Abundant Milk Protein Research Articles

Multiscale modelling of biomolecular corona formation on metallic surfaces.

In the realm of food industry, the choice of non-consumable materials used plays a crucial role in ensuring consumer safety and product quality. Aluminum is widely used in food packaging and food processing applications, including dairy products. However, the interaction between aluminum and milk content requires further investigation to understand its implications. In this work, we present the results of multiscale modelling of the interaction between various surfaces, that is (100), (110), and (111), of fcc aluminum with the most abundant milk proteins and lactose. Our approach combines atomistic molecular dynamics, a coarse-grained model of protein adsorption, and kinetic Monte Carlo simulations to predict the protein corona composition in the deposited milk layer on aluminum surfaces. We consider a simplified model of milk, which is composed of the six most abundant milk proteins found in natural cow milk and lactose, which is the most abundant sugar found in dairy. Through our study, we ranked selected proteins and lactose adsorption affinities based on their corresponding interaction strength with aluminum surfaces and predicted the content of the naturally forming biomolecular corona. Our comprehensive investigation sheds light on the implications of aluminum in food processing and packaging, particularly concerning its interaction with the most abundant milk proteins and lactose. By employing a multiscale modelling approach, we simulated the interaction between metallic aluminum surfaces and the proteins and lactose, considering different crystallographic orientations. The results of our study provide valuable insights into the mechanisms of lactose and protein deposition on aluminum surfaces, which can aid in the general understanding of protein corona formation.

Subclinical Mastitis from Streptococcus agalactiae and Prototheca spp. Induces Changes in Milk Peptidome in Holstein Cattle.

Early detection of bovine subclinical mastitis may improve treatment strategies and reduce the use of antibiotics. Herein, individual milk samples from Holstein cows affected by subclinical mastitis induced by S. agalactiae and Prototheca spp. were analyzed by untargeted and targeted mass spectrometry approaches to assess changes in their peptidome profiles and identify new potential biomarkers of the pathological condition. Results showed a higher amount of peptides in milk positive on the bacteriological examination when compared with the negative control. However, the different pathogens seemed not to trigger specific effects on the milk peptidome. The peptides that best distinguish positive from negative samples are mainly derived from the most abundant milk proteins, especially from β- and αs1-casein, but also include the antimicrobial peptide casecidin 17. These results provide new insights into the physiopathology of mastitis. Upon further validation, the panel of potential discriminant peptides could help the development of new diagnostic and therapeutic tools.

In silico analysis of bioactive peptide of goat milk based on beta-casein protein

The composition, interactions, and properties of bioactive peptides found in the most abundant milk protein, CSN2, are the focus of goat milk peptidomics. The peptidome of goat milk is regarded as a valuable source of a large number of biologically active peptides as well as health-promoting activities. Through data mining for bioactive peptide selection associated with current bioactive peptide data algorithms resulting in bioactive peptide profiles and visualization of three-dimensional protein structure, in silico analysis on bioinformatic approaches has led to enhancements of global knowledge regarding the health benefits of dairy products. Biological potentials for cardiovascular and nervous diseases, as well as metabolic and immune treatments, are identified using these techniques, in order to increase the commercialization of goat milk bioactive peptides as a functional food and promising natural source treatments.

Effect of pH and protein composition on proteolysis of goat milk proteins by pepsin and pancreatin

The roles of protein composition, pH and enzymes in goat milk protein hydrolysis is still unclear and the proteolysis of low abundant goat milk proteins has received limited attention. The aim of this study was to study the impact of protein composition and proteolytic conditions on goat milk protein hydrolysis in a simplified digestion model. Both whole milk and infant formula were hydrolyzed at pH 2 and 4, using pepsin as well as pepsin combined with pancreatin. Intact proteins were separated from digests using spin filters, followed by bottom-up proteomics of the separated proteins. Results show that under all conditions, caseins are hydrolyzed quickly. Goat casein hydrolysis in infant formula was slightly faster than in goat whole milk, possibly due to less casein coagulation during pepsin hydrolysis at both pH 2 and 4. Several low abundant immunoactive goat milk proteins, especially immunoglobulins, GLYCAM-1 and osteopontin, resisted proteolysis more than high abundant proteins, independent of the pH and enzyme used for hydrolysis. Fast hydrolysis of casein and slow hydrolysis of immunoactive proteins may indicate a good balance between protein utilization and protection of the infant by goat milk proteins.

Soft Matter Engineering for the Fabrication of Functionalized Casein Microparticles

AbstractThe structural features of the most abundant milk protein casein can be exploited for the targeted production of microparticles with desired properties. After the addition of pectin, the so‐called casein micelles reversibly form aggregates of several µm in size, which change their shape due to shear and elongation forces. The soft properties are crucial for the subsequent solidification into stable casein microparticles during film drying. Based on pH‐dependent experiments, we show how we analyze and tailor the swelling and disintegration properties of the microparticles for future controlled and instant release applications.

SIRT1 Mediates Neuroprotective and Neurorescue Effects of Camel α-Lactalbumin and Oleic Acid Complex on Rotenone-Induced Parkinson's Disease.

Parkinson's disease (PD) is the second most common devastating neurodegenerative disorder. Presently used therapies for PD have severe side effects and are limited to only temporary improvement. Therefore, a new therapeutic approach to treat PD urgently needs to be developed. α-Lactalbumin, the most abundant milk protein in camel milk, has been attributed to various medicinal properties. This study intended to investigate the neuroprotective efficacy of the camel α-lactalbumin and oleic acid (CLOA) complex. One mechanism postulated to underlie neuroprotection by the CLOA complex is the induction of silent information regulatory protein (SIRT1). SIRT1 is known to be involved in several pathological and physiological processes, and it has been suggested that SIRT1 plays a protective role in PD. Oxidative stress, inflammation, mitochondrial dysfunction, and apoptosis are involved in PD pathogenesis. Our results revealed that SIRT1 inhibits oxidative stress by maintaining HIF-1α in a deacetylated state. SIRT1 upregulates the expression of FOXO3a and HSF-1, thus inhibiting apoptosis and maintaining the homeostasis of cellular proteins. Increased SIRT1 expression reduces the levels of TNF-α, IL-6, and IL-8, which in turn inhibits neuroinflammation. In addition to SIRT1, the CLOA complex also enhances the expression of survivin and leptin and promotes the survival of neuroblastoma cells. Altogether, our results suggest that the CLOA complex might be a novel therapeutic molecule that could ameliorate neuronal cell damage in PD.

Identifying glycation hot-spots in bovine milk proteins during production and storage of skim milk powder

We investigated protein glycation in a complex milk system under controlled conditions representative of real-life consumer products, analysing intermediate and final products from skim milk powder production, and aged powder samples. We combined protein-centric LC-MS(/MS) with peptide-centric multi-protease LC-MS/MS focusing on the six most abundant bovine milk proteins. This strategy resulted in the identification of glycated proteoforms and of the extent of glycation per protein, high protein sequence coverage, and identification and relative occupancy of the glycation sites. We identified new glycation hot-spots additionally to the ones already described in literature. Primary sequence motif analysis revealed that glycation hot-spots were preceded N-terminally by a stretch rich in basic amino acids, and followed C-terminally by a stretch enriched in aliphatic and hydrophobic amino acids. Our study considerably extends the current understanding of milk protein glycation, discussing glycation hot-spots and their localisation in relation to the primary sequences and higher-order protein structures.

First Insight into the Variation of the Milk Serum Proteome within and between Individual Cows

Milk contains all nutrients needed for development of calves. One important group of components responsible for this are the milk proteins. Variation due to feed or animal health, has been studied for the most abundant milk proteins. The aim of this study was to determine the variation between and within cows for their milk serum proteome. Sample Set 1 was collected from Holstein Friesian (HF) cows between November 2011 and March 2012 and prepared using filter aided sample preparation (FASP) followed by LC-MS/MS for protein identification and quantification. The results showed that the milk serum proteome was very constant in mid lactation (four cows at five time points, p > 0.05) between 3 and 6 months in lactation. Sample Set 2 was collected from HF cows in Dec 2012 and analyzed using FASP and dimethyl labeling followed by LC-MS/MS. Significant variation in the milk serum proteome (p < 0.05) between 17 individual cows was found in Sample Set 2. The most variable proteins were immune-related proteins, which may reflect the health status of the individual cow. On the other hand, proteins related to nutrient synthesis and transport were relatively constant, indicating the importance of milk in providing a stable supply of nutrients to the neonate. In conclusion, the milk serum proteome was stable over mid lactation, but differed significantly between individuals, especially in immune-related proteins.

A complete proteomic profile of human and bovine milk exosomes by liquid chromatography mass spectrometry

Background The current study investigates the proteomic content of milk-derived exosomes. A detailed description of the content of milk exosomes is essential to improve our understanding of the various components of milk and their role in nutrition. Methods The exosomes used in this study were isolated as previously described and characterized by their morphology, particle concentration, and the presence of exosomal markers. Human and bovine milk exosomes were evaluated by Information Dependent Acquisition (IDA) Mass Spectrometry. A direct comparison was made between their proteomic profiles. Results IDA analyses revealed similarities and differences in protein content. 229 and 239 proteins were identified in the human and bovine milk exosome proteome respectively, of which 176 and 186 were unique to each species. Fifty-three proteins were common to both groups. These include proteins associated with specific biological processes and molecular functions. Most notably, the 4 abundant milk proteins lactadherin, butyrophillin, perilipin-2 and xanthine dehydrogenase/oxidase were present in the top 20 list for both human and bovine milk exosomes. Conclusion The milk exosome protein profiles we have provided are crucial new information for the field of infant nutrition. They provide new insight into the components of milk from both humans and bovines.

Two-dimensional high-performance thin-layer chromatography for the characterization of milk peptide properties and a prediction of the retention behavior – a proof-of-principle study

High-performance thin-layer chromatography (HPTLC) is a suitable method for the analysis of peptides and proteins due to a wide selection of stationary and mobile phases and various detection options. Especially, two-dimensional HPTLC (2D-HPTLC) enables a higher resolution compared to one-dimensional HPTLC in the separation of complex peptide mixtures. Similar to 2D electrophoresis, characteristic peptide patterns can be obtained, allowing a differentiation of ingredients based on varying protein origins.The aim of this study was to evaluate 2D-HPTLC with regard to its suitability for the characterization of proteins/peptides and to verify whether it is possible to predict the retention behavior of peptides based on their properties. As models, the five most abundant milk proteins α-lactalbumin, β-lactoglobulin, α-, β-, and κ-Casein were used. In order to determine the repeatability of the peptide separation by 2D-HPTLC, each tryptic protein hydrolyzate was separated eight times. The standard deviations of the retardation factors for the separated peptides varied between 1.0 and 11.1 mm for the x-coordinate and 0.5–7.3 mm for the y-coordinate. It was also shown that after the chromatographic separation, peptides of the individual protein hydrolyzates were located in specific areas on the HPTLC plate, so that a clustering could be obtained for the whey proteins‘ as well as the caseins‘ hydrolyzates. For establishing correlations between the properties of the peptides and their retardation factors, 51 of 85 selected peptides were identified by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS). On this basis, statistically significant correlations (α = 0.05) between the retardation factors of the peptides and their isoelectric points, as well as the percentage of anionic and non-polar amino acids in the peptides were established. Finally, it was investigated, whether the retardation factors for peptides can be predicted on the basis of a linear regression of the percentage of non-polar amino acids in a peptide. For this purpose, a mixture of artifical (synthetic) peptides (n = 14) was separated by 2D-HPTLC and the measured retardation factors were compared with the corresponding retardation factors calculated. Absolute deviations of 0.3–17.9 mm were obtained. In addition, the universal applicability of the method to other protein sources other than milk proteins (animal protein) was tested using a mixture of pea peptides (plant protein, n = 3) resulting in absolute deviations of 0.7–8.6 mm.

The influence of lactoferrin on the epigenetic characteristics of mammalian cells of different types

Despite the huge amount of accumulated data, the study of the main mechanisms of interaction between proteins and epigenetic mechanisms in health and various pathologies remains one of the most important problems of molecular biology. The search for various endogenous and exogenous factors affecting the epigenome of eukaryotes continues to be relevant. Lactoferrin is the second most abundant milk protein and has proven to be a very promising anti-inflammatory, antifungal, antibacterial, and anti-cancer agent. This protein can act as a transcription factor regulating the expression of some genes. However, little attention has been paid to the use of lactoferrin as an epigenetic modulating factor. This review demonstrates that lactoferrin can directly and/or indirectly influence epigenetic mechanisms (DNA methylation, histone modification, chromatin compaction, and microRNA pathways) in different types of cells, in particular cancer cells.

Post translational modifications of milk proteins in geographically diverse goat breeds

Goat milk is a source of nutrition in difficult areas and has lesser allerginicity than cow milk. It is leading in the area for nutraceutical formulation and drug development using goat mammary gland as a bioreactor. Post translational modifications of a protein regulate protein function, biological activity, stabilization and interactions. The protein variants of goat milk from 10 breeds were studied for the post translational modifications by combining highly sensitive 2DE and Q-Exactive LC-MS/MS. Here we observed high levels of post translational modifications in 201 peptides of 120 goat milk proteins. The phosphosites observed for CSN2, CSN1S1, CSN1S2, CSN3 were 11P, 13P, 17P and 6P, respectively in 105 casein phosphopeptides. Whey proteins BLG and LALBA showed 19 and 4 phosphosites respectively. Post translational modification was observed in 45 low abundant non-casein milk proteins mainly associated with signal transduction, immune system, developmental biology and metabolism pathways. Pasp is reported for the first time in 47 sites. The rare conserved peptide sequence of (SSSEE) was observed in αS1 and αS2 casein. The functional roles of identified phosphopeptides included anti-microbial, DPP-IV inhibitory, anti-inflammatory and ACE inhibitory. This is first report from tropics, investigating post translational modifications in casein and non-casein goat milk proteins and studies their interactions.

On the utility of fluorescence-detection analytical ultracentrifugation in probing biomolecular interactions in complex solutions: a case study in milk.

β-Lactoglobulin is the most abundant protein in the whey fraction of ruminant milks, yet is absent in human milk. It has been studied intensively due to its impact on the processing and allergenic properties of ruminant milk products. However, the physiological function of β-lactoglobulin remains unclear. Using the fluorescence-detection system within theanalytical ultracentrifuge, we observed an interaction involving fluorescently labelled β-lactoglobulin in its native environment, i.e. cow and goat milk, for the first time. Co-elution experiments support that these β-lactoglobulin interactions occur naturally in milk and provide evidence that the interacting partners are immunoglobulins, while further sedimentation velocity experiments confirm that an interaction occurs between these molecules. The identification of these interactions, made possible through the use of fluorescence-detected analytical ultracentrifugation, provides possible clues to the long debated physiological function of this abundant milk protein.

Casein micelles from bovine Milk exerts Neuroprotection by stalling metabolic complications linked to oxidative brain injury.

Caseins are the most abundant milk proteins in mammalian species and are assembled in supra-macromolecular structures called micelles. In this study, the microstructural properties, particle size, and elemental composition of isolated casein from bovine milk and its therapeutic effect on oxidative and cholinergic activities linked to dementia in oxidative brain injury were investigated. SEM analysis of the isolated casein micelles from skimmed fresh bovine milk revealed spherical colloid aggregates, while TEM analysis revealed dispersed spherical particles with a mean size of 63.15 ± 4.77nm. SEM-EDX analysis revealed clusters of carbon, oxygen, sulfur, copper, sodium, magnesium, potassium, iron, and selenium. Treatment of oxidative brain injury with the isolated casein micelles led to elevated levels of GSH, SOD, catalase, ENTPDase, 5'NTPase activities, while concomitantly suppressing MDA, cholesterol, HDL-c levels, acetylcholinesterase and lipase activities. Treatment with the isolated casein micelles led to complete depletion of oxidative generated lipid metabolites, while deactivating oxidative-activated lipid metabolic pathways. These results indicate the microstructural properties, particle size, elemental composition, and antioxidant neuroprotective effect of casein micelles from bovine milk. Thus, demonstrating the nutraceutical properties of milk in the management of oxidative induced cognitive impairment.

Development and evaluation of a milk protein transcript depletion method for differential transcriptome analysis in mammary gland tissue

BackgroundIn the mammary gland transcriptome of lactating dairy cows genes encoding milk proteins are highly abundant, which can impair the detection of lowly expressed transcripts and can bias the outcome in global transcriptome analyses. Therefore, the aim of this study was to develop and evaluate a method to deplete extremely highly expressed transcripts in mRNA from lactating mammary gland tissue.ResultsSelective RNA depletion was performed by hybridization of antisense oligonucleotides targeting genes encoding the caseins (CSN1S1, CSN1S2, CSN2 and CSN3) and whey proteins (LALBA and PAEP) within total RNA followed by RNase H-mediated elimination of the respective transcripts. The effect of the RNA depletion procedure was monitored by RNA sequencing analysis comparing depleted and non-depleted RNA samples from Escherichia coli (E. coli) challenged and non-challenged udder tissue of lactating cows in a proof of principle experiment. Using RNase H-mediated RNA depletion, the ratio of highly abundant milk protein gene transcripts was reduced in all depleted samples by an average of more than 50% compared to the non-depleted samples. Furthermore, the sensitivity for discovering transcripts with marginal expression levels and transcripts not yet annotated was improved. Finally, the sensitivity to detect significantly differentially expressed transcripts between non-challenged and challenged udder tissue was increased without leading to an inadvertent bias in the pathogen challenge-associated biological signaling pathway patterns.ConclusionsThe implementation of selective RNase H-mediated RNA depletion of milk protein gene transcripts from the mammary gland transcriptome of lactating cows will be highly beneficial to establish comprehensive transcript catalogues of the tissue that better reflects its transcriptome complexity.

Bioactive peptides from milk: animal determinants and their implications in human health.

Milk is an important protein source in human diets, providing around 32 g protein/l (for bovine milk, which constitutes some 85% of global consumption). The most abundant milk proteins are α-lactalbumin, β-lactoglobulin, αs-casein, β-casein, and κ-casein. Besides their nutritional value, milk proteins play a crucial role in the processing properties of milk, such as solubility, water bonding, heat stability, renneting and foaming, among others. In addition, and most importantly for this review, these proteins are the main source of bioactive components in milk. Due to the wide range of proposed beneficial effects on human health, milk proteins are considered as potential ingredients for the production of health-promoting functional foods. However, most of the evidence for bioactive effects comes from in vitro studies, and there is a need for further research to fully evaluate the true potential of milk-derived bioactive factors. Animal genetics and animal nutrition play an important role in the relative proportions of milk proteins and could be used to manipulate the concentration of specific bioactive peptides in milk from ruminants. Unfortunately, only a few studies in the literature have focused on changes in milk bioactive peptides associated to animal genetics and animal nutrition. The knowledge described in the present review may set the basis for further research and for the development of new dairy products with healthy and beneficial properties for humans.

Profiling of Multiphosphorylated Peptides in Kefir and Their Release During Simulated Gastrointestinal Digestion

Casein phosphopeptides are multiphosphorylated milk peptides, which can have anticariogenic activity and improve mineral absorption by binding bivalent metal ions. The present study investigated phosphopeptides in kefir because fermentation may lead to their enhanced release from milk proteins. After selective enrichment by hydroxyapatite extraction, phosphopeptides and their phosphorylation degree were identified by matrix-assisted desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) before and after enzymatic dephosphorylation. Peptide structures were determined by ultrahigh-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (UHPLC–ESI–MS/MS) revealing 27 phosphopeptides in kefir, including nine peptides containing the motif pSpSpSEE, which binds minerals most efficiently. The majority (18) of phosphopeptides were derived from β-casein, but only three were derived from the most abundant milk protein αs1-casein. After simulated gastrointestinal digestion, MALDI-TOF-MS analysis detected eight putative phosphopeptides in kefir, four of which were assigned by UHPLC–ESI–MS/MS to αs2-casein124–133, αs2-casein137–146, β-casein30–40, and κ-casein147–161. These results indicate that kefir is a good dietary source of multiphosphorylated peptides.

Impact of protein-nanoparticle interactions on gastrointestinal fate of ingested nanoparticles: Not just simple protein corona effects

The interaction of nanoparticles (NPs) with proteins in their surroundings is a key factor determining their biological fate. The formation of a protein corona around ingested NPs in complex food matrices and in the gastrointestinal tract (GIT) is known to impact their gastrointestinal fate. In this study, the interaction between titanium dioxide (TiO2) NPs from a common food additive (E171) and casein, an abundant milk protein was characterized. The impact of these interactions on the potential gastrointestinal fate of the NPs was examined using a simulated GIT model. Our results showed that a series of complex molecular events occurred when the TiO2 NPs were added to casein solutions, including dissociation of casein micelles, NP-protein complex formation, and complex aggregation. Extensive aggregation was observed under conditions where charge neutralization occurred, which was attributed to a reduction in the electrostatic repulsion between the NP-protein complexes and to a polymer bridging effects. There was a pronounced decrease in the rate and extent of gastric digestion of the casein in the presence of the TiO2 NPs, indicating that complex formation reduced the access of pepsin to the peptide bonds. These findings stress the importance of taking into account the interactions of ingested NPs with food matrix and gastrointestinal components. Moreover, our findings show that the simple concept of a protein corona does not adequately explain the complex structural changes that occur when proteins and NPs interact.

Aggregation of α- and β- caseins induced by peroxyl radicals involves secondary reactions of carbonyl compounds as well as di-tyrosine and di-tryptophan formation

The present work examined the role of Tyr and Trp in oxidative modifications of caseins, the most abundant milk proteins, induced by peroxyl radicals (ROO•). We hypothesized that the selectivity of ROO• and the high flexibility of caseins (implying a high exposure of Tyr and Trp residues) would favor radical-radical reactions, and di-tyrosine (di-Tyr) and di-tryptophan (di-Trp) formation. Solutions of α- and β-caseins were exposed to ROO• from thermolysis and photolysis of AAPH (2,2′-azobis(2-methylpropionamidine)dihydrochloride). Oxidative modifications were examined using electrophoresis, western blotting, fluorescence, and chromatographic methodologies with diode array, fluorescence and mass detection. Exposure of caseins to AAPH at 37 °C gave fragmentation, cross-linking and protein aggregation. Amino acid analysis showed consumption of Trp, Tyr, Met, His and Lys residues. Quantification of Trp and Tyr products, showed low levels of di-Tyr and di-Trp, together with an accumulation of carbonyls indicating that casein aggregation is, at least partly, associated with secondary reactions between carbonyls and Lys and His residues. AAPH photolysis, which generates a high flux of free radicals increased the extent of formation of di-Tyr in both model peptides and α- and β- caseins; di-Trp was only detected in peptides and α-casein. Thus, in spite of the high flexibility of caseins, which would be expected to favor radical-radical reactions, the low flux of ROO• generated during AAPH thermolysis disfavours the formation of dimeric radical-radical cross-links such as di-Tyr and di-Trp, instead favoring other O2-dependent crosslinking pathways such as those involving secondary reactions of initial carbonyl products.

Application of Response Surface Methodology in the Preparation of Pectin-Caseinate Nanocomplexes for Potential Use as Nutraceutical Formulation: A Statistical Experimental Design Analysis

Background: The formation of electrostatic complexes between two types of biopolymers, sodium Caseinate (a derivative from most abundant milk protein) and Pectin (a natural hetro polysaccharide), was studied as a function of biopolymers concentrations and pH of solutions (3.9- 4.3). Method: The size and morphology of the resulted complexes were investigated by using of laser light scattering and transmission electron microscopy, respectively. Response surface methodology (A three-factor, three levels Box-Behnken design) was used for the optimization procedure with pH, pectin and sodium Caseinate concentrations as independent variables. Particle size and polydispersity index of nanocomplexes were considered as dependent variables. Results: Negatively charged nanocomplexes were produced below the isoelectric point of protein (5.4), at pH 4.1 with a suitable colloidal stability and average particle size of about 100 nm. It was found that the particle size of nanocomplexes could be controlled by changing in variables. Conclusion: In conclusion response surface methodology are simple, rapid and beneficial approach for preparation, optimization and investigation of the effect of independent variables on the properties of products.