeses were manufactured by adding starter, rennet and Munida extracts to milk. Reverse-phase HPLC and MALDI-ToF mass spectrometry showed a more complex pattern of proteolytic products in cheeses made using Munida instead of chymosin. Munida extracts were found to degrade the chymosin-derived β-casein fragment f193–209, one of the peptides associated with bitterness in cheese. In conclusion, Munida digestive enzymes represent a promising tool for development of new cheese products and shorten cheese ripening when used either alone or in addition to calf rennet.

The hepatopancreas enzymes of the crustaceans Munida and their potential application in cheese biotechnology

Proteolytic and peptidase activities were extracted from the hepatopancreas of the crustaceans Munida and characterized by enzymatic assay, 2D zymography and mass spectrometry. Results showed the presence of several isotrypsin-like and isochymotrypsin-like enzymes, aminopeptidases and carboxypeptidases A and B. Six different acidic forms of trypsin were detected using specific inhibitors and 2D zymography. Trypsin-like activity was higher than chymotrypsin-like activity. On the basis of previous evidences in food biotechnology and cheese production, the digestive enzymes of the crustaceans Munida were tested for their ability to degrade casein, a process involved in cheese production. As a result, the Munida enzymes were found to degrade the chymosin-derived β-casein fragment f193–209, one of the peptides associated with bitterness in cheese, revealing their possible application in cheese technology to lower the unpleasant bitter flavour in some cheeses.

Derivatives of human β-Casein fragments (54–59) exhibit highly potent immunosuppressant activity

Human β-casein fragment (54–59) having the amino acid sequence Val–Glu–Pro–Ile–Pro–Tyr, has shown potent immunostimulant activity. Several analogs of this hexapeptide have been synthesized with modification at the N-terminal region and two analogs, viz. peptide I and peptide II have shown significant immunosuppressant activity in-vivo mouse model. Effect on cell mediated immunity (CMI) and humoral immunity was studied in mouse/SRBC model. Both the peptides failed to stimulate immune response in vivo and showed inhibition of CMI and humoral response to sheep red blood cells (SRBC). Peptides showed inhibition in alloantigen induced lymphocyte proliferation, i.e., mixed lymphocyte reaction (MLR) in vitro. Treatment with peptides inhibited the production of interferon-γ (IFN-γ), and increased the production of interleukin-4 (IL-4) as well as improved the skin graft survival. Cyclosporine a known immunosuppressant showed similar effect on mouse model. Present study thus provides a lead for the development of safe and effective immunosuppressant.

Occurrence of β-casein fragments in cold-stored and curdled river buffalo (Bubalus bubalis L.) milk

The safeguard of river buffalo Mozzarella cheese, a Protected Designation of Origin dairy product, has prompted an analytical study to trace the milk and curd used as raw material in cheesemaking. This is to prevent the illegal use of milk or curd from different geographical areas outside of those indicated in the official production protocol. For this purpose, we studied primary proteolysis occurring in fresh and frozen milk and curd to identify a molecular marker that could indicate the raw material used. Whole casein from frozen river buffalo milk was separated using cation-exchange chromatography and sodium dodecyl sulfate-PAGE, and a protein component with an estimated molecular weight of 15.3 kDa was detected. This protein component was revealed in fresh river buffalo milk as a faint electrophoresis band, which drastically increased in intensity in refrigerated and frozen milk as well as in curd and was found to be associated with β-CN through hydrophobic interaction. By using matrix-assisted laser desorption/ionization-time of flight peptide mass mapping, this component was identified as the C-terminal fragment f(69-209) of β-CN (expected molecular weight of 15,748.8Da). β-Casein f(69-209), originating from the early hydrolysis of Lys68-Ser69 by plasmin, has no counterpart in bovine milk. The increased rate of hydrolysis by plasmin toward the cleavage site Lys68-Ser69 has to be ascribed to the elevated proline content of the peptide 61-73. The favored production of β-CN f(69-209) has also drawn attention to the complementary proteose peptone β-CN f(1-68) that is presumed to play a physiological role in inducing milk secretion similar to that of β-CN f(1-29). The higher in vivo and in vitro production rate, compared with γ1-CN formation, indicates that β-CN f(69-209) and its complementary fragment are candidate molecular markers to evaluate milk and curd freshness. We used indirect ELISA analysis based on the determination of remaining nonhydrolyzed β-CN to perform a quantitative evaluation of proteolysis.

Role of food-derived opioid peptides in regulation of thermosensitivity of Periplaneta americana cockroach

The ability of exorphines (relative δ-selective opioid fragments of food-derived peptides) to change the defense response of American cockroaches Periplaneta americana placed in a hot chamber (T= 50°C) was studied. It was found that the ED50 for a fragment of D-ribulose-1,5-bisphosphate carboxylase rubiscolin-5 (YPLDL) doubling the stay time for the insects in the hot chamber was 340 nM/g. The ED50 for wheat gluten exorphine C (YPISL) was 573 nM/g. Cytochropin-4, a fragment of cytochrome B, did not have a significant effect. Comparison of the obtained data with the results of previous experiments with μ-selective exorphines—milk β-casein fragments—led to the conclusion that opioid receptors of various types are equally involved in regulation of cockroach protective behavior. The significance of phytogenous exorphines in formation of the opioid system of insects in the course of evolution is discussed.

Effect of Minor Milk Proteins in Chymosin Separated Whey and Casein Fractions on Cheese Yield as Determined by Proteomics and Multivariate Data Analysis

The objective of this work was to find regressions between minor milk proteins or protein fragments in the casein or sweet whey fraction and cheese yield because the effect of major milk proteins was evaluated in a previous study. Proteomic methods involving 2-dimensional gel electrophoresis and mass spectrometry in combination with multivariate data analysis were used to study the effect of variations in milk protein composition in chymosin separated whey and casein fractions on cheese yield. By mass spectrometry, a range of proteins significant for the cheese yield was identified. Among others, a C-terminal fragment of β-casein had a positive effect on the cheese yield expressed as grams of cheese per 100g of milk, whereas several other minor fragments of β-, αs1-, and αs2-casein had positive effects on the transfer of protein from milk to cheese. However, the individual effect of each identified protein was relatively low. Therefore, further studies of the relations between different proteins/peptides in the rennet casein or sweet whey fractions and cheese yield are needed for advanced understanding and prediction of cheese yield.

Hydrolysis of caprine and ovine milk proteins, brought about by aspartic peptidases from Silybum marianum flowers

The flowers of cardoon ( Asteraceae) are a rich source of aspartic peptidases which possess milk clotting activity – and are thus used in traditional cheesemaking in the Iberian Peninsula. This study was aimed at characterizing the enzymatic action of the aspartic peptidases present in flowers of Silybum marianum (L.) Gaertn. ( Asteraceae), specifically upon degradation of caseins. The proteolytic activities toward Na-caseinates previously prepared from caprine and ovine milks were studied, in a comparative fashion, using urea-PAGE, tricine-SDS-PAGE, densitometry, electroblotting and sequencing. Caprine α s1- and β-caseins were degraded up to 68% and 40%, respectively, during 24 h of incubation. Only one important and well-defined band corresponding to a molecular weight of 14.4 kDa – i.e. a fragment of β-casein, was observed by 12 h of hydrolysis. By 24 h of incubation, ovine α s- and β-caseins were degraded up to 76% and 19%, respectively. In what concerns specificity, the major cleavage site in ovine caseinate was Leu99-Arg100 in α s1-casein.

Characterization of the protein profile of donkey's milk whey fraction

Characterization of the protein profile of the whey fraction from a milk sample taken from an individual donkey belonging to the 'Ragusana' species of the East of Sicily is reported. Direct RP-HPLC/electrospray ionization (ESI)-MS analysis of the whey fraction allowed the detection of some unknown components, together with the identification of already known whey proteins. Matrix-assisted laser desorption/ionization (MALDI)-TOF/MS and RP-HPLC/ESI-MS/MS analysis of the enzymatic digests of the unknown components resulted the identification and characterization of (1) two beta-casein fragments; (2) the sequence of donkey's serum albumin; and (3) the oxidized methionine forms of lysozyme B and alpha-lactoalbumin. One of the two beta-casein fragments corresponds to the sequence Val(176)-Arg(189) of the horse's beta-casein. The second one corresponds the C-terminal sequence Tyr(199)-Val(226) of the horse's beta-casein, with four amino acid substitutions (Q --> R(203), L/I --> P(206), F --> L(210) and P --> A(219)). Both fragments, reasonably arising by endogenous proteases cleavage of the donkey's beta-casein, could be potential biologically active peptides. Direct mass spectrometric sequence characterization of the detected donkey's serum albumin reveals the presence of the amino acid substitution Val --> Ile at position 497 with respect to the cDNA deduced sequence. The oxidized forms of lysozyme B and alpha-lactoalbumin are selectively oxidized at methionine 79 and methionine 90, respectively.