Milk Protein Fractions Research Articles

Milk processing increases the allergenicity of cow's milk-Preclinical evidence supported by a human proof-of-concept provocation pilot.

BackgroundSeveral studies demonstrated the adverse effect of milk processing on the allergy‐protective capacity of raw cow's milk. Whether milk processing also affects the allergenicity of raw milk is hardly investigated.ObjectiveTo assess the allergenicity of raw (unprocessed) and processed cow's milk in a murine model for food allergy as well as in cow's milk allergic children.MethodsC3H/HeOuJ mice were either sensitized to whole milk (raw cow's milk, heated raw cow's milk or shop milk [store‐bought milk]) and challenged with cow's milk protein or they were sensitized and challenged to whey proteins (native or heated). Acute allergic symptoms, mast cell degranulation, allergen‐specific IgE levels and cytokine concentrations were determined upon challenge. Cow's milk allergic children were tested in an oral provocation pilot with organic raw and conventional shop milk.ResultsMice sensitized to raw milk showed fewer acute allergic symptoms upon intradermal challenge than mice sensitized to processed milk. The acute allergic skin response was low (103 ± 8.5 µm vs 195 ± 17.7 µm for heated raw milk, P < 0.0001 and vs 149 ± 13.6 µm for shop milk, P = 0.0316), and there were no anaphylactic shock symptoms and no anaphylactic shock‐induced drop in body temperature. Moreover, allergen‐specific IgE levels and Th2 cytokines were significantly lower in raw milk sensitized mice. Interestingly, the reduced sensitizing capacity was preserved in the isolated native whey protein fraction of raw milk. Besides, native whey protein challenge diminished allergic symptoms in mice sensitized to heated whey proteins. In an oral provocation pilot, cow's milk allergic children tolerated raw milk up to 50 mL, whereas they only tolerated 8.6 ± 5.3 mL shop milk (P = 0.0078).Conclusion and Clinical RelevanceThis study demonstrates that raw (unprocessed) cow's milk and native whey proteins have a lower allergenicity than their processed counterparts. The preclinical evidence in combination with the human proof‐of‐concept provocation pilot provides evidence that milk processing negatively influences the allergenicity of milk.

Variation of Detailed Protein Composition of Cow Milk Predicted from a Large Database of Mid-Infrared Spectra.

Simple SummaryMilk proteins are one of the most valuable milk components. The objective of the present study was to assess sources of variation of detailed protein composition predicted from infrared spectra in milk of dairy and dual-purpose cattle breeds. Results showed that protein fractions were primarily influenced by days in milk, and the relative proportion of each fraction through lactation was not constant. Protein fractions correlated with crude protein, total casein, fat and milk urea nitrogen. In perspective, mid-infrared predictions of milk fractions could be useful for the dairy sector to improve nutritional and technological properties of milk.This study aimed to investigate factors affecting protein fractions, namely α-casein (α-CN), β-casein (β-CN), κ-casein (κ-CN), β-lactoglobulin (β-LG) and α-lactalbumin (α-LA) predicted from milk infrared spectra in milk of dairy and dual-purpose cattle breeds. The dataset comprised 735,328 observations from 49,049 cows in 1782 herds. Results highlighted significant differences of protein fractions in milk of the studied breeds. Significant variations of protein fractions were found also through parities and lactation, with the latter thoroughly influencing protein fractions percentage. Interesting correlations (r) were estimated between β-CN, κ-CN and β-LG, expressed as percentage of crude protein, and milk urea nitrogen (r = 0.31, −0.20 and −0.26, respectively) and between α-LA and fat percentage (r = 0.41). The present study paves the way for future studies on the associations between protein fractions and milk technological properties, and for the estimation of genetic parameters of predicted protein composition.

Study of milk coagulation induced by chymosin using atomic force microscopy

Caseins (αs1-, αs2, β-, and κ-caseins) form the major protein fraction of milk, irrespective of their origins. They are able to form well-ordered colloidal structures in association with colloidal calcium phosphate, named casein micelles. Chymosin-mediated milk coagulation takes place through loss of casein micelles’ stability by hydrolyzing κ-caseins. This process is critical for the quality of cheese and other milk derivatives. Therefore, many microscopy techniques have been used to understand the structural aspects underlying the integrity of casein micelles during chymosin action. However, these technologies can be costly and laborious. In this study, atomic force microscopy (AFM) and dynamic light scattering (DLS) were used to study milk coagulation by chymosin. Following 15 min of chymosin action, the AFM images showed the start of the formation of casein micelle aggregates. After 30–45 min, the micelles continued aggregating, forming structures such as bunches of grapes. Finally, after 45–60 min, these structures formed large clusters of casein micelles. After 60 min, the zeta potential did not drop to zero when the milk clotted, but still had a negative value, suggesting that the κ-caseins on the micelle surface were not totally hydrolyzed. The results corroborate those previously described and suggest this tool as an alternative to study the milk-clotting process at the ultrastructural level induced by proteases.

Casein nanomicelle as an emerging biomaterial-A comprehensive review.

Casein nanomicelles, a major fraction of milk protein, are emerging as a novel drug delivery system owing to their various structural and functional properties. Casein is further divided into α-, β- and κ-casein, and to date various models have been proposed to describe casein structure, but still no definite structure presenting a detailed assembly of the casein micelle has been found. Thus far, the submicellar model and Horne and Holt model are the most accepted models. This article presents a detailed review of casein micelles and their fractions, and the physicochemical properties that account for their numerous applications in nutraceutics, pharmaceutics and cosmetics. Due to their nanosize and self-assembling nature, casein nanomicelles are considered as excellent delivery carriers to provide better bioavailability and stability of various compounds such as vitamins, oils, polyphenols, fattyacids and minerals. Their amphiphilic nature also provides a great opportunity to deliver hydrophobic bioactives in various drug delivery systems such as nanoparticles, nanomicelles, nanogels and nanoemulsions to improve drug binding and targeting.

Milk protein fractions strongly affect the patterns of coagulation, curd firming, and syneresis

The aim of this study was to assess the role of milk protein fractions in the coagulation, curd firming, and syneresis of bovine milk. Analyses were performed on 1,271 individual milk samples from Brown Swiss cows reared in 85 herds classified into 4 types of farming systems, from the very traditional (tied cows, feed manually distributed, summer highland pasture) to the most modern (loose cows, use of total mixed rations with or without silage). Fractions αS1-casein (CN), αS2-CN, β-CN, κ-CN, β-lactoglobulin (LG), and α-lactalbumin (LA) and genotypes at CSN2, CSN3, and BLG were obtained by reversed-phase HPLC. The following milk coagulation properties were measured with a lactodynamograph, with the testing time extended to 60 min: rennet coagulation time (RCT, min), curd firming time (min), and curd firmness at 30 and 45 min (mm). All the curd firmness measures recorded over time (total of 240 observations/sample) were used in a 4-parameter nonlinear model to obtain parameters of coagulation, curd firming, and syneresis: RCT estimated from the equation (min), asymptotic potential curd firmness (mm), the curd firming and syneresis instant rate constants (%/min), and the maximum curd firmness value (CFmax, mm) and the time taken to reach it (min). All the aforementioned traits were analyzed with 2 linear mixed models, which tested the effects of the protein fractions expressed in different ways: in the first, quantitative model, each protein fraction was expressed as content in milk; in the second, qualitative model, each protein fraction was expressed as a percentage of total casein content. Besides proteins, additional nuisance parameters were herd (included as a random effect), daily milk production (only for the quantitative model), casein content (only for the qualitative model), dairy system, parity, days in milk, the pendulum of the lactodynamograph, and the CSN2, CSN3, and BLG genotypes. Both αS1-CN and β-CN showed a clear and favorable effect on CFmax, where the former effect was almost double the latter. Milk coagulation ability was favorably affected by κ-CN, which reduced both the RCT and RCT estimated from the equation, increased the curd firming and syneresis instant rate constants, and allowed a higher CFmax to be reached. In contrast, αS2-CN delayed gelation time and β-LG worsened curd firming, both resulting in a low CFmax. The results of this study suggest that modification of the relative contents of specific protein fractions can have an enormous effect on the technological behavior of bovine milk.

Novel trends in fermented dairy technology

Novel trends in fermented dairy technology are presented in this review paper. The application of new starter cultures (probiotics, kombucha), as well as quality improving ingredients like transglutaminase (TGase), milk protein fractions, and functional components of plant origin have been investigated by the authors worldwide. New processing techniques such as: high-pressure processing (HPP), high pressure homogenization (HPH), and ultrasonic processing (USP) are interesting because of their potential to achieve a specific and/or novel functionality or to improve the efficiency. Novel trends in fermented dairy technology contribute to the creation of various products with high nutritive value, possessing also specific functional properties. Basic health benefits of functional fermented dairy products are: biologically active peptides - ACE inhibitors and antioxidative activity. Due to the mentioned functional characteristics, these dairy products are considered to be among the most precious functional foods.

QUALIDADE FÍSICO-QUÍMICA E MICROBIOLÓGICA DO LEITE BOVINO EM DIFERENTES SISTEMAS DE PRODUÇÃO E ESTAÇÕES DO ANO

Resumo O objetivo deste trabalho foi avaliar a interação entre sistemas produtivos e estações do ano sobre a qualidade do leite bovino. Três propriedades foram avaliadas mensalmente, durante as estações, em quatro graus de especialização dos sistemas de produção: altamente especializado, especializado, semiespecializado e não especializado. Em termos de qualidade do leite, consideraram-se os teores de gordura, proteína, sólidos totais, contagem de células somáticas, contagem bacteriana total, acidez titulável em graus Dornic, densidade, índice crioscópico, estabilidade ao alizarol, potencial de hidrogênio (pH) e teste de redução do azul de metileno. Além destes, devido à sua importância para o rendimento industrial dos produtos lácteos de base proteica, foi realizado o fracionamento da proteína total do leite em equivalente proteico do nitrogênio não proteico, da proteína verdadeira e da caseína. A acidez Dornic foi maior na primavera e no inverno, enquanto os valores mais elevados de contagem de células somáticas foram no verão. Os teores de proteína total e proteína verdadeira foram menores durante o verão e elevaram-se os sólidos totais no outono. Com a diminuição dos níveis de especialização das propriedades rurais, observaram-se aumento na contagem bacteriana total e diminuição do nitrogênio não proteico. A densidade apresentou comportamento inverso ao da gordura, enquanto o potencial de hidrogênio (pH) se expressou de modo antagônico à acidez titulável. Na análise multivariada dos dados, foi possível identificar as combinações entre estratos produtivos e estações do ano que apresentavam a melhor qualidade da proteína do leite. Nesse sentido, todos os arranjos de sistemas de produção com o outono, o sistema altamente especializado no inverno, o semiespecializado no verão e o especializado na primavera tiveram qualidade proteica positiva, destacando-se pelas menores contagens de células somáticas a combinação entre a estação primavera e o sistema semiespecializado de produção.

Raw cow’s milk consumption and allergic diseases – The potential role of bioactive whey proteins

The prevalence of allergic diseases has increased significantly in Western countries in the last decades. This increase is often explained by the loss of rural living conditions and associated changes in diet and lifestyle. In line with this ‘hygiene hypothesis’, several epidemiological studies have shown that growing up on a farm lowers the risk of developing allergic diseases. The consumption of raw, unprocessed, cow’s milk seems to be one of the factors contributing to this protective effect. Recent evidence indeed shows an inverse relation between raw cow’s milk consumption and the development of asthma and allergies. However, the consumption of raw milk is not recommended due to the possible contamination with pathogens. Cow’s milk used for commercial purposes is therefore processed, but this milk processing is shown to abolish the allergy-protective effects of raw milk. This emphasizes the importance of understanding the components and mechanisms underlying the allergy-protective capacity of raw cow’s milk. Only then, ways to produce a safe and protective milk can be developed. Since mainly heat treatment is shown to abolish the allergy-protective effects of raw cow’s milk, the heat-sensitive whey protein fraction of raw milk is an often-mentioned source of the protective components. In this review, several of these whey proteins, their potential contribution to the allergy-protective effects of raw cow’s milk and the consequences of heat treatment will be discussed. A better understanding of these bioactive whey proteins might eventually contribute to the development of new nutritional approaches for allergy management.

Genetic variation in serum protein pattern and blood β-hydroxybutyrate and their relationships with udder health traits, protein profile, and cheese-making properties in Holstein cows

The aim of this study was to investigate in Holstein cows the genetic basis of blood serum metabolites [i.e., total protein, albumin, globulin, albumin:globulin ratio (A:G), and blood β-hydroxybutyrate (BHB)], a set of milk phenotypes related to udder health, milk quality technological characteristics, and genetic relationships among them. Samples of milk were collected from 498 Holstein cows belonging to 28 herds. All animal welfare and milk phenotypes were assessed using standard analytical methodology. A set of Bayesian univariate and bivariate animal models was implemented via Gibbs sampling, and statistical inference was based on the marginal posterior distributions of parameters of concern. We observed a small additive genetic influence for serum albumin concentrations, moderate heritability (≥0.20) for total proteins, globulins, and A:G, and high heritability (0.37) for blood BHB. Udder health traits (somatic cell score, milk lactose, and milk pH) showed low or moderate heritabilities (0.15-0.20), whereas variations in milk protein fraction concentrations were confirmed as mostly under genetic control (heritability: 0.21-0.71). The moderate and high heritabilities observed for milk coagulation properties and curd firming modeling parameters provided confirmation that genetic background exerts a strong influence on the cheese-making ability of milk, largely due to genetic polymorphisms in the major milk protein genes. Blood BHB showed strong negative genetic correlations with globulins (-0.619) but positive correlations with serum albumin (0.629) and A:G (0.717), which suggests that alterations in the serum protein pattern and BHB blood levels are likely to be genetically related. Strong relationships were found between albumin and fat percentages (-0.894), between globulin and αS2-CN (-0.610), and, to a lesser extent, between serum protein pattern and milk technological characteristics. Genetic relationships between blood BHB and traits related to udder health and milk quality and technological characteristics were mostly weak. This study provides evidence that there is exploitable additive genetic variation for traits related to animal health and welfare and throws light on the shared genetic basis of these traits and the phenotypes related to the quality and cheese-making ability of milk.

Improving Fractionation of Human Milk Proteins through Calcium Phosphate Coprecipitation and Their Rapid Characterization by Capillary Electrophoresis.

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.

Variations in milk protein fractions affect the efficiency of the cheese-making process

The aim of this study was to assess the influence of the amounts of the αS1-, αS2-, β-, and κ-casein (CN) and the α-lactalbumin and β-lactoglobulin protein fractions on the efficiency of the cheese-making process independently of their genetic polymorphisms. The study was carried out on milk samples from 1,271 Brown Swiss cows from 85 herds classified into 4 categories according to management, feeding, and housing characteristics (traditional and modern systems). To assess the efficiency of the cheese-making process, we processed the milk samples according to a laboratory cheese-making procedure (1,500 mL/sample) and obtained the following measures: (1) 3 percentage cheese yields (%CYcurd, %CYsolids, %CYwater), (2) 2 daily cheese yields obtained by multiplying %CY (curd and total solids) by daily milk yields (dCYcurd, dCYsolids), (3) 4 measures of nutrient recovery in the curd (RECfat, RECprotein, RECsolids, RECenergy), and (4) 2 measures of cheese-making efficiency in terms of the ratio between the observed and theoretical %CY (Ef-%CYcurd, Ef-%CYsolids). All the aforementioned traits were analyzed by fitting 2 linear mixed models with protein fractions as fixed effects expressed as percentage in the milk (model M-%milk) and as percentage of the total casein content (model M-%cas) together with the effects of total casein content (only in model M-%cas), daily milk yield (only in model M-%milk; not for dCY traits), dairy system, herd (random effect), days in milk, parity, and vat. The efficiency of overall cheese yield (Ef-%CYcurd) was mostly positively associated with β-CN content in the milk, whereas Ef-%CYsolids was greater with higher amounts of κ-CN and αS1-CN (M-%milk) due to the strong influence of both fractions on the recovery rate of milk components in the curd (fat and total solids, protein with αS1-CN only) when expressed as percentage of milk and of total casein; only β-CN was more important for RECprotein. In contrast, we found β-lactoglobulin to be highly negatively related to all the traits related to the cheese-making process and to the daily cheese yield per cow, whereas α-lactalbumin was positively associated with the latter traits. Additional research on this topic is needed, with particular focus on the genetic and genomic aspects of the role of protein fractions in the cheese-making process and on the associations between genetic polymorphisms in milk protein and milk nutrient recovery in the curd.

In vitro immunogenicity of various native and thermally processed bovine milk proteins and their mixtures

In vitro immunogenicity of various native and thermally processed (72°C/15 s and 100°C/30 s) bovine milk protein fractions, their mixtures, whey, and skim milk, was studied by analyzing the immune response of T helper (Th) cells in human peripheral blood mononuclear cells. The secretion of Th type cytokines induced by the protein stimulants was quantified while determining the heat-induced protein denaturation. Purified whey proteins, caseins and whey fraction, and skim milk provoked substantial immune responses at various degrees, indicating their potent immunogenicity. The protein mixtures prepared using the fractionated whey proteins with or without caseins appeared less immunogenic in both native and heat-treated forms, implying their potential of producing less immunogenic dairy products. The 100°C/30 s treatment significantly altered the immunogenicity of most of the potent protein stimulants, which mostly coincided with their levels of protein denaturation. The 72°C/15 s treatment caused the least protein denaturation but altered the immunogenicity of several protein stimulants notably, including heat-stable caseins and α-lactalbumin.

Concentration of Immunoglobulins in Microfiltration Permeates of Skim Milk: Impact of Transmembrane Pressure and Temperature on the IgG Transmission Using Different Ceramic Membrane Types and Pore Sizes.

The use of bioactive bovine milk immunoglobulins (Ig) has been found to be an alternative treatment for certain human gastrointestinal diseases. Some methodologies have been developed with bovine colostrum. These are considered in laboratory scale and are bound to high cost and limited availability of the raw material. The main challenge remains in obtaining high amounts of active IgG from an available source as mature cow milk by the means of industrial processes. Microfiltration (MF) was chosen as a process variant, which enables a gentle and effective concentration of the Ig fractions (ca. 0.06% in raw milk) while reducing casein and lactose at the same time. Different microfiltration membranes (ceramic standard and gradient), pore sizes (0.14–0.8 µm), transmembrane pressures (0.5–2.5 bar), and temperatures (10, 50 °C) were investigated. The transmission of immunoglobulin G (IgG) and casein during the filtration of raw skim milk (<0.1% fat) was evaluated during batch filtration using a single channel pilot plant. The transmission levels of IgG (~160 kDa) were measured to be at the same level as the reference major whey protein β-Lg (~18 kDa) at all evaluated pore sizes and process parameters despite the large difference in molecular mass of both fractions. Ceramic gradient membranes with a pore sizes of 0.14 µm showed IgG-transmission rates between 45% to 65% while reducing the casein fraction below 1% in the permeates. Contrary to the expectations, a lower pore size of 0.14 µm yielded fluxes up to 35% higher than 0.2 µm MF membranes. It was found that low transmembrane pressures benefit the Ig transmission. Upscaling the presented results to a continuous MF membrane process offers new possibilities for the production of immunoglobulin enriched supplements with well-known processing equipment for large scale milk protein fractionation.

Potential antioxidant bioactive peptides from camel milk proteins

Camel milk is traditionally considered to have medicinal characteristics that it has potential health benefits and could help to treat several illnesses. Particularly, it is closest to human breast milk and has high levels of nutrients and bioactive components. The aim of this study was to explore the antioxidant peptides derived from protein fractions of camel milk. Camel milk proteins (CMP) were fractionated into camel casein protein (CCP) and camel whey protein (CWP), which were hydrolyzed with pepsin to produce peptic digests P-CCP and P-CWP, respectively. RP-HPLC was used for fractionation of the peptides from the P-CCP and P-CWP. The antioxidant activities were evaluated using superoxide anion generating system of xanthine oxidase (XOD) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging assay. Active peptides were analyzed using matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) whereas a number of antioxidant peptides, with masses ranging from 913 to 2,951 Da, derived mainly from alpha-casein, lactophorin and lactoferrin, were identified. When yeast cells are used as a system for modeling mitochondrial disease, the peptides in caseins and whey fractions significantly enhanced the tolerance of yeast cells against peroxide-induced oxidative stress. The results show that both caseins and whey proteins of camel milk possess bioactive peptides with significant radical-scavenging activities and thus herald a fascinating opportunity for their potential as nutraceuticals or therapeutic peptides for prevention and treatment of oxidative stress-associated diseases.

Novel method for metalloproteins determination in human breast milk by size exclusion chromatography coupled to inductively coupled plasma mass spectrometry

Levels of essential metals in human breast milk (HBM) have been determined by different analytical techniques, but there is few woks about human whey milk fractions. However, the current trend lies in metalloproteomic and identification of different metalloproteins. In this sense, native separative techniques (N-PAGE and SEC) coupled to ICP-MS provide us with valuable information. Besides it is necessary the development of new methodologies in order to determine with accuracy and precision the profile of such metals and metalloproteins in the different whey protein fractions of HBM. Thus, the aim of this work was to develop a new method for metals and metalloproteins determination by SEC-ICP-MS in whey protein fractions of HBM. Human whey fractions were obtained of HBM samples by ultracentrifugation. Then, protein fractions of whey milk were separated by SEC coupled to ICP-MS for metalloproteins and Mn, Co, Cu and Se quantification. Besides, protein profile of whey milk was determined by N-PAGE and computer assisted image analysis. SEC-ICP-MS results indicated that first and second protein fractions showed detectable levels of the Mn, Co, Cu, and Se. Protein profile determined by N-PAGE and image analysis showed that molecular weight of protein fractions ranged between 68,878–1,228.277 Da. In this work, metalloproteins were analyzed by SEC coupled to ICP-MS, with adequate sensitivity and accuracy. Our study has shown the presence of Mn, Co, Cu and Se bound to two protein fractions in whey milk of HBM. Metals levels analyzed were within the ranges reported in the literature.

Impact of a thermisation treatment on oxytetracycline spiked ovine milk: Fate of the molecule and technological implications

A few studies have been published on the distribution of oxytetracyline (OTC) residues present in milk among cheese, whey, and milk protein fractions, throughout cheese-making, most of them, focused on the effect of pasteurization and Ultra High Temperature (UHT) treatments, and carried out on cow milk. This study aimed to investigate the impact of a thermisation treatment of ovine milk spiked with oxytetracycline at MRL (maximum residue limit) and half MRL, on the fate of the molecule, and the effect of OTC residues on starter culture development and cheese composition. The antibiotic recovery and partition from milk into whey and cheese were assessed by liquid chromatography-high resolution mass spectrometry (LC-HRMS). Starter and non-starter microflora development was monitored by viable plate counts. Milk thermisation did not affect OTC recovery, partition and cheese chemical composition. On a dry matter basis, an OTC reduction between 15 and 19% was calculated in 60-day cheese, at MRL and half MRL, respectively. OTC caused a dose-dependent difference in the time required to reach pH 5.60, which was significantly higher (P < 0.05) at MRL level compared to half MRL and control, allowing coliform bacteria to reach 6 log CFU g−1 in 1-day MRL OTC cheeses.

Combining different proteomic approaches to resolve complexity of the milk protein fraction of dromedary, Bactrian camels and hybrids, from different regions of Kazakhstan.

Nutritional suitability of milk is not only related to gross composition, but is also strongly affected by the microheterogeniety of the protein fraction. Hence, to go further into the evaluation of the potential suitability of non-bovine milks in human/infant nutrition it is necessary to have a detailed characterization of their protein components. Combining proven proteomic approaches (SDS-PAGE, LC-MS/MS and LC-ESI-MS) and cDNA sequencing, we provide here in depth characterization of the milk protein fraction of dromedary and Bactrian camels, and their hybrids, from different regions of Kazakhstan. A total 391 functional groups of proteins were identified from 8 camel milk samples. A detailed characterization of 50 protein molecules, relating to genetic variants and isoforms arising from post-translational modifications and alternative splicing events, belonging to nine protein families (κ-, αs1-, αs2-, β-; and γ-CN, WAP, α-LAC, PGRP, CSA/LPO) was achieved by LC-ESI-MS. The presence of two unknown proteins UP1 (22,939 Da) and UP2 (23,046 Da) was also reported as well as the existence of a β-CN short isoform (946 Da lighter than the full-length β-CN), arising very likely in both genetic variants (A and B) from proteolysis by plasmin. In addition, we report, for the first time to our knowledge, the occurrence of a αs2-CN phosphorylation isoform with 12P groups within two recognition motifs, suggesting thereby the existence of two kinase systems involved in the phosphorylation of caseins in the mammary gland. Finally, we demonstrate that genetic variants, which hitherto seemed to be species- specific (e.g. β-CN A for Bactrian and β-CN B for dromedary), are in fact present both in Camel dromedarius and C. bactrianus.

A new insight into cystatin C contained in milk basic protein to bone metabolism: Effects on osteoclasts and osteoblastic MC3T3-E1 cells invitro.

A milk protein fraction possessing alkaline isoelectric points (milk basic protein [MBP]) improves bone metabolism invivo, and it inhibits bone resorption by osteoclasts and promotes mouse osteoblastic MC3T3-E1 cells proliferation invitro. Cystatin C (CysC) is a component of MBP and shows bone resorption inhibitory activity. Therefore, it is likely that MBP with higher CysC content improves bone metabolism more effectively. In this study, we prepared MBP with low and high contents of CysC and compared its effects on bone metabolism with standard MBP invitro. Our results showed that the CysC content in MBP was positively related to not only bone resorption inhibitory activity but also MC3T3-E1 cells proliferative activity. Furthermore, purified CysC stimulated MC3T3-E1 cells proliferation. These results indicate that CysC contributes to promotion of MC3T3-E1 cells proliferation, and MBP with higher CysC content shows enhanced bone resorption inhibitory activity and MC3T3-E1 cells proliferative activity. CysC is considered an important factor in the effect on bone metabolism of MBP.

Milk nutrition and childhood epilepsy: An ex vivo study on cytokines and oxidative stress in response to milk protein fractions

We present a pilot study on the effects of milk protein fractions [αS1-casein (CN), αS2-CN, κ-CN, β-CN, and a mix of α-lactalbumin (α-LA) and β-lactoglobulin (β-LG)] from different animal species (bovine, ovine, and caprine) on pro- and anti-inflammatory cytokines and oxidative status in cultured peripheral blood mononuclear cells from children with generalized epilepsy. Peripheral blood mononuclear cells (PBMC) were obtained by density gradient from blood of 10 children with generalized epilepsy (5 males; mean age 33.6 ± 5.4 mo) and 10 controls (5 males; mean age 35.6 ± 6.8 mo). Children with epilepsy were grouped according to cytokine levels as follows: children with epilepsy having low levels of cytokines not different from those of control children (LL-EC); children with epilepsy having cytokine levels at least 5-fold higher (medium levels) than those of control children (ML-EC); and children with epilepsy having cytokine levels at least 10-fold higher (high levels) than those of control children (HL-EC). The production of tumor necrosis factor-α (TNF-α), IL-10, IL-6, and IL-1β was studied in cultured PBMC incubated with αS1-CN, αS2-CN, κ-CN, β-CN, and a mix of α-LA and β-LG from bovine, caprine, and ovine milks. The levels of reactive oxygen and nitrogen species (ROS/RNS) and catalase activity were assessed in cultured supernatant. In the HL-EC group, β-CN from small ruminant species (ovine and caprine) induced the highest levels of TNF-α, whereas PBMC incubated with αS2-CN from ovine milk and the mix of β-LG and α-LA from all tested milk species had the lowest levels of TNF-α. Within the HL-EC group, production of IL-1β was higher for bovine and ovine αS2-CN fractions and lower for caprine and ovine β-CN and κ-CN. In the HL-EC group, IL-6 was higher in cultured PBMC incubated with αS2-CN from bovine and ovine milk than from caprine milk. The cytokine IL-10 did not differ among milking species. The highest levels of ROS/RNS were found after incubation of PBMC with the β-CN fraction in bovine milk. Catalase activity was higher in PBMC cultured with β-CN isolated from bovine and caprine milk and with αS1-CN from ovine milk.

Size Separation Techniques for the Characterisation of Cross-Linked Casein: A Review of Methods and Their Applications

Casein is the major protein fraction in milk, and its cross-linking has been a topic of scientific interest for many years. Enzymatic cross-linking has huge potential to modify relevant techno-functional properties of casein, whereas non-enzymatic cross-linking occurs naturally during the storage and processing of milk and dairy products. Two size separation techniques were applied for characterisation of these reactions: gel electrophoresis and size exclusion chromatography. This review summarises their separation principles and discusses the outcome of studies on cross-linked casein from the last ~20 years. Both methods, however, show limitations concerning separation range and are applied mainly under denaturing and reducing conditions. In contrast, field flow fractionation has a broad separation range and can be easily applied under native conditions. Although this method has become a powerful tool in polymer and nanoparticle analysis and was used in few studies on casein micelles, it has not yet been applied to investigate cross-linked casein. Finally, the principles and requirements for absolute molar mass determination are reviewed, which will be of increased interest in the future since suitable calibration substances for casein polymers are scarce.