Β-casein Variants Research Articles

Genotypic profiling of A1 and A2 β-casein alleles in Punganur cattle using PCR-RFLP

Milk is regarded as nature’s perfect food due to the presence of essential nutrients required for the growth and development of mammals. The consumption of milk with a specific β casein variant either A1 or A2 had a relationship with the risk of diseases has been identified. Considering the importance of β casein variants with the health significance, the present study was undertaken in Punganur cattle (N=175) by using PCR-RFLP with Dde I restriction enzyme that showed the distribution of A1A2 (121 bp, 86 bp, and 35 bp) in 5 animals, A2A2 (86 bp and 35 bp) in 170 animals with no evidence of A1A1 genotype. The genotypic frequency of the A1A2 heterozygous β-casein variant of exon7 of the CSN2 gene was 0.029, whereas, for the A2A2 homozygous β-casein variant was 0.971 and the A1A1 homozygous β-casein variant is absent in Punganur cattle. The A1 allelic frequency of exon7 of the CSN2 gene was 0.014, whereas, the A2 gene frequency is 0.986 in Punganur cattle. The study indicates that there is a minimum genetic variability in the tested population of the Punganur cattle breed suggesting less or lower level of crossbreeding activities with exotic animals.

Effects of A1 Milk, A2 Milk and the Opioid-like Peptide β-Casomorphin-7 on the Proliferation of Human Peripheral Blood Mononuclear Cells.

Special attention is given to cow's milk and its variants, with ongoing discussions about health-related impacts primarily focusing on the A1 variant in contrast to the A2 variant. The difference between these variants lies in a single amino acid alteration at position 67 of β-casein. This alteration is presumed to make the A1 variant more susceptible to enzymatic breakdown during milk digestion, leading to an increased release of the peptide β-casomorphin-7 (BCM-7). BCM-7 is hypothesized to interact with µ-opioid receptors on immune cells in humans. Although BCM-7 has demonstrated both immunosuppressive and inflammatory effects, its direct impact on the immune system remains unclear. Thus, we examined the influence of A1 and A2 milk on Concanavalin A (ConA)-stimulated human peripheral blood mononuclear cells (PBMCs), as well as the effect of experimentally digested A1 and A2 milk, containing different amounts of free BCM-7 from β-casein cleavage. Additionally, we evaluated the effects of pure BCM-7 on the proliferation of ConA-stimulated PBMCs and purified CD4+ T cells. Milk fundamentally inhibited PBMC proliferation, independent of the β-casein variant. In contrast, experimentally digested milk of both variants and pure BCM-7 showed no influence on the proliferation of PBMCs or isolated CD4+ T cells. Our results indicate that milk exerts an anti-inflammatory effect on PBMCs, regardless of the A1 or A2 β-casein variant, which is nullified after in vitro digestion. Consequently, we deem BCM-7 unsuitable as a biomarker for food-induced inflammation.

BCM-7: Opioid-like Peptide with Potential Role in Disease Mechanisms.

Bovine milk is an essential supplement due to its rich energy- and nutrient-rich qualities. Caseins constitute the vast majority of the proteins in milk. Among these, β-casein comprises around 37% of all caseins, and it is an important type of casein with several different variants. The A1 and A2 variants of β-casein are the most researched genotypes due to the changes in their composition. It is accepted that the A2 variant is ancestral, while a point mutation in the 67th amino acid created the A1 variant. The digestion derived of both A1 and A2 milk is BCM-7. Digestion of A2 milk in the human intestine also forms BCM-9 peptide molecule. The opioid-like characteristics of BCM-7 are highlighted for their potential triggering effect on several diseases. Most research has been focused on gastrointestinal-related diseases; however other metabolic and nervous system-based diseases are also potentially triggered. By manipulating the mechanisms of these diseases, BCM-7 can induce certain situations, such as conformational changes, reduction in protein activity, and the creation of undesired activity in the biological system. Furthermore, the genotype of casein can also play a role in bone health, such as altering fracture rates, and calcium contents can change the characteristics of dietary products. The context between opioid molecules and BCM-7 points to a potential triggering mechanism for the central nervous system and other metabolic diseases discussed.

High-resolution mass spectrometer-based identification of β-Casein variant (A2/A1) in the milk of Indian Holstein Friesian crossed cows

Foodomics tools are ameliorating the compositional and quality analysis part of various domains of food products. The use of proteomics in the polymorphic product investigation of beta-casein (β-Cn) is intriguing because of the fast and reliable results from the accurate mass spectrometry. This study delineates the LC-MS/MS-based sequential analysis of gel fractionated β-Cn of eighteen Holstein Friesian (HF) crossed cow milk for the identification of A2 milk. The probe of genetically induced polymorphism of β-Cn milk protein in the crossed animals is essential for the quality concern and fulfilling the demand of the consumers. Several in-vivo studies have shown that proteolytic digestion of the A1 milk secreted an opioid, i.e., β-casomorphin-7 that can cause many types of non-communicable diseases. SDS-PAGE was done on 15% resolving gel and an orbitrap mass spectrometer equipped with UniProt database search was used for the detection of the genetic variants in the HF crossed cows have shown ten animals are lactating milk with A2 β-Cn (Pro67), whereas only eight animals have A1 β-Cn (His67). The high number of HF-crossed cows are producing A2 milk. Among the molecular biology methods, top-down proteomics is the most efficient, accurate, and sustainable technique of milk protein genetic variant analysis.

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.

Novel details on the dissociation of casein micelle suspensions as a function of pH and temperature

Membrane filtration is a widespread process for fractionation and recombination of milk components. Although the dissociation of micellar caseins has been studied in detail in skim milk, it is important to better understand the dissociation dynamics occurring between the colloidal and noncolloidal fractions in systems of modified composition. This research aimed at understanding the dissociation of casein proteins in micellar fractions depleted of whey proteins. Casein micelle dispersions were tested at neutral pH and pH 6 (using glucono-δ-lactone as acidulant), after incubation at 4°C or 22°C, and compared with skim milk. The ionic composition of the serum phase was measured using inductively coupled plasma-mass spectrometry, and the protein distribution analyzed using reversed phase-HPLC coupled with mass spectrometry. When incubated at 22°C, there were no differences in casein micelle dissociation between skim milk and whey protein-depleted micelles (∼2.6% dissociated casein). No additional dissociation occurred by lowering the pH from 6.8 to 6 at 22°C, albeit there were more soluble ions at low pH (71% Ca and 65% P). At 4°C, there was an increased amount of β-casein found in the serum phase (23-33% of total β-casein). In addition, there was an uneven dissociation behavior of the various genetic β-casein variants, whereof A2 was more readily released with cooling. In skim milk, approximately 22%, 18%, and 14% of κ-, αS2, and αS1-caseins, respectively, were dissociated from the micellar phase upon cooling and acidification to pH 6.0. This was in contrast to whey protein-depleted casein suspensions, in which only 6%, 5%, and 3% of κ-, αS2, and αS1-caseins, respectively, had dissociated. The results suggested that the whey proteins in the serum phase play a role in the equilibrium between colloidal and soluble caseins in milk. This is of great relevance in processes such as cold membrane fractionation, where more attention should be given to the protein composition in the serum phase, especially when concentration is combined with fractionation of the serum proteins.

Influence of β-casein genotype on physicochemical properties and functionality of bovine milk

Several studies have been focused on the effect of milk protein genetic variants on milk physicochemical properties and functionality in recent years. β-casein, an important protein related to milk processibility, has been reported to have 2 main genetic variants A1 and A2, for which cows may be homozygous or heterozygous. In this study, several physicochemical properties of milk with β-casein variants A1A1, A1A2, and A2A2 from 3 collection occasions were analyzed. Higher manganese content and lower pH were found to be associated with the A1A1 variant compared with the other 2 genotypes. Better rennet and acid coagulation were found in A1A1 milk compared with A1A2 and A2A2 milk (although P > 0.05), whereas A2A2 milk was more stable to creaming compared with the other 2 genotypes, which may be linked to its smaller fat globule size. Thus, milk from cows with A1A1 genotype could be preferable for cheese making, while that with A2A2 variant can be used in formulations requiring good stability against creaming, and for example, yogurt making, where the softer yogurt texture may be easier to digest.

SIGNIFICANCE OF GENETIC CASEIN POLYMORPHISM IN ANIMAL HUSBANDRY

: Milk proteins are a rich source of peptides which are bioactive. Beta-casomorphin-7 ( BCM-7) is produced by successive proteolytic ingestion of beta- casein variants A1 and B, while not observed in A2 variant. The large amount of proteins in bovine’s milk are caseins, which consist of four categories ( αs1, αs2, β, and κ) encrypted by corresponding genes ( CSN1S1, CSN1S2, CSN2, and CSN3, respectively). Ingestion of A1 beta-casein showed the way for the emergence of beta-casomorphin-7 (BCM-7), a peptide that has been proposed to be a major cause of various human hazards.The relative profuseness of proline, methionine, and α-lactose were the highest in A2 variant of β-casein, while glycine, citric acid, choline, and cAMP (cyclic Adenosine Monophosphate) exhibited the highest abundance in A1 variant. These results thus come up with novel insights into the effect of casein variants, further expediting research into the biogenesis of milk ingredients and the prospective physiological activity of milk correlated with variants of β-casein.

Effects of Feeding A1 and A2 Cow Milk-based Diet on Hematological Parameters in Diabetic Mice Model

Background: Milk is essential part of diet across the globe and is a rich source of protein and calcium. Major protein component of milk is casein with beta-casein (β-casein) as the second most prevalent protein in cow milk. β-casein has 15 different genetic variants and of these A1 and A2 have gained research focus. All livestock as well as well human have proline at amino acid position 67 of β-casein, which is referred as A2 variant, but in cattle breeds, other genetic variant called A1 with histidine at amino acid position 67 is also present. This A1 type variant of â-casein or A1 type milk has been implicated as a potential etiological factor in several pathologies. Methods: The objective of the present study was to evaluate the A1and A2 β-casein variants of cow milk as factors affecting different hematological parameters and other parameters like glucose and insulin in streptozotocin (STZ)-induced diabetic C57/BL6 mice after the induction of diabetes. Diabetes was induced by injecting STZ intraperitoneally at dose of 45mg per kg of body weight for consecutive 5 days. Milk powder prepared from milk with A1A1 and A2A2 genotypes was used for feeding for three months. Result: After 3 months of feeding trial, it was observed that diabetic mice fed with A1A1 milk (STZ+A1A1) exhibited significantly elevated levels of glucose and insulin. Similarly, the levels of white blood cells, lymphocytes and neutrophils showed significant changes in STZ+A1A1 group compared to control and STZ+A2A2 group indicating the probable association A1A1 milk with inflammatory reaction. However, no significant changes were observed in parameters like red blood cells, hemoglobin, hematocrit or mean cell volume. In the mice group fed with A2A2 milk powder-based diet, no significant change was observed in the observed parameters except lymphocyte percentage which was lower compared to control group. In summary, our results show that A1 form of cow milk might have a proinflammatory effect.

Modulatory Effects of A1 Milk, A2 Milk, Soy, and Egg Proteins on Gut Microbiota and Fermentation

Milk can be divided into A1 and A2 types according to β-casein variants, and there is a debate about whether A1 milk consumption exacerbates gut environments. This study examined the cecum microbiota and fermentation in mice fed A1 casein, A2 casein, mixed casein (commercial casein), soy protein isolate, and egg white. The cecum acetic acid concentration was higher, and the relative abundances of Muribaculaceae and Desulfovibrionaceae were greater in mice fed A1 versus A2 casein. The other parameters of cecum fermentation and microbiota composition were similar among the mice fed A1, A2, and mixed caseins. The differences were more distinctive among the three caseins, soy, and egg feedings. Chao 1 and Shannon indices of the cecum microbiota were lowered in egg white-fed mice, and the microbiota of mice fed milk, soy, and egg proteins were separately grouped by principal coordinate analysis. Mice fed the three caseins were characterized by a high abundance of Lactobacillaceae and Clostridiaceae, those fed soy were characterized by Corynebacteriaceae, Muribaculaceae, and Ruminococcaceae, and those fed egg white were characterized by Eggerthellaceae, Rikenellaceae, and Erysipelatoclostridiaceae. Thus, although several differences can arise between A1 and A2 caseins in terms of their modulatory effects on gut environments, the differences between milk, soy, and egg proteins can be more distinctive and are worth further consideration.

Modification of a previously patented method to unequivocally score A2-like and A1-like bovine β-casein variants

A growing interest in the production and commercialization of A2 cow's milk has been observed in many countries in the last few years due to the beneficial properties for human health attributed to A2 β-casein variant. Methods of varying complexity and different equipment requirements have been proposed for the determination of the β-casein genotype of individual cows. We proposed herein a modification of a previously patented method based on an amplification-created restriction site PCR followed by restriction fragment length polymorphism analysis. This method allows to identify and differentiate A2-like from A1-like β-casein variants, after differential endonuclease cleavage flanking the nucleotide that determines the amino acid at position 67 of β-casein. The advantages of this method are that it:• enables to unequivocally score A2-like as well as A1-like β-casein variants,• can be performed at low cost in simply equipped molecular biology laboratories, and• can be scaled up to analyze hundreds of samples per day.For these reasons, and based on the results obtained from the analysis carried out in this work, it showed to be a reliable method for the screening of herds to selective breeding of homozygous cows and bulls for A2 or A2-like alleles.

Bovine beta casein polymorphism and environmental sustainability of cheese production: The case of Grana Padano PDO and mozzarella cheese

Several genetic variants of β-casein have been identified but A1 and A2 are the most common. Bovine β-casein variants may play an important role on cheese yield and quality, besides milk production and composition, and, thereby, affect environmental sustainability of cheese production processes. The aim of the study was to investigate the possible effect on environmental sustainability of cheese production, related to bovine β-casein polymorphism. A Life Cycle Assessment (LCA) was performed, considering Grana Padano PDO and mozzarella cheese, made with either A1A1, A1A2 or A2A2 β-casein milk and using economic and dry matter allocation methods for cheese and co-products. Additionally to characterization, normalization and weighting (endpoints method), were also performed. Results on the environmental impact of 1 kg of packaged cheese showed that, among the β-casein genetic variants, A1A1 seemed to be the most impactful, only due to the lower individual daily milk production of cows belonging to A1A1 group, compared to the cows belonging to A1A2 and A2A2 groups, i.e. 29.6, 37.1 and 34.6 kg of fat and protein corrected milk (FPCM) day cow −1 , respectively. Allocation method strongly affected the impacts per kg of cheese product and, consequently, of co-products. The normalization allowed to understand the relative importance of different impact categories and the result obtained indicated that the notable impact categories of the cheese industry were natural land transformation, aquatic eutrophication and terrestrial acidification. Results of the weighting highlighted that greater damage was given to the ecosystem quality, followed by human health and, finally, resource scarcity. Overall, biggest differences were detected for the two cheeses, rather than for the β-casein genetic variants and the differences in environmental sustainability of cheese made with A1A1, A1A2 and A2A2 milk were mainly due to the different cow milk production, rather than cheese yield. Therefore considering only the technological properties useful for cheese making the selection of milk with A2A2 β-casein may be not so convenient. Normalization and weighting results allow to identify the most impactful categories and so can help decision-makers to determine where to prioritize efforts aimed at reducing cheese environmental impact. • Effect of β-casein polymorphism on environmental impact of cheese was investigated. • LCA of Grana Padano PDO and mozzarella made with different β-casein milk was performed. • A1A1 cows produced slightly lower amounts of milk then A1A2 and A2A2 cows. • Natural land transformation was the most notably affected of the studied categories. • The greatest damage related to cheese production was given to the ecosystem quality.

Frequency of β-Casein Gene Polymorphisms in Jersey Cows in Western Japan.

Simple SummaryReports presenting survey results for β-casein gene polymorphisms have increased, but none have been about Jersey cows in Asia. This study examined the CSN2 gene variants for 590 Jersey cows in Okayama Prefecture, located in the western region of Japan. Blood samples were collected at eight farms, and nucleotide substitutions were determined by sequencing exon 7 regions of chromosome 6 of the CSN2 gene. Blood biochemical analyses were also performed to clarify if A1A1, A1A2, and A2A2 cows differ in their metabolic profiles. The frequency of the A2 allele found in this study was numerically higher than those reported for Holsteins, crossbreeds, and Mexican and Danish Jerseys. The β-casein genotypes did not affect the metabolism of the major nutrients.This study aimed to investigate β-casein gene polymorphisms in Jersey cows in Japan. Blood samples were collected from 590 cows from eight Jersey farms in Okayama Prefecture, western Japan. Sequence analysis of exon 7 regions in chromosome 6 of the CSN2 gene revealed the genotype and allele frequencies of the β-casein variants. Considering that variant B belongs to the A1 group and variant I to the A2 group, plasma metabolite concentrations were compared among the A1A1, A1A2, and A2A2 group-based genotypes. The most frequent genotype was A2A2 (0.558), followed by A2B (0.190) and A2I (0.103). No variants of A3, F, G, H1, or H2 were found. The frequencies of group-based genotypes were A1A1 (0.032), A1A2 (0.303), and A2A2 (0.665). Although farm-to-farm differences were observed in the plasma concentrations of urea nitrogen, calcium, and phosphorus, no differences were found between the A1A1, A1A2, and A2A2 group-based genotypes; hence, the β-casein genotypes did not affect the metabolism of major nutrients. Owing to the high frequency of the A2 variant, Jersey cows can be considered an attractive breed for marker-assisted selection to create A2A2 herds.

Effects of A1 and A2 variants of β-casein on human health—is β-casomorphin-7 really a harmful peptide in cow milk?

Effects of A1 and A2 variants of β-casein on human health—is β-casomorphin-7 really a harmful peptide in cow milk?

A2 Milk and BCM-7 Peptide as Emerging Parameters of Milk Quality.

Beta-casein makes up about 30% of the total protein contained in milk and can be present in cows' milk in two distinct forms (A1 or A2) or as a combination of the two. The only difference between these two variants of β-casein (β-CN) is a single amino acid substitution. This results in a different behavior of the protein upon enzymatic cleavage, following human consumption or due to microbial action. In most of the commercially available milk containing A1 or A1/A2 β-CN variants, the β-casomorphin-7 peptide (BCM-7) is released upon digestion and during cheese manufacturing/ripening, while this does not happen with A2 milk. BCM-7 is a known μ-opioid receptor agonist that may influence the gastro-intestinal physiology directly and may also exert effects elsewhere in the body, such as on the cardiovascular, neurological and endocrine systems. The present article is aimed at a revision of prior review papers on the topic, with a focus on the impact of ingestion of A1 β-CN milk and A2 β-CN milk on any health-related outcomes and on the impact of A1 or A2 β-CN variant on technological properties of cows' milk. When systematic reviews were considered, it was possible to conclude that A2 β-CN exerts beneficial effects at the gastrointestinal level compared with A1 β-CN, but that there is no evidence of A1 β-CN having negative effects on human health. Physicochemical differences among cows' milk containing either β-CN A2 or β-CN A1 and their effects on technological properties are discussed.

Marker-assisted selection of dairy cows for β-casein gene A2 variant

Many studies highlighted potential associations of β-casein A1 with specific human diseases and a minor digestibility of milk, due to the bioactive peptide β-casomorphin 7 (BCM-7) release during digestion. Conversely, the ancestral β-casein A2 variant seems to be a favorable trait because it is not associated with BMC-7 release. The aim of this work was to evaluate frequencies of β-casein variants in offspring of previously genotyped cows inseminated with A2 homozygous semen. The frequency of the A2/A2 animals has almost doubled from 37 to 69%. These are encouraging results with the perspective of reaching the goal of producing A2 milk.

Genotyping of HF Crossbred Breeding Bulls for A1/A2 Variant of β-Casein Gene

Background: The variants of β-casein (CSN 2) gene viz. A1 and A2 are supposed to be related with human health. Hence, to know the status of breeding bulls used for AI services were screened for A1 and A2 variants. Total 47 blood samples of crossbred breeding bulls from Frozen Semen Laboratory, Pune and Aurangabad were used for present work. Methods: The extraction of genomic DNA by phenol-chloroform method were carried out from the blood samples of 47 breeding bulls. Further, DNA based PCR-RFLP method was used to identify the A1, A2 variants. Pure DNA fragments of b-casein (CSN 2) gene were amplified by PCR using suitable pairs of primer to amplify 121 bp and 244 bp products. The primer used to amplify 244 bp was allele specific. The 121 bp fragment then subjected to restriction enzyme digestion. The restriction enzyme digestions resulted into two types of band pattern. The 244 bp fragment was used for validation of A1 and A2 allele of 121 bp. Result: Two types of genotypes i.e. A1A2 and A2A2 were revealed in the experimental population of HF crossbred breeding bulls. The genotypic frequency of A1A2 and A2A2 was estimated 65 and 35 per cent, respectively. The present frequency status of A1 and A2 allele in the breeding bulls may be suggested for planning future breeding strategy in dairy cattle.

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

β-Casein variants differently affect bulk milk mineral content, protein composition, and technological traits

Detailed milk protein composition has strong influence on milk quality. This study quantitatively investigated the effect of β-casein variants A1, A2 and B on composition, and technological traits of bulk milk. A total of 171 commercial herds located in northern and central Italy were visited, and 299 bulk milk samples were collected. Traditional milk quality traits, major minerals, milk coagulation properties were assessed. Detailed milk protein composition and β-casein genetic variants were determined by reversed-phase HPLC. Effects of β-casein variants were estimated by fitting a mixed model with variants concentration as a covariate. Results demonstrated that greater concentration of β-casein B is linked with greater protein, casein, and fat content. Likewise, greater concentration of variants A1 and B of β-casein had a significant positive effect on rennet coagulation time and curd firmness, and was linked to a greater amount of Ca and P content than was the A2 variant.

Conformational and physicochemical characteristics of bovine skim milk obtained from cows with different genetic variants of β-casein

This study highlights differences in conformational and physicochemical characteristics of bovine skim milk and micellar casein from cows of different β-casein phenotypes. These genetic variants have been one of the predominant topics among dairy researchers due to their differences in β-casein structure, and thus their potential effects on dairy processing and human health. For characterising differences in milk protein structure, Fourier Transform Infrared (FTIR) and Nuclear Magnetic Resonance (1H NMR) spectroscopies combined with chemometric analysis were used. Additionally, physiochemical properties such as mineral content, particle size, and electrostatic charge in skim milk and micellar casein samples were analysed at 4 and 20 ᵒC. Results showed variation in the secondary structure of all three genetic variants independent of temperature. Moreover, the main differences involved a higher level of β-turn and α-helical structures in A1/A1 β-casein milk, while intermolecular β-sheets were more numerous in A1/A2 β-casein milk, whereas random or polyproline II (PPII) structures were more common in A2/A2 β-casein milk. Temperature slightly affected these differences, which was due to the dissociation of β-casein from the micelle at low temperature. In addition, A2/A2 β-casein milk and its micellar casein had a larger average particle size, which resulted in a lower negative ζ-potential. The A2/A2 β-casein samples contained greater amounts of phosphorus and less calcium compared to the other genetic variants of milk and their micellar caseins. The results also indicated that a combination of FTIR and 1H NMR spectroscopies could be used to establish conformational differences in milk and micellar caseins of different genetic variants.