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Sample preparation is the most critical step in proteomics as it directly affects the subset of proteins and peptides that can be reliably identified and quantified. Although a variety of efficient and reproducible sample preparation strategies have been developed, their applicability and efficacy depends much on the biological sample. Here, three approaches were evaluated for the human milk and plasma proteomes. Protein extracts were digested either in an ultrafiltration unit (filter-aided sample preparation, FASP) or in-solution (ISD). ISD samples were desalted by solid-phase extraction prior to nRPC-ESI-MS/MS. Additionally, milk and plasma samples were directly digested by FASP without prior protein precipitation. Each strategy provided inherent advantages and disadvantages for milk and plasma. FASP appeared to be the most time efficient procedure with a low miscleavage rate when used for a biological sample aliquot, but quantitation was less reproducible. A prior protein precipitation step improved the quantitation by FASP due to significantly higher peak areas for plasma and a much better reproducibility for milk. Moreover, the miscleavage rate for milk, the identification rate for plasma, and the carbamidomethylation efficiency were improved. In contrast, ISD of both milk and plasma resulted in higher miscleavage rates and is therefore less suitable for targeted proteomics.

Age gelation is a major quality defect in ultra-high-temperature (UHT) pasteurized milk during extended storage. Changes in plasmin (PL)-induced sedimentation were investigated during storage (23 °C and 37 °C, four weeks) of UHT skim milk treated with PL (2.5, 10, and 15 U/L). The increase in particle size and broadening of the particle size distribution of samples during storage were dependent on the PL concentration, storage period, and storage temperature. Sediment analysis indicated that elevated storage temperature accelerated protein sedimentation. The initial PL concentration was positively correlated with the amount of protein sediment in samples stored at 23 °C for four weeks ( r = 0.615; p < 0.01), whereas this correlation was negative in samples stored at 37 °C for the same time ( r = -0.358; p < 0.01) due to extensive proteolysis. SDS-PAGE revealed that whey proteins remained soluble over storage at 23 °C for four weeks, but they mostly disappeared from the soluble phase of PL-added samples after two weeks' storage at 37 °C. Transmission electron micrographs of PL-containing UHT skim milk during storage at different temperatures supported the trend of sediment analysis well. Based on the Fourier transform infrared spectra of UHT skim milk stored at 23 °C for three weeks, PL-induced particle size enlargement was due to protein aggregation and the formation of intermolecular β-sheet structures, which contributed to casein destabilization, leading to sediment formation.

Ti 4+ -immobilized hierarchically porous zirconium-organic frameworks (denoted as THZr-MOFs) was prepared for phosphopeptide enrichment. The THZr-MOFs showed high specific surface area of 185.28 m 2  g -1 , wide pore-size distribution of 3 ~ 20 nm, good chemical stability and excellent hydrophilicity. Introduction of hierarchical pores in MOFs not only facilitated the accessibility of phosphopeptides to the internal metal affinity sites and reduce their mass transfer resistance, but also increased the exposure sites of metal affinity interaction and binding energies of Zr and Ti elements. Benefited from these advantages, the THZr-MOFs showed high adsorption capacity (79.8 μg mg -1 ) towards standard phosphopeptide. A low detection limit (0.05 fmol μL -1 ) and high enrichment selectivity (β-casein/BSA with a molar ratio of 1:5000) were also obtained by MALDI-TOF MS. The THZr-MOFs were applied to analyze complex samples including nonfat milk, human serum, and HeLa cell lysate. In total, 1432 phosphopeptides derived from 762 phosphoproteins were identified from human HeLa cell lysate. Schematic representation of the application of Ti 4+ -immobilized hierarchically porous zirconium-organic frameworks (denoted as THZr-MOFs) in high-efficiency and selective enrichment of low-abundance phosphopeptides from the tryptic digest of human HeLa cell lysate.

Two-dimensional electrophoretic (2DE)-based proteomics remains a powerful tool for allergenomic analysis of goat's milk but requires effective extraction of proteins to accurately profile the overall causative allergens. However, there are several current issues with goat's milk allergenomic analysis, and among these are the absence of established standardized extraction method for goat's milk proteomes and the complexity of goat's milk matrix that may hamper the efficacy of protein extraction. This study aimed to evaluate the efficacies of three different protein extraction methods, qualitatively and quantitatively, for the 2DE-proteomics, using milk from two commercial dairy goats in Malaysia, Saanen, and Jamnapari. Goat's milk samples from both breeds were extracted by using three different methods: a milk dilution in urea/thiourea based buffer (Method A), a triphasic separation protocol in methanol/chloroform solution (Method B), and a dilution in sulfite-based buffer (Method C). The efficacies of the extraction methods were assessed further by performing the protein concentration assay and 1D and 2D SDS-PAGE profiling, as well as identifying proteins by MALDI-TOF/TOF MS/MS. The results showed that method A recovered the highest amount of proteins (72.68% for Saanen and 71.25% for Jamnapari) and produced the highest number of protein spots (199 ± 16.1 and 267 ± 10.6 total spots for Saanen and Jamnapari, respectively) with superior gel resolution and minimal streaking. Six milk protein spots from both breeds were identified based on the positive peptide mass fingerprinting matches with ruminant milk proteins from public databases, using the Mascot software. These results attest to the fitness of the optimized protein extraction protocol, method A, for 2DE proteomic and future allergenomic analysis of the goat's milk.

Purpose: Human milk (HM) proteins are known as important factors in growing and development of neonates. For longer and easier storage of HM, freeze-drying is suggested as one of the promising methods for HM banks. However, the effects of freeze-drying on HM proteins were not evaluated yet. The purpose of this study is to analyze and compare proteomic data before and after the freeze-drying. Material and methods: Totally nine fresh HM samples were collected from three healthy mothers at 15 and 60 days of lactation period. The samples were freeze-dried and the proteomic analysis was performed by shotgun proteomic method with mass spectrometry. The results were compared between samples of different lactation periods, and before and after the freeze-drying using Wilcoxon signed-rank test for paired comparisons. Moreover, the functional grouping and analysis were performed for the detected proteins by bioinformatics analysis. Results: Totally, 245 proteins were detected in the HM samples. The expression of proteins was not affected by both of the different lactation periods and the freeze-drying status ( P >.050). Moreover, the functional analysis of proteomic data revealed no significant difference between both groups as well. Conclusion: HM proteins were found not to be significantly affected by the lactation periods (15 and 60 days) and freeze-drying status. As significant changes of HM proteins were not found after the freeze-drying, we hope that the present study would support introducing freeze-drying in the HM banks. However, the number of samples was quite small to provide strong evidence. Moreover, the evaluation of the safe storage length in the view of infectious agents and the composition changes after freeze-drying is warranted in the further study.

There has been a great deal of interest in developing isolated dairy lipid fractions that are rich in phospholipids (PL), due to their health benefits and functional properties. Dairy by-products that contain elevated levels of PL and milk fat globule membrane (MFGM) proteins can be an excellent source for these isolates. The β stream, a by-product of anhydrous milk fat production, is an excellent candidate because it contains a higher concentration of PL than many other dairy by-products. In this study, we investigated an economically feasible processing method to obtain these valuable components from the β stream. The use of zinc acetate and calcium acetate, along with mild heat treatment and pH adjustment, was effective in precipitating PL and proteins into a pellet fraction. With an additional extraction from the pellet using ethanol (90% at 70°C), a PL-enriched lipid fraction was obtained. The effective precipitation conditions were zinc acetate of 25 mM concentration at pH greater than 6.5 at 30°C, and calcium acetate of greater than 75 mM concentration at pH greater than 6.5 at 60°C. With ethanol extraction, PL recovery of 97.7 ± 1.7% from the zinc acetate precipitate and 94.9 ± 3.7% from calcium acetate precipitate were achieved., (Copyright © 2020 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Accurate and comprehensive immunochromatographic assay (ICA) data are urgently required in the daily supervision of plants, schools, testing institutions, and law-enforcing departments. Through pretreatment-integration and device-facilitated operation, a quantitative ICA with high sensitivity and throughput was realized on the basis of a commercialized semi-quantitative ICA strip. Three pretreatment methods, namely, acid base, heavy metal salt, and organic solvent methods, have less than three steps. The pretreatment was established for protein removal. A total of 17 pretreated ICA items in milk were considered for the identification of the most suitable pretreatment method. The items are composed of six items pretreated by the acid-base method, six by the heavy salt method, and five by the organic solvent method. Then, the ICA results with pretreatment were compared with those without pretreatment. After pretreatment, the signal intensity increased by 39%, the detection limit decreased to 12%, the half maximal inhibitory concentration decreased to 18%, and the detection range increased fourfold. A device with mixing and centrifugation functions was designed for the pretreatment-related operations. A pre-incubation sampling device was used to facilitate incubation in batch and high-throughput detection. An ICA reader was used. The detection throughput reached 8 samples per batch or 32 samples per hour. The designed devices were printed through 3D printing and rapid prototyping.

In this study, an in-tube solid-phase microextraction column packed with mesoporous TiO 2 nanoparticles, coupled with MALDI-TOF-MS, was applied to the selective enrichment and detection of phosphopeptides in complex biological samples. The mesoporous TiO 2 nanoparticles with high specific surface areas, prepared by a sol-gel and solvothermal method, were injected into the capillary using a slurry packing method with in situ polymerized monolithic segments as frits. Compared with the traditional solid-phase extraction method, the TiO 2 -packed column with an effective length of 1 cm exhibited excellent selectivity (α-casein/β-casein/BSA molar ratio of 1:1:100) and sensitivity (10 fmol of a β-casein enzymatic hydrolysis sample) for the enrichment of phosphopeptides. These performance characteristics make this system suitable for the detection of phosphorylated peptides in practical biosamples, such as nonfat milk., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The objective of this work was to evaluate the impact of changes during membrane filtration on the heat stability of milk protein concentrates. Dairy protein concentrates have been widely employed in high protein drinks formulations and their stability to heat treatment is critical to ensure quality of the final product. Pasteurized milk was concentrated three-fold by membrane filtration, and the ionic composition was modified by addition of water or permeate from filtration (diafiltration). Diafiltration with water did not affect the apparent diameter of the casein micelles, but had a positive effect on heat coagulation time (HCT), which was significantly longer (50 min), compared to the non diafiltered concentrates (about 30 min). UHT treatments increased the particle size of the casein micelles, as well as the turbidity of retentates. Differences between samples with and without diafiltration were confirmed throughout further analysis of the protein composition of the unsedimentable fraction, highlighting the importance of soluble protein composition on the processing functionality of milk concentrates.

Background: Sodium caseinate (SC) is not considered suitable for use as an emulsifier in organic processed food in the food industry because of the use of prohibited synthetic chemical substances during its production. Casein mixture preparation (CMP), one of the permissible substances specified in the regulations, was isolated from organic milk using citric acid and dibasic potassium phosphate for organic processed foods., Results: To compare CMP and SC, model emulsions stabilized with each substance were prepared at various concentrations and their physicochemical properties were analyzed. The emulsions' stability was determined using Turbiscan under various environmental stresses. The zeta potential of SC and CPM showed a high surface charge (≤ 30 mV) at all protein concentrations. Because the concentration of the protein preparation increased to 0.75% (w/v), the particle size of the CMP emulsion decreased with the surface load increased as much as that of SC. The CMP and SC emulsions were stable at neutral pH and room temperature. However, at acidic pH and high temperature, both emulsions were destabilized by creaming and flocculation and increased the creaming migration rates., Conclusion: Overall, our data suggest the use of CMP as an emulsifier substitute for SC in organic processed foods. © 2018 Society of Chemical Industry., (© 2018 Society of Chemical Industry.)

Modified Fe 3 O 4 magnetic nanoparticles (magnetic nanocarrier) technology have found the proper place in separation and purification techniques, such as protein and enzyme purification, mostly due to its easy and fast operational procedure by using an external permanent magnet. Herein, Fe 3 O 4 magnetic nanoparticles were prepared, and surface modification was performed with vanadate and phosphate salts to yield four various model of magnetic nanocarriers. Affinity ligands which are used for immobilization on the nanocarriers leading to the development of appropriative nanocarriers for the affinity separation of alkaline phosphatase from the bovine milk. The findings showed that the use of sodium hexametaphosphate affinity ligand attached to the carrier with an 18-atom linker leads to better separation of alkaline phosphatase from the bovine milk with 14.1-fold purification efficiency. All results confirmed that our designed nanocarriers can purify alkaline phosphatase using a fast and low-cost approach., (Copyright © 2018 Elsevier B.V. All rights reserved.)

High-milk-protein concentrates (>80% on a dry weight basis) are typically produced by ultrafiltration (UF) with constant-volume diafiltration (DF). To maximize protein retention at a commercial scale, polymeric spiral-wound UF membranes with a molecular weight cut-off (MWCO) of 10 kDa are commonly used. Flux decline and membrane fouling during UF have been studied extensively and the selection of an optimal UF-DF sequence is expected to have a considerable effect on both the process efficiency and the volumes of by-products generated. The objective of this study was to characterize the performance of the UF-DF process by evaluating permeate flux decline, fouling resistance, energy and water consumption, and retentate composition as a function of MWCO (10 and 50 kDa) and UF-DF sequence [3.5×-2 diavolumes (DV) and 5×-0.8DV]. The UF-DF experiments were performed on pasteurized skim milk using a pilot-scale filtration system operated at 50°C under a constant transmembrane pressure of 465 kPa. The results showed that MWCO had no effect on permeate flux for the same UF-DF sequence. Irreversible resistance was also similar for both sequences, whatever the MWCO, suggesting that soluble protein deposition within the pores is similar for all conditions. Despite lower permeate fluxes and greater reversible resistance for the 5×-0.8DV sequence, the overall energy consumption of the 2 UF-DF sequences was similar. However, the 3.5×-2DV sequence required more water for DF and generated larger volumes of permeate to be processed, which will require more membrane area and lead to greater environmental impact. A comparative life cycle assessment should however be performed to confirm which sequence has the lowest environmental impact., (Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

This work describes a simple sample pretreatment method for the fractionation of human milk proteins (into their two main groups, whey and caseins) prior to their analysis. The protein-extraction protocol is based on the addition of calcium phosphate to nonadjusted pH human milk. The combination of calcium ions with phosphate results in an effective coprecipitation of caseins. To assess the suitability of this fractionation protocol, the protein extracts were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), LC-MS/MS, and capillary electrophoresis (CE) analysis. The results evidence a significant decrease in contamination of casein fraction with whey proteins and vice versa compared with the conventional isoelectric precipitation of caseins. In addition, CE fraction collection coupled to LC-MS/MS (off-line coupling) has been successfully applied to the identification of minor proteins in this complex matrix. The methodology presented here constitutes a promising tool to enlarge the knowledge of human milk proteome.

We have previously derived a novel antimicrobial peptide, LPcin-YK3(YK3), based on lactophoricin and have successfully studied and reported on the relationship between its structure and function. In this study, antimicrobial peptides with improved antimicrobial activity, less cytotoxicity, and shorter length were devised and characterized on the basis of YK3, and named YK5, YK8, and YK11. The peptide design was based on a variety of knowledge, and a total of nine analog peptides consisted of one to three amino acid substitutions and C-terminal deletions. In detail, tryptophan substitution improved the membrane perturbation, lysine substitution increased the net charge, and excessive amphipathicity decreased. The analog peptides were examined for structural characteristics through spectroscopic analytical techniques, and antimicrobial susceptibility tests were used to confirm their activity and safety. We expect that these studies will provide a platform for systematic engineering of new antibiotic peptides and generate libraries of various antibiotic peptides.

Xanthine oxidase (XO) found in all mammals and excess activity leads to urolithiasis. The cow milk XO was purified to 305-fold with a specific activity of 8.76 EU/mg and overall yield of 87% by using DEAE-Sepharose chromatography. The phenolics showed potent XO inhibitory effect with K i , P1 (0.412), P2 (0.632), P3 (0.585), P4 (0.886), P5 (1.633), P6 (0.503), P7 (2.882), P8 (3.761), P9 (4.487), and P10 (5.841) μM. The phenolics P9 and P10 exhibited uncompetitive inhibition; the phenolics P1, P2, P3, P4, and P6 showed competitive inhibition, and other phenolic acids showed noncompetitive inhibition. The studied phenolic compounds showed potent antioxidant activity and expressed as EC 50 , ranged from, DPPH (4.2-25.8 μg mL -1 ), ABTS (10.2-42.5 mmol TE 100 g -1 ), and FRAP (6.3-36.8 mol Fe (II) 100 g -1 ). The results obtained from this study might be utilized for design of XO inhibitors and as antigout agent., (© 2017 Wiley Periodicals, Inc.)

Modulating conditions during ultrafiltration of skim milk appears to be a feasible strategy to obtain milk protein concentrates (MPC) with tailored functionalities. Adjustment of pH and process temperature attenuated properties of casein micelle resulting in enhanced emulsification capacity. Additional pre-treatment options such as addition of calcium chelators can further impact on the functionality of MPC by modifying the calcium distribution and casein micelle integrity. The objective of the project was to establish effects of pre-treating skim milk with calcium chelators (EDTA or citrate) in concentrations between 10 to 30 mm prior to UF on the physical properties of the feed, corresponding retentates and dried MPC, including particle size, zeta potential and calcium distribution in skim milk and the corresponding retentates, as well as the physical functionalities such as solubility, heat stability and emulsifying properties. Addition of calcium chelators (EDTA or citrate), at levels 20-30 mm concentrations reduced casein micelle size as well as total, soluble and ionic calcium contents that resulted in MPC with enhanced solubility and heat stability. The emulsion capacity was, however, improved only with EDTA at 10 mm concentration. The enhanced functionality is attributed to the reduced particle size resulting from the removal of calcium from the retentate that could modify micellar casein to an extent sufficient to cause such improvements.

Microfiltration is largely used to separate casein micelles from milk serum proteins (SP) to produce a casein-enriched retentate for cheese making and a permeate enriched in native SP. Skim milk microfiltration is typically performed with ceramic membranes and little information is available about the efficiency of spiral-wound (SW) membranes. We determined the effect of SW membrane pore size (0.1 and 0.2 µm) on milk protein separation in total recirculation mode with a transmembrane pressure gradient to evaluate the separation efficiency of milk proteins and energy consumption after repeated concentration and diafiltration (DF). Results obtained in total recirculation mode demonstrated that pore size diameter had no effect on the permeate flux, but a drastic loss of casein was observed in permeate for the 0.2-µm SW membrane. Concentration-DF experiments (concentration factor of 3.0× with 2 sequential DF) were performed with the optimal 0.1-µm SW membrane. We compared these results to previous data we generated with the 0.1-µm graded permeability (GP) membrane. Whereas casein rejection was similar for both membranes, SP rejection was higher for the 0.1-µm SW membrane (rejection coefficient of 0.75 to 0.79 for the 0.1-µm SW membrane versus 0.46 to 0.49 for the GP membrane). The 0.1-µm SW membrane consumed less energy (0.015-0.024 kWh/kg of permeate collected) than the GP membrane (0.077-0.143 kWh/kg of permeate collected). A techno-economic evaluation led us to conclude that the 0.1-µm SW membranes may represent a better option to concentrate casein for cheese milk; however, the GP membrane has greater permeability and its longer lifetime (about 10 yr) potentially makes it an interesting option., (Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Identification of bioactive milk peptides could improve food technology through improved selection of food supplements with a focus on antihypertensive properties. We hypothesized that angiotensin I-converting enzyme (ACE) inhibitory activities of milk di- and tripeptides could be predicted using 3-dimensional quantitative structure activity relationship methods and that these activities could be explained through evaluation of structural features (hydrogen bond donor/acceptor, hydrophobic, steric, and electrostatic) that are responsible for this bioactivity. We aimed to build comparative molecular field analysis (CoMFA) models combined with in silico digestion to predict the peptide sequences released from enzymatic digestion and to evaluate peptides without experimental data. Furthermore, molecular docking simulation was performed with the aim to evaluate structural features. Molecular docking simulations revealed that the most potent inhibitory peptides contain hydrophobic amino acids that enter deep into the hydrophobic pocket of the ACE active site and make interactions with its residues. CoMFA results point out favorable steric interactions and electronegativity at the C-terminus of the milk dipeptides. The CoMFA model appears to favor electropositive amino acids at the second place in tripeptides and electronegative interaction with Tyr520. Furthermore, predicted values of ACE inhibitory activity of dipeptides obtained by peptide cutter are relatively high, which recommend them for application as functional food supplements and natural alternatives to ACE inhibitory drugs. This research suggests that obtained 3-dimensional quantitative structure activity relationship models are able to successfully identify milk-derived di- and tripeptides that have significant antihypertensive activity and provide information for screening and design of novel ACE inhibitors that could be used as supplements in human nutrition., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Lactophoricin (LPcin), which is a part of proteose peptone isolated from bovine milk, is a cationic amphipathic α-helical antimicrobial peptide. Its truncated variants and mutated analogs were designed and their antimicrobial activities were evaluated by using various assays, like broth dilution methods and disk diffusion methods as well as hemolysis assay. Three analogs, LPcin-C8 (LPcin-YK1), LPcin-T2&6W (LPcin-YK2), and LPcin-T2&6W-C8 (LPcin-YK3), which showed better antibiotic activities than LPcin, were selected. Their secondary structures were also characterized by using CD spectropolarimetry. These three analogs of LPcin could be used as an alternative source of powerful antibacterial agents.

Human milk is the optimal mode of infant feeding for the first several months of life, and infant formulas serve as an alternative when breast-feeding is not possible. Milk proteins have a balanced amino acid composition and some of them provide beneficial bioactivities in their intact forms. They also encrypt a variety of bioactive peptides, possibly contributing to infant health and growth. However, there is limited knowledge of how milk proteins are digested in the gastrointestinal tract and bioactive peptides are released in infants. A peptidomic analysis was conducted to identify peptides released from milk proteins in human milk and infant formula, using a suckling rat pup model. Among the major milk proteins targeted, α-lactalbumin and β-casein in human milk, and β-lactoglobulin and β-casein in infant formula were the main sources of peptides, and these peptides covered large parts of the parental proteins' sequences. Release of peptides was concentrated to specific regions, such as residues 70-92 of β-casein in human milk, residues 39-55 of β-lactoglobulin in infant formula, and residues 57-96 and 145-161 of β-CN in infant formula, where resistance to gastrointestinal digestion was suggested. In the context of bioactive peptides, release of fragments containing known bioactive peptides was confirmed, such as β-CN-derived opioid and antihypertensive peptides. It is therefore likely that these fragments are of biological significance in neonatal health and development., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Lactoperoxidase (LPO) plays a key role in immune response against pathogens. In this study, we examined the effects of some phenolic acids on LPO. For this purpose, bovine milk LPO was purified 380.85-fold with a specific activity of 26.66 EU/mg and overall yield of 73.33% by using Amberlite CG-50 H + resin and CNBr-activated Sepharose-4B-l-tyrosine-sulfanilamide affinity chromatography. After purification, the in vitro effects of phenolic acids (tannic acid, 3,4-dihydroxybenzoic acid, 3,5- dihydroxybenzoic acid, chlorogenic acid, sinapic acid, 4-hydroxybenzoic acid, vanillic acid, salicylic acid, and 3-hydroxybenzoic acid) were investigated on LPO. These phenolic acids showed potent inhibitory effect on LPO. K i values for these phenolic acids were found as 0.0129 nM, 0.132 μM, 0.225 μM, 0.286 μM, 0.333 μM, 2.33 μM, 10.82 μM, 0.076 mM, and 0.405 mM, respectively. Sinapic acid and 4-hydroxybenzoic acid exhibited noncompetitive inhibition; 3,4-dihydroxybenzoic acid showed uncompetitive inhibition, and other phenolic acids showed competitive inhibition., (© 2016 Wiley Periodicals, Inc.)

Donkey milk is a valuable product for the food industry due to its nutraceutical, nutritional, and functional properties. In this work, the endogenous peptides from donkey milk were investigated for their antioxidant and ACE-inhibitory activities, combining a two-dimensional peptide fractionation strategy with high-resolution mass spectrometry, bioinformatics analysis, and in vitro assays. After extraction, the endogenous peptides were fractionated twice, first by polymeric reversed phase and then by hydrophilic interaction chromatography. Fractions were screened for the investigated bioactivities and only the most active ones were finally analyzed by nanoRP-HPLC-MS/MS; this approach allowed to reduce the total number of possible bioactive sequences. Results were further mined by in silico analysis using PeptideRanker, BioPep, and PepBank, which provided a bioactivity score to the identified peptides and matched sequences to known bioactive peptides, in order to select candidates for chemical synthesis. Thus, five peptides were prepared and then compared to the natural occurring ones, checking their retention times and fragmentation patterns in donkey milk alone and in spiked donkey milk samples. Pure peptide standards were finally in vitro tested for the specific bioactivity. In this way, two novel endogenous antioxidant peptides, namely EWFTFLKEAGQGAKDMWR and GQGAKDMWR, and two ACE-inhibitory peptides, namely REWFTFLK and MPFLKSPIVPF, were successfully validated from donkey milk. Graphical Abstract Analytical workflow for purification and identification of bioactive peptides from donkey milk sample.

The objective of this study was to investigate how ceramic membrane pore size and filtration temperature influence the protein fractionation of skim milk by cross flow microfiltration (MF). Microfiltration was performed at a uniform transmembrane pressure with constant permeate flux to a volume concentration factor of 2.5. Three different membrane pore sizes, 0.05, 0.10, and 0.20µm, were used at a filtration temperature of 50°C. Furthermore, at pore size 0.10µm, 2 different filtration temperatures were investigated: 50 and 60°C. The transmission of proteins increased with increasing pore size, giving the permeate from MF with the 0.20-µm membrane a significantly higher concentration of native whey proteins compared with the permeates from the 0.05- and 0.10-µm membranes (0.50, 0.24, and 0.39%, respectively). Significant amounts of caseins permeated the 0.20-µm membrane (1.4%), giving a permeate with a whitish appearance and a casein distribution (αS2-CN: αS1-CN: κ-CN: β-CN) similar to that of skim milk. The 0.05- and 0.10-µm membranes were able to retain all caseins (only negligible amounts were detected). A permeate free from casein is beneficial in the production of native whey protein concentrates and in applications where transparency is an important functional characteristic. Microfiltration of skim milk at 50°C with the 0.10-µm membrane resulted in a permeate containing significantly more native whey proteins than the permeate from MF at 60°C. The more rapid increase in transmembrane pressure and the significantly lower concentration of caseins in the retentate at 60°C indicated that a higher concentration of caseins deposited on the membrane, and consequently reduced the native whey protein transmission. Optimal protein fractionation of skim milk into a casein-rich retentate and a permeate with native whey proteins were obtained by 0.10-µm MF at 50°C., (Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Milk fat globule-epidermal growth factor-factor 8 (MFG-E8), as its name suggests, is a major glycoprotein component of milk fat globules secreted by the mammary epithelium. Although its role in milk fat production is unclear, MFG-E8 has been shown to act as a bridge linking apoptotic cells to phagocytes for removal of these dying cells. MFG-E8 is capable of bridging these two very different cell types via interactions through both its epidermal growth factor (EGF)-like domain(s) and its lectin-type C domains. The EGF-like domain interacts with αVβ3 and αVβ5 integrins on the surface of phagocytes, whereas the C domains bind phosphatidylserine found on the surface of apoptotic cells. In an attempt to purify full-length, recombinant MFG-E8 expressed in either insect cells or CHO cells, we find that it is highly aggregated. Systematic truncation of the domain architecture of MFG-E8 indicates that the C domains are mainly responsible for the aggregation propensity. Addition of Triton X-100 to the conditioned cell culture media allowed partial recovery of non-aggregated, full-length MFG-E8. A more comprehensive detergent screen identified CHAPS as a stabilizer of MFG-E8 and allowed purification of a significant portion of non-aggregated, full-length protein. The CHAPS-stabilized recombinant MFG-E8 retained its natural ability to bind both αVβ3 and αVβ5 integrins and phosphatidylserine suggesting that it is properly folded and active. Herein we describe an efficient purification method for production of non-aggregated, full-length MFG-E8., (Copyright © 2016 Elsevier Inc. All rights reserved.)

A two-dimensional liquid chromatography system coupled to triple quadrupole tandem mass spectrometer (2D LC-MS/MS) was employed for the simultaneously quantification of fluoxetine (FLX) and norfluoxetine (NFLX) enantiomers in human milk by direct injection of samples. A restricted access media of bovine serum albumin octadecyl column (RAM-BSAC18) was used in the first dimension for the milk proteins depletion, while an antibiotic-based chiral column was used in the second dimension. The results herein described show good selectivity, extraction efficiency, accuracy, and precision with limits of quantification in the order of 7.5ngmL(-1)for the FLX enantiomers and 10.0ngmL(-1) for NFLX enantiomers. Furthermore, it represents a practical tool in terms of sustainability for the sample preparation of such a difficult matrix., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The objective of this study was to investigate the milk protein profiles of normal milk and those of milk during the course of subclinical mastitis, caused by natural Streptococcus agalactiae infection. Two-dimensional gel electrophoresis and liquid chromatography mass spectrometry were used to assess protein profiles and to identify the proteins. The results showed that S. agalactiae subclinical mastitis altered the protein profiles of milk. Following Mascot database matching, 11 and 12 protein types were identified in the milk collected from healthy and S. agalactiae subclinical mastitic udders, respectively. The distinct presence of the antibacterial protein cathelicidin-1 was detected in infected milk samples, which in turn was highly correlated to the severity of subclinical mastitis as represented by the milk somatic cell count (r = 0.616), but not the bacterial count. The protein profile of milk reveals changes in the host response to S. agalactiae intramammary infection; cathelicidin-1 could therefore serve as a biomarker for the detection of subclinical mastitis in dairy cows., (© 2015 Japanese Society of Animal Science.)

Year to year obesity prevalence, reduced physical activities, bad habits/or stressful lifestyle, and other environmental and physiological impacts lead to increase in diseases such as coronary heart disease, stroke, cancer, diabetes, and hypertension worldwide. Hypertension is considered as one of the most common serious chronic diseases; however, discovery of medications with high efficacy and without side effects for treatment of patients remains a challenge for scientists. Recent trends in functional foods have evidenced that food bioactive proteins play a major role in the concepts of illness and curing; therefore, nutritionists, biomedical scientists, and food scientists are working together to develop improved systems for the discovery of peptides with increased potency and therapeutic benefits. This review presents a recent research carried out to date for the purpose of isolation and identification of bioactive hydrolyzates and peptides with angiotensin I converting enzyme inhibitory activity and antihypertensive effect from animal, marine, microbial, and plant food proteins. Effects of food processing and hydrolyzation conditions as well as some other impacts on formation, activity, and stability of these hydrolyzates and peptides are also presented.

The separation and determination of proteins in food is an important aspect in food industry. Inspired by the self-polymerization of dopamine under alkaline conditions and the natural adhesive properties of polydopamine, in this paper, a simple and economical method was developed for the preparation of polydopamine-coated open tubular column, in which ammonium persulfate was used as the source of oxygen to induce and facilitate the polymerization of dopamine to form polydopamine. In comparison with a naked fused-silica capillary, the direction and magnitude of the electro-osmotic flow of the as-prepared polydopamine-coated open tubular column could be manipulated by varying the pH values of background solutions due to the existence of amine and phenolic hydroxyl groups on polydopamine coating. The surface morphology of the polydopamine-coated open tubular column was studied by scanning electron microscopy, and the thickness of polydopamine coating was 106 nm. The performance of the polydopamine-coated open tubular column was validated by analysis of proteins. The relative standard deviations of migration times of proteins representing run-to-run, day-to-day, and column-to-column were less than 3.5%. In addition, the feasibility of the polydopamine-coated open tubular column for real samples was verified by the separation of proteins in chicken egg white and pure milk., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Allergy to cow's milk increases the risk of sensitization to other foods in young children., Objectives: We sought to evaluate the effect of early epicutaneous immunotherapy (EPIT) on further sensitization to peanut or house dust mite (HDM) in a murine model of sensitization to cow's milk., Methods: BALB/c mice orally sensitized to milk were epicutaneously treated with a Viaskin patch (DBV Technologies) loaded with milk proteins for 8 weeks. Mice were then sensitized to peanut or HDM. After sensitization to peanut, mice were exposed to a peanut regimen known to induce eosinophilic esophageal inflammation. After sensitization to HDM, mice were challenged with aerosols to HDM, and airway hyperresponsiveness was evaluated by using plethysmography. Humoral response was also analyzed. The role of regulatory T (Treg) cells was evaluated by adoptively transferring Treg cells from milk EPIT-treated mice to naive mice before sensitization to peanut. Protection against anaphylaxis was also investigated. Methylation of the promoter region of transcription factors was analyzed by using PCR assays., Results: In milk-sensitized mice specific EPIT prevented further sensitization to peanut or HDM. EPIT significantly modified the humoral response, reduced TH2 cytokine levels, decreased eosinophilic esophageal infiltration, and suppressed airway hyperresponsiveness. The protective effect was sustained over 2 months. Moreover, the adoptive transfer of milk EPIT Treg cells completely prevented sensitization to peanut and peanut-induced anaphylaxis. Milk EPIT enhanced methylation of the GATA-3 promoter region., Conclusions: Our results showed that EPIT influences the natural history of allergy and reduces the risk of further sensitization through a Treg cell-dependent mechanism., (Copyright © 2014 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

The crystal structure of the triclinic form of the milk protein β-lactoglobulin from sheep (Ovis aries) at 1.1 Å resolution is described together with a comparison of the triclinic structures of the low-pH bovine and high-pH ovine proteins. All three structures are remarkably similar, despite the well known pH-dependent conformational transition described for the bovine and porcine proteins that occurs in solution. The high resolution of the present structure determination has allowed a more accurate description of the protein than has hitherto been possible, but it is still not clear whether flexibility changes in the external loops can compensate for the presence of a significant void in the unliganded interior of the structure.

The anti-inflammatory cytokines (interleukin (IL)-4 and IL-10) and the pro-inflammatory cytokines (IL-1β, IL-6, and tumor necroses factor-alpha (TNF-α)) have important functions in wound healing. Thus, the aim of this study was to determine whether dietary supplementation with whey protein could enhance normal inflammatory responses during wound healing in diabetic rats. In this study, male albino rats were divided into a wounded control group, a wounded diabetic group, and a wounded diabetic group supplemented with whey protein orally at a dose of 100 mg/kg body weight. Tested rats showed increasing wound closure in rats treated with whey protein. In addition, after 4 days of wound, modulation in IL-4, IL-10, IL-1β, IL-6, and TNF-α levels were detected. Statistical analysis of data showed significant difference between the whey-protein-treated group and either control or diabetic groups (P < 0.05). Dietary supplementation with whey protein enhances the normal inflammatory responses during wound healing in diabetic rats by modulating the levels of some anti-inflammatory and inflammatory cytokines.

There is a raising demand for sensitive and high throughput MS based methods for screening purposes especially tailored to the detection of allergen contaminants in different food commodities. A challenging issue is represented by complex food matrices where the antibody-based kits commercially available might encounter objective limitations consequently to epitope masking phenomena due to a multitude of interfering compounds arising from the matrix. The performance of a method duly optimized for the extraction and simultaneous detection of soy, egg and milk allergens in a cookie food matrix by microHPLC-ESI-MS/MS, is herein reported. Thanks to the innovative configuration and the versatility shown by the dual cell linear ion trap MS used, the most intense and reliable peptide markers were first identified by untargeted survey experiment, and subsequently employed to design an ad hoc multi-target SRM method, based on the most intense transitions recorded for each selected precursor peptide. A sample extraction and purification protocol was optimized also including an additional step based on sonication, which resulted in a considerable improvement in the detection of milk allergen peptides. Data Dependent™ Acquisition scheme allowed to fill out a tentative list of potential peptide markers, which were further filtered upon fulfilling specific requirements. A total of eleven peptides were monitored simultaneously for confirmation purposes of each allergenic contaminant and the two most sensitive peptide markers/protein were selected in order to retrieve quantitative information. Relevant LODs were found to range from 0.1μg/g for milk to 0.3μg/g for egg and 2μg/g for soy., (Copyright © 2014 Elsevier B.V. All rights reserved.)

To determine the adulteration of soybean proteins in dairy product, a microchip-CE device was developed to isolate selected fraction of soybean and milk proteins in pI range from 5.5 ∼ 7.0 by 1D IEF, followed by ITP/CZE in the embedded capillary for preconcentration, separation and UV detection at 280 nm. Compared to IEF-CZE without ITP preconcentration, the enhancement factor (EF) in detection of soybean proteins was 20 times. Adulteration of 0.1% soybean protein in total dairy proteins can be detected in less than 10 min., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The 50-amino acid protein medin is the main fibrillar component of human aortic medial amyloid (AMA), the most common form of localised amyloid which affects 97% of Caucasians over the age of 50. Structural models for several amyloid assemblies, including the Alzheimer's amyloid-β peptides, have been defined from solid-state nuclear magnetic resonance (SSNMR) measurements on (13)C- and (15)N-labelled protein fibrils. SSNMR-derived structural information on fibrillar medin is scant, however, because studies to date have been restricted to limited measurements on site-specifically labelled protein prepared by solid-phase synthesis. Here we report a procedure for the expression of a SUMO-medin fusion protein in Escherichia coli and IMAC purification yielding pure, uniformly (13)C,(15)N-labelled medin in quantities required for SSNMR analysis. Thioflavin T fluorescence and dynamic light scattering measurements and transmission electron microscopy analysis confirm that recombinant medin assembles into amyloid-like fibrils over a 48-h period. The first (13)C and (15)N SSNMR spectra obtained for uniformly-labelled fibrils indicate that medin adopts a predominantly β-sheet conformation with some unstructured elements, and provide the basis for further, more detailed structural investigations., (Copyright © 2014. Published by Elsevier Inc.)

The epitheliochorial nature of the porcine placenta prevents the transfer of maternal immunity. Therefore, ingestion of the colostrum immediately after birth is crucial for neonatal piglets to acquire passive immunity from the sow. We performed a shotgun proteomic analysis of porcine milk to reveal in detail the protein composition of porcine milk. On the basis of the Swiss-Prot database, 113 and 118 proteins were identified in the porcine colostrum and mature milk, respectively, and 50 of these proteins were common to both samples. Some immune-related proteins, including interleukin-18 (IL-18), were unique to the colostrum. The IL-18 concentration in the colostrum and mature milk of four sows was measured to validate the proteomic analysis, and IL-18 was only detected in the colostrum (191.0 ± 53.9 pg/mL) and not in mature milk. In addition, some proteins involved in primary defense, such as azurocidin, which has never been detected in any other mammal's milk, were also identified in the colostrum., (© 2014 Japanese Society of Animal Science.)

The worldwide production of whey increases by around 186 million tons each year and it is generally considered as a waste, even when several whey proteins have important economic relevance. For its valorization, inexpensive ligands and integrated chromatography methods need to be developed for specific and low-cost protein purification. Here, we describe a novel affinity process with the dye Yellow HE-4R immobilized on Sepharose for bovine lactoferrin purification. This approach based on a low-cost ligand showed an efficient performance for the recovery and purification of bovine lactoferrin directly from whey, with a yield of 71% and a purification factor of 61., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Liposomes containing magnetic gold nanoparticles (AuNPs) and an enzymatic substrate (4-methylumbelliferyl-phosphate) have been used as on-flow microcontainers for reagent preconcentration in a flow injection method for the determination of alkaline phosphatase (ALP) activity. The dynamic range of the calibration graph was 6.4 × 10(-3)-0.25 U L(-1) ALP, and the detection limit was 1.9 × 10(-3) U L(-1). The precision, expressed as relative standard deviation (RSD%), was in the range of 0.7-2.4%. The overall method showed a sampling frequency of 10 h(-1). The method was applied to the determination of ALP in milk samples with recovery values ranging between 87.5 and 104.6%. The residual ALP activity in milk samples subjected to temperature treatments was also determined. The results obtained in the analysis of all milk samples were compared with those obtained by applying a previously described flow injection method.

An integrated expanded bed adsorption process was developed in this study to purify lactoferrin and immunoglobulin G (IgG) from crude sweet whey. The process used two sequential expanded beds packed with a cationic exchanger (Fastline SP) and a mixed-mode resin (Streamline Direct CST-1), respectively. Lactoferrin was isolated in the first expanded bed packed with Fastline SP, and the flow through was loaded into the second expanded bed packed with Streamline Direct CST-1 to separate IgG. Three integration strategies were compared to improve the separation efficiency, especially for the purification of IgG in the second expanded bed. The purities of IgG obtained from these three strategies were 91.9%, 83.8% and 92.4%, and the recoveries were 14.3%, 63.7% and 29.7%, respectively. By efficient integration of the two expanded beds, lactoferrin and IgG were successfully recovered from crude sweet whey with high purities and reasonable recoveries. Moreover, the stream flowed out of the integrated process was collected and separated by ultrafiltration to produce whey protein concentrate. Therefore, sweet whey resource could be fully utilized. The results demonstrated that it is possible to purify multiple proteins from untreated crude resource with a rationally designed expanded bed adsorption process., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: Xanthine oxidoreductase (XOR) existing in two interconvertible forms, xanthine dehydrogenase (XDH) and xanthine oxidase (XO), catabolises xanthine to uric acid that is further broken down to antioxidative agent allantoin. XOR also produces free radicals serving as second messenger and microbicidal agent. Large variation in the XO activity has been observed among various species. Both hypo and hyper activity of XOR leads to pathophysiological conditions. Given the important nutritional role of buffalo milk in human health especially in south Asia, it is crucial to understand the functional properties of buffalo XOR and the underlying structural basis of variations in comparison to other species., Methods and Findings: Buffalo XO activity of 0.75 U/mg was almost half of cattle XO activity. Enzymatic efficiency (k cat/K m) of 0.11 sec(-1) µM(-1) of buffalo XO was 8-10 times smaller than that of cattle XO. Buffalo XOR also showed lower antibacterial activity than cattle XOR. A CD value (Δε430 nm) of 46,000 M(-1) cm(-1) suggested occupancy of 77.4% at Fe/S I centre. Buffalo XOR contained 0.31 molybdenum atom/subunit of which 48% existed in active sulfo form. The active form of XO in buffalo was only 16% in comparison to ∼30% in cattle. Sequencing revealed 97.4% similarity between buffalo and cattle XOR. FAD domain was least conserved, while metal binding domains (Fe/S and Molybdenum) were highly conserved. Homology modelling of buffalo XOR showed several variations occurring in clusters, especially close to FAD binding pocket which could affect NAD(+) entry in the FAD centre. The difference in XO activity seems to be originating from cofactor deficiency, especially molybdenum., Conclusion: A major fraction of buffalo milk XOR exists in a catalytically inactive form due to high content of demolybdo and desulfo forms. Lower Fe/S content and structural factors might be contributing to lower enzymatic efficiency of buffalo XOR in a minor way.

Sialic acid is a carbohydrate moiety of k-casein glycomacropeptide (GMP), which is a 64 amino acid residue C-terminal sialylated phosphorylated glycopeptide released from k-casein by the action of chymosin during cheese making. GMP lacks aromatic amino acids including phenylalanine, tyrosine, and tryptophan. Because of its unique amino acid composition and various biological activities, GMP is thought to be a potential ingredient for dietetic foods (e.g., a food for PKU patients) and pharmaceuticals. Thus, increased attention has been given to the development of techniques to purify GMP. In this review, techniques of GMP purification described in patents and scientific research papers were introduced. A sialic acid assay is the important method to track GMP isolation and purification processes, for which the thiobarbituric acid reaction with 1-propanol as a chromophore extracting solvent is an inexpensive, practical and specific technique. Sephacryl S-200 gel filtration chromatography, cellulose acetate electrophoresis, and sodium dodecyl sulfate polyacrylamide gel electrophoresis are the major techniques to identify sialic acid specific to GMP. Sephacryl S-200 chromatography and cellulose acetate electrophoresis are also used to detect GMP sialic acid in whey pearmeate and whey added commercial margarine samples. Future research includes development of an economical industrial scale method to produce high purity GMP.

The high nutritive value and diverse functional properties of milk proteins are well known. Beyond these qualities, milk proteins have attracted growing scientific and commercial interest as a source of biologically active molecules. Such proteins are found in abundance in colostrum which is the initial milk secreted by mammalian species during late pregnancy and the first few days after birth of the offspring. The best characterized colostrum-based bioactive proteins include alpha-lactalbumin, beta-lactoglobulin, immunoglobulins, lactoferrin, lactoperoxidase and growth factors. All of them can nowadays be enriched and purified on an industrial scale from bovine colostral whey or cheese whey. These native proteins exhibit a wide range of biological activities that are known to affect the digestive function, metabolic responses to absorbed nutrients, growth and development of organs and disease resistance. Also, some of these proteins may prove beneficial in reduction of the risks of chronic human diseases reflected by the metabolic syndrome. It is speculated that such potentially beneficial effects are partially attributed to bioactive peptides derived from intact proteins. These peptides can be liberated during gastrointestinal digestion or fermentation of milk by starter cultures. The efficacy of a few peptides has been established in animal and human studies and the number of commercial products supplemented with specific milk peptides is envisaged to increase on global markets. Bovine colostrum appears as a highly potential source of biologically active native proteins and peptide fractions for inclusion as health-promoting ingredients in various food applications.

Sample preparation is a bottleneck in systems for chemical analysis and it is a required step in order to remove interference and preconcentrate the target analytes. Much research in recent years has focused on porous monolithic materials since they are highly permeable to liquid flow and show high mass transfer compared with common packed beds. This study has focused on the use of a glass microchip containing an inorganic silica-based monolithic material modified with octadecyl groups for preconcentration of milk proteins from skimmed cows' milk that vary in molecular weight, hydrophobicity, and abundance. Comparison between the fabricated device and a commercial cartridge for the preconcentration of proteins in skimmed cows' milk showed the ability of the device to successfully enrich protein mixtures from the sample. The three replicate experiments showed that the RSD of the mass to charge ratio of milk proteins ranged from 0.01 to 0.46%. In addition, it was found that there were no significant differences between the observed and reported masses of the milk proteins and the relative percentage error of the molecular masses ranged between 0.03 and 0.90%. The fact that the small amounts of sample required and short sample preparation time suggest that this new microfluidic device may be a viable alternative to existing procedures for protein extraction from real samples., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Our objective was to determine the effect of concentration factor (CF) on the removal of serum protein (SP) from skim milk during microfiltration (MF) at 50 °C using a 0.3-μm-pore-size spiral-wound (SW) polymeric polyvinylidene fluoride (PVDF) membrane. Pasteurized (72°C for 16 s) skim milk was MF (50 °C) at 3 CF (1.50, 2.25, and 3.00×), each on a separate day of processing starting with skim milk. Two phases of MF were used at each CF, with an initial startup-stabilization phase (40 min in full recycle mode) to achieve the desired CF, followed by a steady-state phase (90-min feed-and-bleed with recycle) where data was collected. The experiment was replicated 3 times, and SP removal from skim milk was quantified at each CF. System pressures, flow rates, CF, and fluxes were monitored during the 90-min run. Permeate flux increased (12.8, 15.3, and 19.0 kg/m(2) per hour) with decreasing CF from 3.00 to 1.50×, whereas fouled water flux did not differ among CF, indicating that the effect of membrane fouling on hydraulic resistance of the membrane was similar at all CF. However, the CF used when microfiltering skim milk (50°C) with a 0.3-μm polymeric SW PVDF membrane did affect the percentage of SP removed. As CF increased from 1.50 to 3.00×, the percentage of SP removed from skim milk increased from 10.56 to 35.57%, in a single stage bleed-and-feed MF system. Percentage SP removal from skim milk was lower than the theoretical value. Rejection of SP during MF of skim milk with SW PVDF membranes was caused by fouling of the membrane, not by the membrane itself and differences in the foulant characteristic among CF influenced SP rejection more than it influenced hydraulic resistance. We hypothesize that differences in the conditions near the surface of the membrane and within the pores during the first few minutes of processing, when casein micelles pass through the membrane, influenced the rejection of SP because more pore size narrowing and plugging occurred at low CF than at high CF due to a slower rate of gel layer formation at low CF. It is possible that percentage removal of SP from skim milk at 50 °C could be improved by optimization of the membrane pore size, feed solution composition and concentration, and controlling the rate of formation of the concentration polarization-derived gel layer at the surface of the membrane during the first few minutes of processing., (Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

To improve resolution of important minor proteins and eliminate time-consuming precipitation of major protein with associated analyte co-precipitation risk, a multi-dimension strategy is adopted in the 2D microchip-CE device to isolate major proteins on-chip, enrich minor proteins in capillary before their separation in CE for UV quantitation. A standard fluorescent protein mixture containing FITC-BSA, myoglobin and cytochrome as specific pI markers has prepared to demonstrate capability of the device to fractionate minor proteins by IEF. The results using a standard protein mixture with profile resembling infant milk formula show a complete isolation of high abundance proteins by a 2-min 1D IEF run. The subsequent t-ITP/CZE run by on-chip high voltage switching delivers a high stacking ratio, realizing 60 folds enrichment of isolated protein fractions. All five important functional proteins (LF, IgG, α-LA, β-LgA and β-LgB) known to fortify infant milk formula are isolated and determined using two consecutive t-ITP-CZE runs within a 18-min total assay time, a significant saving compared to several hours conventional pretreatment. For a 100g infant milk formula sample, working ranges of 20-8000mg, repeatability 3.8-5.3% and detection limits 2.3-10mg have been achieved to meet government regulations. Method reliability is established by 100% recoveries and agreeable results within expected ranges and labeled values. The capability of the device for field operation, rapid assay with quick results, label-free universal detection, simple operation by aqueous dissolution before injection, and the demanding matching in 2D separation based on isolated fractions at specified pI ranges, closely matched migration time and baseline-resolved peak shape makes the device a general tool to detect unknown proteins and determine known minor proteins in protein-rich samples with interfering constituents., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Lactoferrin (LF) is known as an iron-binding antimicrobial protein present in exocrine secretions such as milk and releases the potent antimicrobial peptide lactoferricin (LFcin) by hydrolysis with pepsin. The antimicrobial activity of LF and LFcin has been studied well; however, their cooperative action with other milk proteins remains to be elucidated. In this study, we identified milk proteins enhancing the antimicrobial activity of bovine LF and LFcin against gram-negative bacteria, gram-positive bacteria, and fungi. As the target fraction, we isolated a minor milk protein fraction around 15 kDa, which was identified as bovine RNase 5 (angiogenin-1), RNase 4, and angiogenin-2 by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. As these proteins are collectively known as the RNase A family, we referred to the target protein fraction as milk RNase of 15 kDa (MR15). The number of colony-forming units of Escherichia coli and other pathogenic microorganisms with the addition of MR15 to LF (MR15:LF ratio=16:1,000) was dramatically lowered than that with LF alone. On the other hand, MR15 itself did not show any reductions in the number of colony-forming units at the concentrations tested. Similarly, the antimicrobial activities of LFcin against various microorganisms were significantly enhanced by the addition of MR15. These results suggest that LF and MR15 may be concomitantly acting antimicrobial agents in milk., (Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

As one of several biologically active compounds in milk, glycoproteins have been indicated to be involved in the protection of newborns from bacterial infection. As much of the physical and immune development of the tammar wallaby (Macropus eugenii) young occurs during the early phases of lactation and not in utero, the tammar is a model species for the characterization of potential developmental support agents provided by maternal milk.In the present study, the N- and O-linked glycans from tammar wallaby milk glycoproteins from six individuals at different lactation time points were subjected to glycomics analyses using porous graphitized carbon liquid chromatography electrospray ionization mass spectrometry. Structural characterization identified a diverse range of glycan structures on wallaby milk glycoproteins including sialylated, sulphated, core fucosylated and O-fucosylated structures. 30 % of N-linked structures contained a core (α1-6) fucose. Several of these structures may play roles in development, and exhibit statistically significant temporal changes over the lactation period. The N-glycome was found to contain structures with NeuGc residues, while in contrast the O-glycome did not. O-fucosylated structures were identified in the early stages of lactation indicating a potential role in the early stages of development of the pouch young. Overall the results suggest that wallaby milk contains structures known to have developmental and immunological significance in human milk and reproduction in other animals, highlighting the importance of glycoproteins in milk.

The article presents a new concept of preparative solution IEF where time requirements and efficiency are similar to gel-based IEF whereas simple fraction handling as well as quick and complete protein recovery typical for solution-based IEF methods are maintained. The presented method is based on the IEF in separation medium soaked in a segmented strip of nonwoven fabric. The strip is positioned in an open horizontal V-shaped trough. Suggested focusing method combines free solution IEF under continuous evaporation and whole channel dispensing. Separation medium based on ethylene glycol/water mixture enhances viscosity enough to reduce electroosmosis and prevents the medium from completely drying out. Generation of pH gradient and final local pH is visually traced by colored low-molecular pI markers added to input mixture, which enables an optimization of focusing process and collection of individual fractions at desired pH range. The proposed method was tested by fractionation of the proteins and bioactive peptides originating from raw whey. Moreover, subsequent HPLC analysis of the individually collected solution IEF fractions was used for identification of whey components. We confirmed that the method is capable to process directly few tenths of milliliters of raw samples including the salty ones., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Alcohol consumption is a leading cause of liver disease worldwide; thus, there is an urgent need to develop novel therapeutic interventions. Key events for the onset and progression of alcoholic liver disease result in part from the gut-to-liver interaction. Osteopontin is a cytokine present at high concentration in human milk, umbilical cord, and infants' plasma with beneficial potential. We hypothesized that dietary administration of milk osteopontin could prevent alcohol-induced liver injury perhaps by maintaining gut integrity and averting hepatic inflammation and steatosis. Wild-type mice were fed either the control or the ethanol Lieber-DeCarli diets alone or in combination with milk osteopontin for 3 wk, and parameters of gut and liver damage were measured. Milk osteopontin protected the stomach and the gut by increasing gland height, crypt cell plus enterocyte proliferation, and mucin content in addition to lowering macrophages, plasmacytes, lymphocytes, and neutrophils in the mucosa and submucosa in alcohol-fed mice. Milk osteopontin targeted the gut-liver axis, preserving the expression of tight-junction proteins in alcohol-fed mice thus maintaining intestinal integrity and permeability. There was protection from liver injury since transaminases, the activity scores, triglyceride levels, neutrophil infiltration, 3-nitrotyrosine residues, lipid peroxidation end products, translocation of gram-negative bacteria, lipopolysaccharide levels, and tumor necrosis factor-α were lower in cotreated than in ethanol-fed mice. Furthermore, milk osteopontin diminished ethanol-mediated liver injury in OPN knockout mice. Milk osteopontin could be a simple effective nutritional therapeutic strategy to prevent alcohol hepatotoxicity due, among others, to gut protective, anti-inflammatory, and anti-steatotic actions.

Small pore microfiltration (MF) can be used to remove serum proteins (SP) from skim milk. The process's SP removal efficiency directly influences the technology's economic feasibility. Our objective was to quantify the capacity of 0.14μm ceramic Isoflux MF membranes (TAMI, Nyons, France) to remove SP from skim milk. A 3-stage, 3×, feed-and-bleed MF study with diafiltration in the latter 2 stages was conducted at 50°C using Isoflux membranes to determine cumulative SP removal percentages and SP removal rates at each processing stage. The experiment was replicated 3 times starting with 3 separate lots of raw milk. In contrast to 3× MF theoretical cumulative SP removal percentages of 68, 90, and 97% after 1, 2, and 3 stages, respectively, the 3× Isoflux MF process removed only 39.5, 58.4, and 70.2% of SP after 1, 2, and 3 stages, respectively. Previous research has been published that provides the skim milk SP removal capacities of 3-stage, 3× 0.1μm ceramic Membralox (Pall Corp., Cortland, NY) uniform transmembrane pressure (UTP), 0.1μm ceramic Membralox graded permeability (GP), and 0.3μm polymeric polyvinylidene fluoride spiral-wound (PVDF-SW) MF systems (Parker-Hannifin, Process Advanced Filtration Division, Tell City, IN) at 50°C. No difference in cumulative SP removal percentage after 3 stages was detected between the Isoflux and previously published PVDF-SW values (70.3%), but SP removal was lower than published GP (96.5%) and UTP (98.3%) values. To remove 95% of SP from 1,000kg of skim milk in 12h it would take 7, 3, 3, and 7 stages with 6.86, 1.91, 2.82, and 17.98m(2) of membrane surface area for the Isoflux, GP, UTP, and PVDF-SW systems, respectively. The MF systems requiring more stages would produce additional permeate at lower protein concentrations. The ceramic MF systems requiring more surface area would incur higher capital costs. The authors hypothesize that SP removal with the Isoflux membranes was lower than theoretical for the following reasons: a range of membrane pore sizes existed (i.e., some pores were too small to pass SP), the selective layer modification and reverse flow conditions at the membrane outlet combined to reduce the effective membrane surface area, and the geometric shape of the Isoflux flow channels promoted early fouling of the membrane and rejection of SP by the foulant., (Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)