Β-casein Variants Research Articles

The Global Scenario of A1, A2 β-Casein Variant in Cattle and its Impact on Human Health

Beta casein (β-casein) has gained considerable interest among scientists and farmers as it provides promising factors to milk that enhance its quality and concentration. It is believed that the A1 β-casein variant shares an association with numerous human diseases. Since there are numerous hypotheses concerning the link between variants of β-casein and human diseases, many researchers across the globe have genotyped the cattle in terms of A1, A2 β-casein variants. This paper catalogs the A1 and A2 β-casein genotypes shared by different cattle breeds across the globe. Various literature encompassing the adverse effects of β-casein variants were assessed adequately. This paper suggests that the effects of undertaking the A1 β-casein variant on human health are ambiguous. Adequate research providing stronger evidence is needed for understanding the effects imparted by the consumption of both A1 and A2 β-casein variants on human health.

“A2 milk” authentication using isoelectric focusing and different PCR techniques

Genetic variants of milk proteins have attracted great interest for decades as they are related to important issues such as the composition and technological properties of milk. More recently, an “A1/A2 hypothesis” was developed saying that β-casein variant A1 may be a dietary risk factor for cardiovascular diseases, type 1 diabetes, sudden infant death syndrome and neurological disorders due to the release of β-casomorphin-7, whereas no evidence for such adverse effects was assumed for β-casein A2. Thus, the aim of this study was to adapt and establish analytical methods for the identification of genetic variants of β-casein using isoelectric focusing of milk proteins as well as appropriate PCR techniques. Allele-specific polymerase chain reaction (AS-PCR) proved to be a reliable method for differentiating most common β-casein variants (A1, A2, B, C), amplification-created restriction site (ACRS)-PCR using three different restriction enzymes allowed also the detection of variant A3, and the restriction fragment length polymorphism (RFLP)-PCR method enabled the reliable discrimination between A2 (homozygote/heterozygote) and non-A2 animals. Since traces of β-casein A1 were also found in commercial “A2 milk” in Austria, the authentication of such expensive dairy products is urgently recommended, both by genotyping of all dairy cows at farm level (to confirm that all cows are homozygous β-casein A2A2) and by screening commercial products on the market (to confirm the absence of β-casein variants A1, B, and C in dairy products labelled “A2 milk”) to protect consumers from this unexpected fraud.

A2 Bovine Milk and Caprine Milk as a Means of Remedy for Milk Protein Allergy

A new type of cow’s milk, called A2 milk, has appeared in the dairy aisles of supermarkets in recent years. Cows’ milk generally contains two major types of beta-casein as A1 and A2 types, although there are 13 genetic variants of β-casein: A1, A2, A3, A4, B, C, D, E, F, H1, H2, I and G. Studies have shown that A1 β-casein may be harmful, and A2 β-casein is a safer choice for human health especially in infant nutrition and health. The A2 cow milk is reportedly easier to digest and better absorb than A1 or other types of milk. The structure of A2 cow’s milk protein is more comparable to human breast milk, as well as milk from goats, sheep and buffalo. Digestion of A1 type milk produces a peptide called β-casomorphin-7 (BCM-7), which is implicated with adverse gastrointestinal effects on milk consumption. In addition, bovine milk contains predominantly αs1-casein and low levels or even absent in αs2-casein, whereby caprine milk has been recommended as an ideal substitute for patients suffering from allergies against cow milk protein or other food sources. Since goat milk contains relatively low levels of αs1-casein or negligible its content, and αs2-casein levels are high in the milk of most dairy goat breeds, it is logical to assume that children with a high milk sensitivity to αs1-casein should tolerate goat milk well. Cow milk protein allergy (CMPA) is considered a common milk digestive and metabolic disorder or allergic disease with various levels of prevalence from 2.5% in children during the first 3 years of life to 12–30% in infants less than 3 months old, and it can go up to even as high as 20% in some countries. CMPA is an IgE-mediated allergy where the body starts to produce IgE antibodies against certain protein (allergens) such as A1 milk and αs1-casein in bovine milk. Studies have shown that ingestion of β-casein A1 milk can cause ischemic heart disease, type-1 diabetes, arteriosclerosis, sudden infant death syndrome, autism, schizophrenia, etc. The knowledge of bovine A2 milk and caprine αs2-casein has been utilized to rescue CMPA patients and other potential disease problems. This knowledge has been genetically applied to milk production in cows or goats or even whole herds of the two species. This practice has happened in California and Ohio, as well as in New Zealand, where this A2 cow milk has been now advanced commercially. In the USA, there have been even promotions of bulls, whose daughters have been tested homozygous for the A2 β-casein protein.

β-casein Variants and Anti-oxidant Profiles of Milk of Siquijor Native Cattle (Bos taurus indicus L.) as Compared to those of Holstein Friesian x Sahiwal Cattle

The study aims to isolate, characterize, and evaluate the antioxidant activity of A1 and A2 β-casein (β-CN) variants from milk of Siquijor native cattle (SN) and compare it to that of Holstein Friesian x Sahiwal (HF). Four milk samples from SN and three milk samples from HF, collected at 60-90 days during the first and second parities, were used in this study. Caseins were isolated from the milk samples by isoelectric precipitation at pH 4.5, urea denaturation, and SDS-PAGE. The fractions were quantified by Bradford assay. Antioxidant activity of the fractions was determined by DPPH scavenging assay. All the samples were analyzed using one-way ANOVA to determine the statistical difference. The concentrations of β-CN variants isolated from the milk of Siquijor native cattle and the milk of Holstein Friesian x Sahiwal were not significantly different (p>0.05). All of the casein samples exhibited DPPH scavenging activity with A2 β-CN exhibiting significantly higher scavenging activity (p<0.05). SN1 A2 β-CN exhibited the highest DPPH scavenging activity at 5.298% ± 0.17 among all of the samples. These results indicate that A2 β-CN may play a vital role in maintaining antioxidant homeostasis in the human body when the milk is consumed. These results also indicate the significance of A2 β-CN in extending the shelf-life of milk and other dairy products. In conclusion, this study successfully fractionated and characterized both A1 and A2 β-CN variants in the milk of Siquijor native cattle and Holstein Friesian x Sahiwal, with A2 β-CN having higher antioxidant activity compared to A1 β-CN.

CRITICAL REVIEW ON GODUGDHA (COW MILK) HEALTH BENEFITS AND HAZARDS

Milk is touted as complete food. Cow milk is said to be one of the most commonly consumed amongst the many kinds available. Human beings are the only species that drink the milk of another species, specially cow’s milk. Besides humans no species drinks milk beyond their natural age of weaning or drinks the milk of another species. Some Constituents of Cow milk may be useful, and some may be harmful to human beings. Depending upon the variants of β-casein (30% of cows’ milk-proteins) found genetically, the Milk is divided into A1 and A2 type. Cow milk is deficient in essential fatty acids as compared to human milk. A1 and A2 milk variants differ by a single amino acid. A1 type of milk has serious health hazards on hu-mans. In order to fulfill the increasing demand and make profitable business, today the farmers are exces-sively using the Chemical drugs, antibiotics, forced feeding plans, Hormones and specialized breeding plan to increase the milk production. All these additives are having serious health adverse effects on both ani-mals and consumers of Godugdha (Cow milk). The present review article is compiled from many articles and research papers collected from different websites along with related materials from ancient Ayurvedic classics.

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.

A1- and A2-Milk and Their Effect on Human Health

Milk proteins are a heterogeneous group of polymeric compounds that have a wide range of different molecular structures and properties. They occur as caseins, whey proteins, enzymes, minor proteins and nitrogen compounds. Caseins constitute about 80% of the total proteins of cow’s milk. β-casein is an important part of the caseins, which makes up about 37% of the total caseins. Within β-casein, there are a number of variants which are genetically determined. However, there are thirteen genetic variants of β-casein found in cow´s milk. A1 and A2 are the most common variants, which are called A1 β-casein (A1-milk) and A2 β-casein (A2-milk). The only difference between A1- and A2-milk is a difference in the 67th amino acid in the chain. At this position, A2-milk has a proline amino acid, while A1-milk has histidine amino acid. Several studies have reported that cow’s milk with a dominant or singular A2-milk may be healthier than A1-milk. These studies are based on digestion of A1-milk which lead to release β-casomorphin-7 (BCM-7). Subsequently increase inflammation, Type-1-diabetes, heart disease, autism, gastrointestinal discomfort and other disease in the consumer. For this reason, there is a growing global interest in A2-milk. In conclusion, the effects of A1-milk compared to A2-milk on human health show mixed results. On the basis of the available results, we cannot conclusively assess the health effects of A1-milk and A2-milk. Therefore, further investigations are needed.

Changes in Metabolites from Bovine Milk with β-Casein Variants Revealed by Metabolomics.

β-casein is a primary protein in milk, and its variants have been associated with changes in the protein content of bovine milk. However, there has been little research focused on the effects of β-casein variants on milk metabolites. In the present study, dairy cows producing milk with β-casein variant A1/A1 (A1), A2/A2 (A2), and their heterozygote A1/A2 (A12) were screened by a high-resolution melting method. Individual milk samples were then collected from each of the cows, and the milk metabolites were separated and analyzed using nuclear magnetic resonance spectroscopy- and liquid-chromatography mass spectrometry-based metabolomics techniques. Differences in metabolites among the variant groups were evaluated by multivariate statistical analysis. The relative abundances of methionine, proline, and α-lactose were the highest in β-casein variant A2 milk, whereas choline, glycine, citric acid, and cyclic adenosine monophosphate (cAMP) showed the highest abundances in variant A1 milk. Metabolic pathways analysis indicated that the differential metabolites between variants A1 and A2 were involved in pantothenate and coenzyme A biosynthesis, butanoate metabolism, and valine, leucine, and isoleucine biosynthesis. Our results reveal the differences in milk metabolites among the β-casein variants A1, A2, and the heterozygote. These findings, thus, provide novel insights into the effects of β-casein variants on milk metabolites, facilitating further research into the mechanism of the biosynthesis of milk components in the mammary gland and the potential physiological function of milk associated with β-casein variants.

Frequencies Evaluation of β-Casein Gene Polymorphisms in Dairy Cows Reared in Central Italy.

Simple SummaryBovine milk contains several β-casein variants, with the A1 and A2 variants occurring most frequently. The presence of some variants, such as A1, B, and C, is considered a risk factor for disease in humans who consume milk. These variants are probably involved in intolerance to milk and some human diseases due to the production of a bioactive peptide with opioid activity during digestion, β-casomorphin 7 (BCM-7). In contrast, the A2 variant is not involved in pathogenetic mechanisms; thus, its presence in milk is a desirable feature. The difference between the A1 and A2 variants is a mutation at position 67 of the β-casein gene (CSN2), which causes an amino acid to change from histidine (in the A1, B, and C variants) to proline (in the A2 variant). To select dairy cows on the basis of the presence of the β-casein variant A2, allele frequencies of CSN2 variants were evaluated in Italian dairy cows reared in central Italy. The results of this study may help with the selection of animals with the β-casein gene variant A2 to produce a more digestible milk that only contains the β-casein variant A2.The majority of proteins in cow’s milk are caseins, which occur in four groups (α-s1, α-s2, β, and k) encoded by different genes (CSN1S1, CSN1S2, CSN2, and CSN3, respectively). In this study, we focused on the β-casein allele variants A1 and A2 due to their influence on milk’s technological characteristics and human health. Digestion of the β-casein variant A1 leads to the formation of β-casomorphin 7 (BCM-7), a bioactive peptide that has been suggested to be a possible cause of various human diseases and associated with low milk digestibility. The potential negative role of the β-casein variant A1 in human health has stimulated the planning of cattle breeding programs based on genetic selection to increase the frequency of the A2 variant, which is associated with increased milk digestibility. The aim of this work was to evaluate the frequencies of the different β-casein variants in Italian Holstein Friesian dairy cows from cattle farms located in central Italy to select a population of A2 homozygous animals. β-casein genotypes were identified by evaluating the presence of single nucleotide polymorphisms (SNPs) of the CSN2 gene using PCR and sequencing analysis. The frequency of the desirable β-casein variant A2 in the studied bovine population was 0.61. The frequency of the undesirable A1 variant in the studied bovine population was 0.30. The frequency of the A2 allele was higher than expected for the breed; therefore, genetic selection for the A2 variant in these animals could be achieved in a fairly short time using A2 homozygous bulls.

Comparison of the impact of bovine milk β-casein variants on digestive comfort in females self-reporting dairy intolerance: a randomized controlled trial

Lactose malabsorption (LM) is a major cause of digestive discomfort from dairy products. Recently, a role for bovine β-casein A1 has been proposed. We examined whether there are distinct symptoms of digestive discomfort due to either lactose or differing bovine β-casein types. Women (n=40; age: 25.2 ± 0.5 y) with self-reported varying dairy tolerance underwent a 50-g lactose challenge. Based on postchallenge LM and digestive discomfort, participants were classified as either lactose intolerant (LI; n=10, self-reported intolerant, diagnosed lactose intolerant), nonlactose dairy intolerant (NLDI; n=20, self-reported intolerant, diagnosed lactose tolerant), or dairy tolerant (DT; n=10, self-reported tolerant, diagnosed lactose tolerant). In a double-blinded randomized sequence, participants consumed 750 mL conventional milk (CON; containing A1 and A2 β-casein and lactose), a2 Milk (A2M; exclusively containing A2 β-casein with lactose), or lactose-free conventional milk (LF-CON; containing A1 and A2 β-casein without lactose). Subjective digestive symptoms and breath hydrogen (measuring LM) were recorded regularly over 3 h, and further ad hoc digestive symptoms over 12h. LI subjects experienced prolonged digestive discomfort with CON milk. A2M reduced (P<0.05) some symptoms (nausea: A2M 8 ± 3 mm compared with CON 15 ± 3mm; fecal urgency: A2M 4 ± 1 compared with CON 10 ± 3 mm), and attenuated the rise in breath hydrogen over 3 h, relative to CON milk (A2M 59 ± 23 compared with CON 98 ± 25 ppm at 150 min; P<0.01). In contrast, NLDI subjects experienced rapid-onset, transient symptoms (abdominal distension, bloating, and flatulence) without increased breath hydrogen, irrespective of milk type. In LI individuals, LM and digestive comfort with lactose-containing milks was improved with milk containing exclusively A2 β-casein. Furthermore, self-reported dairy intolerance without LM (NLDI) is characterized by early-onset digestive discomfort following milk ingestion, irrespective of lactose content or β-casein type. This trial was registered at www.anzctr.org.au as ACTRN12616001694404.

Protein fingerprinting and quantification of β-casein variants by ultra-performance liquid chromatography–high-resolution mass spectrometry

Infant formulations are constantly evolving as novel protein ingredients are added to make them more closely mimic the protein profile of human milk; however, precise analytical methods for characterizing and quantifying the major milk proteins in such formulations are currently lacking. This article describes an ultra-performance liquid chromatography-high-resolution mass spectrometry method for intact proteins that can efficiently detect, identify, and characterize the major milk proteins and their proteoforms (phosphorylation status, degree of glycation, genetic variants among others) in ingredients and final products, with an emphasis on detecting and quantifying specific genetic variants of β-casein in infant formulas. Method sensitivity allows detection of β-casein A1 in A2-based infant formulas with a limit of detection of 2% (grams of β-casein A1 per 100 g of total β-casein). Protein glycation affects signal intensity in a linear fashion, which permits proteins to be quantified from their mass spectrometry signals after correction according to their measured glycation index. The method was validated for the quantification of β-casein in infant formulas. Repeatability ranged from 2 to 3% and intermediate reproducibility from 5 to 9%. Calculated β-casein amounts ranged between 77 and 110% of the values based on formulations and published protein profiles for milk. Altogether, this method can be used for general fingerprinting as well as specific characterization and quantification of individual major milk proteins in dairy-based ingredients and products.

Comparative Evaluation of Antioxidant Potential of In-Vitro Digested Cow Milk Derived A1 and A2 β Casein Variants Using Different Proteases

This study was designed to investigate the antioxidant activity of A1 and A2 β-casein variant with time by applying different enzyme. Pepsin, Trypsin, Alcalase and combination of Pepsin-Trypsin were used for hydrolysis of A1 and A2 β-casein for the duration of 1, 2, 3 and 24 h. All antioxidant parameter including DPPH, ABTS radical-scavenging activity and reducing power assay increasing gradually with time. Enzyme Pepsin-Trypsin combination followed by Alcalsae display comparatively higher antioxidant activity. Among β-casein variant A2 showed relatively higher antioxidant potential over all the entire duration of time but the difference among the A1 and A2 variants was not significant to arrive at a substantial scientific conclusion. It can be concluded from the study that antioxidant potential of the milk depends upon factors such as duration of hydrolysis and enzyme used, during hydrolysis and not alone on the fact that whether the milk is A1 or A2 in nature.

Genetic Polymorphism of β-Casein Gene in Polish Red Cattle-Preliminary Study of A1 and A2 Frequency in Genetic Conservation Herd.

Simple SummaryCurrently, milk and dairy products are considered nutritious foods because they contain bioactive constituents. However, in some cases, milk consumption might cause health-related issues, such as allergic reactions. Among all of the proteins present in milk, caseins deserve special attention. A genetic variant called β-casein is recognized as a potential cause of some health implications. However, although milk protein characteristics have been widely covered, a significant majority of published research focus on one cattle breed: Holstein There are numerous local breeds that have not been widely analyzed to date. One such breed is a dual-purpose breed called Polish Red.Although there is growing interest in Red cow’s milk in Poland, to date there are few reports investigating the characteristics of milk components in the studied population. Particular emphasis on milk proteins is advised, since β-casein is a source of bioactive peptides named β-casomorphins. β-casomorphin 7, which originates mostly from β-casein variants A1, may be a significant risk factor in human ischemic heart disease, arteriosclerosis, type I diabetes, sudden infant death syndrome, and autism. The aim of the present study was to identify CSN2 polymorphism gene in exon 7 using the genomic sequence from GenBank (M55158), g.8101C>A, (codon 67). Blood samples were collected from 201 Polish Red cattle (24 males and 177 females). The genotype of β-casein was determined using PCR-ACRS. The frequency of β-casein A2 in Polish Red population was 0.47. β-casein A2 frequency in Polish Red bulls and in cows was 0.58 and 0.37, respectively.

Genetic association analysis reveals significant effect of β-casein A1/A2 loci on production & reproduction traits in Frieswal crossbred cows

ABSTRACT Milk β-casein has attracted extensive research interest in the dairy industry and animal breeding as a source of high-quality protein for human diet; however, adverse health effects have been ascribed to A1 β-casein variant consumption. A study was conducted to genotype the polymorphism in milk β-casein gene and examines the potential association between β-casein genotypes and milk production and reproduction traits in population of Frieswal cows. β-Casein A1/A2 genotype had significant effect (P ≤ 0.05) on several production traits including Total Milk Yield (TMY) (P ≤ 0.05), Milk Milk yield 300 days (MY300) (P ≤ 0.05), Lactation Length (LL) (P ≤ 0.01) and Peak Yield (PY) (P ≤ 0.01). β-Casein A1A1 genotype cows had significantly lower TMY, MY300, PY and DP as compared to A1A2 and A2A2 genotypes. A1A2 genotype had highest TMY, MY300, PY and DP; however, A2A2 and A1A2 genotypes were not significantly different from each other. The results pointed towards the superiority of A2 β-casein variant for several production traits in crossbreds. Further, with higher price of A2 milk, the inclusion of β-casein A1/A2 genotype in the selection of crossbreds offers higher profitability from dairy enterprise as well as provides a nutritional wellness product for consumers.

Characterisation of African elephant beta casein and its relevance to the chemistry of caseins and casein micelles

Caseins in milk exist as casein micelles, the structure of which is unclear. The combination of all four casein types, together with calcium phosphate clusters, are believed to be important for the stabilisation of casein micelle structure. In African elephant milk, only two major caseins, β- and κ-casein, have been identified, with β-casein forming the greater part, similar to human milk. African elephant β-casein was purified and identified as five isomers with different isoelectric points. A single β-casein isoform in its equal unphosphorylated and singly phosphorylated forms at Ser 9 was characterised. This phosphoprotein seems to be a major β-casein variant with an internal truncation of the peptide sequence ESVTQVNK encoded by exon 5, resulting in a variant with 200 amino acid residues. The other minor β-casein isoforms are possibly different splice variants of exons 3 and 4. This combination of proteins may provide additional insight into casein micelle structure.

Release of β-casomorphin-7 from bovine milk of different β-casein variants after ex vivo gastrointestinal digestion

The release of β-casomorphin-7 (BCM-7) in bovine milk with β-casein genetic variants A1, A2, F and I was assessed. An ex vivo gastrointestinal (GI) digestion model was applied and multiple reaction monitoring method for quantification of BCM-7 was set up using Triple-Q LC-ESI-MS specifically for the BCM-7 peptide. BCM-7 was released after digestion of milk of all variants, although with large differences in concentration between variants. Higher amounts of BCM-7 were found from milk samples of the A1 family (A1 and F) with 1.85–3.28 mg g−1 β-CN digested, whereas the A2 family (A2 and I) only released 0.01–0.06 mg g−1 β-CN digested. Compared with earlier findings on digestion of purified β-CN, this study showed larger differences in quantified BCM-7 between different milk types, potentially due to the different matrixes of the milk. However, all β-CN variants A1, A2, F and I released BCM-7 after GI digestion.

Microstructure and physicochemical properties reveal differences between high moisture buffalo and bovine Mozzarella cheeses

Mozzarella cheese is a classical dairy product but most research to date has focused on low moisture products. In this study, the microstructure and physicochemical properties of both laboratory and commercially produced high moisture buffalo Mozzarella cheeses were investigated and compared to high moisture bovine products. Buffalo and bovine Mozzarella cheeses were found to significantly differ in their microstructure, chemical composition, organic acid and proteolytic profiles but had similar hardness and meltability. The buffalo cheeses exhibited a significantly higher ratio of fat to protein and a microstructure containing larger fat patches and a less dense protein network. Liquid chromatography mass spectrometry detected the presence of only β-casein variant A2 and a single β-lactoglobulin variant in buffalo products compared to the presence of both β-casein variants A1 and A2 and β-lactoglobulin variants A and B in bovine cheese. These differences arise from the different milk composition and processing conditions. The differences in microstructure and physicochemical properties observed here offer a new approach to identify the sources of milk used in commercial cheese products.

Identification of bioactive peptides and quantification of β-casomorphin-7 from bovine β-casein A1, A2 and I after ex vivo gastrointestinal digestion

This study investigated whether different genetic variants of β-casein (β-CN) give rise to different bioactive peptides during digestion. β-CN was purified from bovine milk of genetic variants A1, A2 and I, and digested with human gastrointestinal juices in a static ex vivo model. Mass spectrometry analyses revealed that the peptide 60YPFPGPIPN68 was exclusively identified from variants containing proline at position 67. Most strikingly, the opioid peptide β-casomorphin-7, 60YPFPGPI66, was identified from both variants A1 and A2 after simulated digestion, though with concentration being somewhat higher after digestion of the variant A1, compared with variants A2 and I. The peptides 134HLPLP138 and 133LHLPLP138 were both identified after initial 5 min of duodenal digestion. In conclusion, genetic variation of β-CN may affect proteolysis during digestion; however, the release of β-casomorphin-7 (BCM7) does not seem to be linked solely to variant A1, as earlier suggested by relevant published literature on in vitro digestion.

Probing the internal and external micelle structures of differently sized casein micelles from individual cows milk by dynamic light and small-angle X-ray scattering

Casein micelles vary in size both within and between individual cows depending on the ratios of specific caseins and the level of glycosylation. In this work, milks were obtained from individual cows with known genetic variants of κ- and β-casein, and β-lactoglobulin, containing a range of native micelle diameters. κ- and β-Casein variants are known to influence rennetability of milk. The specific reason for these differences are not known, although, it is clear casein phenotype has an influence. Large and small micelle fractions were also prepared, narrowing the size distribution of micelles at both extremes. When examined by SAXS, all the milk samples were found to have identical scattering patterns in the intermediate and high q regions, implying that the internal structures of casein micelles are the same regardless of their size and genetic variation. When the micelles were exposed to EDTA above 2.7 mM, the inflexion at q ∼0.080 Å−1 in the SAXS curves were diminished indicating changes to internal protein packing due to disruption of the colloidal calcium phosphate nanoclusters within the micelles. Ethanolic buffers caused changes to the micelle diameter, attributed to changes in the micelle internal structure and the collapse of the ‘hairy layer’ at the micelle surface, evident by the appearance of the inflexion at q ∼0.035 Å−1 and changes in the SAXS curves low q. Despite the large natural variation in micelle size and protein genetic variants, the internal organisation of the micelles is not influenced by diameter or protein genetic variation.

Short communication: Inhibition of angiotensin 1-converting enzyme by peptides derived from variants of bovine β-casein upon apical exposure to a Caco-2 cell monolayer

This study investigated the consequence of genetically contingent amino acid substitutions in bovine β-casein (CN) genetic variants A1, A2, B, and I on the structure and bioactive potential of peptides following in vitro digestion. The β-CN variants were digested in vitro using pepsin and pancreatin, and a peptide profile was obtained by liquid chromatography tandem mass spectrometry, revealing among others, the β-casomorphin precursor peptides VYPFPGPIHN and VYPFPGPIPN, derived from variant A1/B and from A2/I, respectively. These 2 peptides were synthesized and assessed for angiotensin 1-converting enzyme (ACE) inhibitory capacity before and after incubation with a monolayer of Caco-2 intestinal cells. The VYPFPGPIHN was a stronger ACE inhibitor than VYPFPGPIPN, with the concentration needed to reach half-maximal inhibition (IC50) of 123 ± 14.2 μM versus 656 ± 7.6 μM. Exposure to a Caco-2 intestinal cell monolayer did not affect ACE inhibition by VYPFPGPIHN, but resulted in an almost 2-fold increase in inhibition by VYPFPGPIPN after incubation. Subsequent tandem mass spectrometric analysis identified the truncated peptide VYPFPGPIP, suggesting hydrolysis by a cell membrane associated peptidase. Thus, genetic variation in bovine β-CN results in the generation of peptides that differ in bioactivity, and are differently affected by intestinal brush border peptidases.