Milk Β-casein Research Articles

Influence of the Maillard reaction on the structure of β-casein using a simulated cooking system

Abstract Glycation during food processing can change the structural characteristics of dietary protein and endow a variety of functionalities. Milk β-casein (MC) with five different types of carbohydrates (i.e., glucose, sucrose, amylose, amylopectin, and soluble starch) in a simulated cooking system at 98 and 115 °C was investigated. The changes in ultraviolet absorption, endogenous fluorescence intensity, surface hydrophobicity, particle size, free amino group, free sulphydryl group, and circular dichroism were measured. For the glucose-added group, the lowest free amino content and most significant spectral changes among all groups were observed after heating at 115 °C for 6 hr (OH-MC-Glu). Results showed that glycation activities of five sugars were glucose > sucrose > soluble starch> amylopectin ≈ amylose. Under the simulated cooking system, the secondary structure and conformation of glycated MC changed to different degrees, which were affected by the glycation degree. Moderate glycation (0–2 hr of heating) may promote the conversion reaction of sulfhydryl-disulfide bond (SH-SS) in MC, causing cross-linking and self-aggregation of MC, and induce the particle size increase of MC. Excessive glycation (2–6 hr of heating) can further reduce the surface hydrophobicity of MC, inhibit self-aggregation of MC, and reduce the particle size of MC.

Proline-Selective Electrochemiluminescence Detecting a Single Amino Acid Variation Between A1 and A2 β-Casein Containing Milks.

The proline amino acid and prolyl residues of peptides/proteins confer unique biological and biochemical properties that motivates the development of proline-selective analysis. The study focuses on one specific class of problem, the detection of single amino acid variants involving proline, and reports a Pro-selective electrochemiluminescence (ECL) method. To develop this method, the A1-/A2- variants of milk's β-casein protein are investigated because it is a well-established example and abundant samples are readily available. Specifically, β-casein has 209 amino acids with 34 (or 35) proline residues: the A1-variant has a Pro-to-His substitution at position 67 (relative to the A2 variant). The study shows that proline's strong luminescence allows the generic discrimination of: Pro from other amino acids; an A2-oligopeptide from an A1-oligopeptide; the A2-β-casein variant from the A1-variant; and commercially-available A2 milks from A1-containing regular milks. The evidence indicates that luminescence depends on proline content and accessibility, as well as signal quenching. Compared to conventional immunoassays, the ECL method is simple, rapid, and inexpensive. Further, the ECL-method is Pro-selective (vs molecularly-selective like typical immunoassays) which should make it broadly useful for studying the role of proline in biology and especially useful for tracking the digestion of proline-rich proteins in the diet.

Determining the influence of raw milk β-casein polymorphism on the efficiency of making soft cheese

One of the ways to increase the profitability of the cheese industry is the genetic selection of dairy cows to obtain milk with excellent cheese-capacity characteristics. The object of this study is the technology of fresh soft cheeses made by the acid-rennet and thermo-acid method from the milk of cows with different β-casein genotypes (A1A1, A1A2, A2A2). The subjects of research are the physicochemical indicators of raw milk from cows with different genotypes for β-casein (A1A1, A1A2, A2A2); as well as the yield of soft cheeses. The study has established that the physicochemical parameters of milk from cows with different β-casein genotypes are typical for fresh cow's milk. The study showed that with the acid-rennet method, the composition of cheeses from A1A1 milk was 51.60 %, 21.63 %, and 23.62 % of moisture, protein, and fat, respectively. A1A2 milk cheeses contained 50.70 % moisture, 20.96 % protein, and 25.12 % fat. A2A2 milk cheeses consisted of 52.50 % moisture, 20.70 % protein, and 23.71 % fat. With the thermo-acid method, cheeses from A1A1 milk were characterized by the moisture content of 55.13 %, proteins – 23.31 %, and fat – 20.21 %. A1A2 milk cheeses contained 58.13 %, 22.62 %, and 17.98 % of moisture, protein, and fat, respectively. A2A2 milk cheeses consisted of 54.03 % moisture, 22.33 % protein, and 22.25 % fat. The calculation of the production efficiency of soft cheeses from the milk of cows with different genotypes according to β-casein with the acid-rennet method of production is 119.3 % on average, which is more compared to milk A1A2 (by 4 %) and A2A2 (by 7 %). With the thermo-acidic method, the efficiency of cheese production from A2A2 milk is 107.5 %, which is higher compared to A1A2 milk (by 9 %) and A1A1 (by 5 %). The conclusions show that changes in the β-casein genotype in raw milk can affect the yield and quality of cheese, and therefore, the profitability of production

Influence of different genotype combinations of β-lactoglobulin and β-casein in cow milk on physicochemical and sensory properties of stirred yoghurt

Influence of different genotype combinations of β-lactoglobulin and β-casein in cow milk on physicochemical and sensory properties of stirred yoghurt

Evaluation of the Toxicological Efficacy of Protein Hydrolysates From Camel Milk β-Casein Against Different Types of Cancer Cell Lines in Vitro

Evaluation of the Toxicological Efficacy of Protein Hydrolysates From Camel Milk β-Casein Against Different Types of Cancer Cell Lines in Vitro

Effect and mechanisms of thermal sterilization methods on the in vitro phenolic bioaccessibility of rose tea with milk

Rose polyphenols, key functional components in roses, require adequate bioaccessibility for their health benefits, subject to influence by food components and processing. Investigating the impact of various thermal sterilization methods on the bioaccessibility of rose polyphenols in rose tea with milk and the underlying mechanisms, our findings indicated a significant increase in bioaccessibility following treatment at 85°C/30min. Conversely, 121°C/15min treatment decreased bioaccessibility. Examining the interaction between β-casein in milk and rose polyphenols under different sterilization conditions, SEM and particle size analysis revealed binding, with fluorescence spectroscopy indicating non-covalent bonds. Binding forces followed the order 121°C>85°C>25°C. Notably, at 85°C, non-covalent binding improved polyphenol bioaccessibility, while the intensified binding at 121°C decreased it. SDS-PAGE and amino acid analysis confirmed no covalent bond. This study establishes a theoretical basis for selecting thermal sterilization temperatures for milk-flower combinations, considering polyphenol bioaccessibility.

Significance of CD10 for Mucosal Immunomodulation by β-Casomorphin-7 in Exacerbation of Ulcerative Colitis.

β-Casomorphin-7 (BCM), a breakdown product of milk β-casein, exhibits opioid activity. Opioids are known to affect the immune system, but the effects of BCM on ulcerative colitis (UC) are not clear. We examined the effects of BCM on mucosal immunity using a mouse dextran sulfate sodium-induced colitis model and an in vitro CD8+ T cell activation model. Human UC patients were examined to reveal the relationship between CD10 and mucosal immunity. Combined treatment of the colitis model with thiorphan (TOP) inhibited BCM degradation by suppressing CD10 in the intestinal mucosa, activating mouse mucosal CD8, and suppressing CD4 and Treg. In the CD8+ T cell in vitro activation assay using mouse splenocytes, BCM inhibited the oxidative phosphorylation (OXPHOS) of CD8+ T cells and induced the glycolytic pathway, promoting their activation. Conversely, in a culture system, BCM suppressed OXPHOS and decreased defensin α production in IEC6 mouse intestinal epithelial cells. In the mouse model, BCM reduced defensin α and butyrate levels in the colonic mucosa. During the active phase of human ulcerative colitis, the downward regulation of ileal CD10 expression by CpG methylation of the gene promoter was observed, resulting in increased CD8 activation and decreased defensin α and butyrate levels. BCM is a potential aggravating factor for UC and should be considered in the design of dietary therapy. In addition, decreased CD10 expression may serve as an indicator of UC activity and recurrence, but further clinical studies are needed.

A discussion on A1-free milk: Nuances and comments beyond implications to the health.

This chapter provides an overarching view of the multifaceted aspects of milk β-casein, focusing on its genetic variants A1 and A2. The work examines the current landscape of A1-free milk versus regular milk, delving into health considerations, protein detection methods, technological impacts on dairy production, non-bovine protein, and potential avenues for future research. Firstly, it discussed ongoing debates surrounding categorizing milk based on A1 and A2 β-casein variants, highlighting challenges in establishing clear regulatory standards and quality control methods. The chapter also addressed the molecular distinction between A1 and A2 variants at position 67 of the amino acid chain. This trait affects protein conformation, casein micelle properties, and enzymatic susceptibility. Variations in β-casein across animal species are acknowledged, casting doubt on non-bovine claims of "A2-like" milk due to terminology and genetic differences. Lastly, this work explores the burgeoning field of biotechnology in milk production.

E-beam irradiation of defatted liquid camel and cow milk fractions: Antiproliferative, antidiabetic and antioxidant activities

E-beam irradiation of defatted liquid camel and cow milk fractions: Antiproliferative, antidiabetic and antioxidant activities

Determination of A1 and A2 β-Casein in Milk Using Characteristic Thermolytic Peptides via Liquid Chromatography-Mass Spectrometry

β-casein, a protein in milk and dairy products, has two main variant forms termed as A1 and A2. A1 β-casein may have adverse effects on humans. The fact that there is only one amino acid variation at the 67th position between A1 and A2 β-casein makes it difficult to distinguish between them. In this study, a novel method using characteristic thermolytic peptides is developed for the determination of A1 and A2 β-casein in milk. Firstly, caseins extracted from milk samples are thermolytic digested at 60 °C without any denaturing reagents required for unfolding proteins, which simplifies the sample pretreatment procedure. The characteristic thermolytic peptides (i.e., fragments 66–76 and 59–76 for A1 and A2 β-casein, respectively) selected to specifically distinguish A1 and A2 β-casein only have eleven or eighteen amino acid moieties. Compared with tryptic characteristic peptides with a length of 49 amino acid moieties, these shorter thermolytic characteristic peptides are more suitable for LC-MS analysis. This novel method, with the advantages of high specificity, high sensitivity, and high efficiency, was successfully applied for the analysis of six milk samples collected from a local supermarket. After further investigation, it is found that this method would contribute to the development of A2 dairy products for a company and the quality inspection of A2 dairy products for a government.

Benefits of A2 Milk for Sports Nutrition, Health and Performance.

Bovine milk is one of the best pre-and pro-workout sources for athletes owing to its rich nutritional content. Even though bovine milk consumption significantly benefits athletes' health and performance, many athletes cannot consume bovine milk since they struggle with gastrointestinal problems caused after milk consumption. Especially, the consumption of regular milk, which contains A1 β-casein, is associated with a variety of diseases ranging from gastrointestinal discomfort to ischemic heart diseases. The main reason behind this is related to β-casomorphine 7 (BCM-7), which is derived from A1 β-casein during the digestion of A1 milk. A1 β-casein is formed as a result of a point mutation in the position of 67th in the amino acid sequence A2 β-casein by changing proline to histidine. Therefore, this mutated form of β-casein in regular milk cannot easily be digested by the human-associated digestion enzymes. A2 milk, which includes A2 β-casein instead of A1 β-casein, is the best substitute for regular milk with the same nutritional content. This natural form of milk positively affects the athlete's health as well as performance without causing any gastrointestinal discomfort or more serious problems which are seen in the consumption of regular milk. In this review, A2 milk and its potential health effects in comparison to diseases related to A1 milk consumption are discussed.

The biological role of casomorphins

The literature review is devoted to biologically active metabolites of casein — the results of its hydrolysis — oligopeptides casomorphins. These peptides with a chain length of 4 to 11 amino acids are derived from milk β-casein and are released during digestion, both in vivo and in vitro. Caseomorphins exhibit opioid and pharmacological activity due to binding to μ-receptors located in the central nervous system, gastrointestinal tract and some immune cells. Understanding the biological role of caseomorphins in the milk of mammals, including humans, and their effect on organs and systems, will bring specialists closer to deciphering the etiology of a whole group of diseases.

STUDY OF BETA-CASEIN GENE POLYMORPHISM AND ITS RELATIONSHIP WITH MILK COMPOSITION IN SIMMENTAL COWS

STUDY OF BETA-CASEIN GENE POLYMORPHISM AND ITS RELATIONSHIP WITH MILK COMPOSITION IN SIMMENTAL COWS

Monitoring Human Milk β-Casein Phosphorylation and O-Glycosylation Over Lactation Reveals Distinct Differences between the Proteome and Endogenous Peptidome.

Human milk is a vital biofluid containing a myriad of molecular components to ensure an infant’s best start at a healthy life. One key component of human milk is β-casein, a protein which is not only a structural constituent of casein micelles but also a source of bioactive, often antimicrobial, peptides contributing to milk’s endogenous peptidome. Importantly, post-translational modifications (PTMs) like phosphorylation and glycosylation typically affect the function of proteins and peptides; however, here our understanding of β-casein is critically limited. To uncover the scope of proteoforms and endogenous peptidoforms we utilized mass spectrometry (LC-MS/MS) to achieve in-depth longitudinal profiling of β-casein from human milk, studying two donors across 16 weeks of lactation. We not only observed changes in β-casein’s known protein and endogenous peptide phosphorylation, but also in previously unexplored O-glycosylation. This newly discovered PTM of β-casein may be important as it resides on known β-casein-derived antimicrobial peptide sequences.

Investigation of genetic polymorphism at β-casein A1/A2 loci and association analysis with production & reproduction traits in Vrindavani crossbred cows

Casein constitutes approximately 80% of the total protein in bovine milk and is regarded as a high-quality dietary protein embracing all the nine essential amino acids. However, the contested physiological effect of a bioactive peptide released upon digestion of a β-casein milk protein variant originating from a cow of a particular genetic makeup has evoked wide interest in research and industry. Present investigations were carried out to genotype the polymorphism in milk β-casein gene, delineate the seasonal, periodic, and parity variations in production and reproduction traits, and examine the genetic association between β-casein genotypes and production, and reproduction traits in Vrindavani crossbred cows. The study revealed that all three types of genotypes viz. A1A1, A2A2 and A1A2 were present in the Vrindavani crossbred population with genotypic frequencies of 12.3%, 39.6% and 48.1% respectively. The least-squares analysis revealed that the season of calving, period of calving, and parity affected several production and reproduction traits of Vrindavani cows significantly. It was found that β–Casein A1/A2 genotype had a significant effect on economic traits viz. LL (p ≤ 0.05), MY/LL (p ≤ 0.05), and Fat% (p ≤ 0.05) in Vrindavani crossbreds. The findings uncover the genetic constitution of the crossbreds for β-casein locus and emphasize its relationship with important economic traits that can aid in devising selection goals.

Analysis of A1-type and A2-type β-casein in Maiwa Yak and Pien-niu milk by HPLC-high-resolution MS and tandem MS.

In this study, a peptide-based method employing ultra high performance liquid chromatography electrostatic field orbitrap high-resolution mass spectrometry and triple quadrupole mass spectrometry was developed for quantification of A1-type and A2-type β-casein in milk from Yak, cows, and their offspring of crosses, Pien-niu. The specific peptides of A1-type and A2-type β-casein were screened and confirmed by protein software after analysis of high-resolution mass spectrometry. The multiple reaction monitoring method was established based on the qualitative results, and isotope-label peptides were used as internal standards. The linear correlation coefficients of this method were>0.99. The relative standard deviations of repeatability test were 0.2-3.6%. The recovery rate ranged from 93.3 to 114.4% with relative standard deviations<6% at three different spiking levels. The method was applied to analyze 45 milk samples from different species. The results showed that β-casein in Yak and Pien-niu milk was about 30% higher than that in cow milk. Furthermore, the β-casein in the Yak milk only contains A2-type β-casein. A1-type and A2-type β-casein coexist in most samples of Pien-niu and cow milk, a few samples contain only one type of β-casein. These results provide further understanding in nutritional value of milk from Yak and Pien-niu.

Determination of A2 β-casein and total β-casein in cow milk and milk powder by capillary zone electrophoresis

β-casein (β-CN) is one of the major casein proteins in cow milk. There are 13 different variants documented for β-CN in cow milk, among which A1 and A2 are the major variants. The separation and quantitation of A2 β-CN are imperative for A2 dairy products. A new capillary zone electrophoresis (CZE) method with UV detection at 214 nm was established for the separation and quantification of the A2 variant and total β-CN content in cow milk and milk powders. The separation of β-CN variants was achieved on bare fused silica capillaries (50 μm×30/40 cm (effective/total length)). The separation buffer was a mixture of 4 mol/L urea, 0.2% (mass fraction) hydroxypropyl methylcellulose, 140 mmol/L citric acid, and 50 mmol/L disodium hydrogen phosphate buffer (pH 2.7). The corrected peak areas and the concentrations of total β-CN and the A2 variant showed good linearity, with correlation coefficients (r2) ranging from 0.9968 to 0.9997. The intra-day precisions for A2 β-CN and total β-CN determination in four samples (two pasteurized milk samples and two milk powder samples) were in the ranges of 2.4%-4.7% and 2.6%-4.8%, respectively. The inter-day precisions for A2 β-CN and total β-CN determination in four samples were in the ranges of 4.0%-6.3% and 3.9%-6.7%, respectively. The recoveries of A2 and total β-CN ranged from 85.5% to 106.4%. With the established CZE method, the A2 β-CN variant and total β-CN protein in liquid and powder bovine milk products could be separated and accurately quantified. By calculating the A2 β-CN content in the total β-CN, the quality of A2 dairy products can be evaluated, and this in turn would aid in the protection of consumer rights.

Determination of Total and A1-Type β-Casein in Milk and Milk-Derived Ingredients by Liquid Chromatography-Mass Spectrometry Using Characteristic Tryptic Peptides.

Gastrointestinal digestion of A1-type β-casein is conducive to β-casomorphin-7 with potential adverse digestive health effects. Monitoring of A1-type β-casein concentration in milk and milk-derived ingredients used in the formulation of A2-type nutritional products with associated health claims is important from a quality standpoint. New analytical methods were developed and validated for total and A1-type β-casein in milk and milk-derived ingredients. Data on total and A1-type β-casein concentrations in milk, nonfat dry milk, and whey protein concentrate was generated. The methods are based on a bottom-up proteomic approach using tryptic marker peptides and stable isotope dilution liquid chromatography-mass spectrometry. The measurement includes all protein sequences (intact, modified, and partial) which are potential sources of β-casomorphin-7. Total β-casein was quantified using a neat calibration curve. Recovery and between-day precision RSD were 98% and 5.8%, respectively. A1-type β-casein was quantified by the method of standard additions. Between-day precision RSD was 7.2% and limit of quantitation was 0.01% in nonfat dry milk. The mass fraction of A1-type β-casein in the β-casein standard was 0.444. Samples manufactured from A2-type milk contained 0.26-5.0% A1-type β-casein relative to total β-casein. The methods described enable the monitoring of the A1-type β-casein concentration in milk and milk-derived ingredients destined for the manufacture of A2-type products with associated health claims. New methods are presented for the analysis of total and A1-type β-casein in milk and milk-derived ingredients. The mass fraction of A1-type β-casein in a commercial β-casein standard was determined to enable its use as a calibrant.

Negative regulation of αS1-casein (CSN1S1) improves β-casein content and reduces allergy potential in goat milk

Compared with other milk proteins, αS1-casein (encoded by CSN1S1) is associated with higher rates of allergies in humans. Although some studies have revealed that CSN1S1 affects a variety of cellular physiological processes such as immune response and proliferation in various cells, whether CSN1S1 regulates other milk proteins in ruminants is not known. In this study, we observed a negative Pearson correlation between the contents of αS1-casein and β-casein in goat milk. Thus, we used isolated primary goat mammary epithelial cells along with adenoviral infection or small interference RNA to alter abundance of CSN1S1 and examine its role in milk protein synthesis regulation. Overexpressing CSN1S1 through adenoviral transfection decreased β-casein mRNA (CSN2) and protein abundance, whereas interference of CSN1S1 resulted in a significant increase in β-casein abundance. CSN1S1 reduced phosphorylation level of Janus kinase 2 (JAK2) and signal transducer and activator of transcription 5a (STAT5a). The transcription factor STAT5a activates CSN2 transcription by binding with its promoter region and then promotes β-casein synthesis. Furthermore, CSN1S1 inhibited CSN2 promoter activity and β-casein synthesis by modulating the JAK2/STAT5a signaling pathway. No changes in abundance were detected for αS2-casein (CSN1S2), κ-casein (CSN3) and major whey proteins (LALBA, BLG). Overall, our results underscored a mechanism whereby CSN1S1 inhibits β-casein synthesis via inhibition of STAT5a. The data suggested that knocking down CSN1S1 not only reduced the content of αS1-casein, but also increased the abundance of major milk proteins including β-casein. Thus, the present study provides a theoretical basis for manipulating αS1-casein mRNA to improve quality of ruminant milk for human consumption.

Development of an LC-MS Method for the Identification of β-Casein Genetic Variants in Bovine Milk

Bovine β-casein is a highly polymorphic protein, with A1 and A2 representing the most frequent variants identified in Western cattle breeds. Depending on the presence of histidine or proline at position 67 of the sequence, β-casein variants can be grouped in two families: those within the A1 family (A1-like), possessing histidine, and those within the A2 family (A2-like), with proline. Upon gastrointestinal digestion, specific peptides endowed with opioid-like activity, called β-casomorphins, may be released from β-casein. Interestingly, the presence of histidine at position 67 seems to favor the release of a peptide called β–casomorphin-7, which has been proposed as a risk factor for the development of some non-communicable diseases. Based on these assumptions, the A2 milk market (i.e., a milk containing only A2 β-casein) is spreading worldwide. In the scientific community, however, the impact of A1 β-casein on human health remains a matter of debate. Aim of the present study was to develop an analytical method based on mass spectrometry to distinguish between bovine A2 milk and commercial milk, typically composed of a mixture of A1-like and A2-like β-casein. By enzymatically hydrolyzing β-casein, a specific peptide containing the critical mutation at position 67 has been identified. This peptide constituted a suitable marker to determine the presence of A1-like and/or A2-like β-casein in bovine milk. The developed method proved appropriate for the detection of β-casein in real milk samples; we estimated that contaminations of A2 milk with up to 1% commercial milk could be successfully detected.