Approximately 30% of milk protein is β-casein. We aimed to determine whether lactose maldigesters who chronically consumed two cups of A1/A2 milk (containing 75% A1 β-casein and 25% A2 β-casein) would adapt to have fewer intolerance symptoms, lower serum inflammatory markers, and/or altered glutathione levels similar to those consuming A2 milk (containing 100% A2 β-casein). A double-blinded, randomized, crossover trial was conducted. Sixteen confirmed lactose maldigesters consumed 250 mL of A1/A2 milk and A2 milk twice daily with meals for two weeks. At the end of the adaptation period on day 15, lactose maldigestion was measured after a challenge with the same milk used for adaptation (0.5 g of lactose per kg of body weight) with a hydrogen breath test. Fecal urgency was higher during the two-week consumption of A1/A2 milk compared to A2 milk (p = 0.04, n = 16). Bloating (p = 0.03, n = 16) and flatulence (p = 0.02, n = 16) were also higher on the 15th day with A1/A2 milk compared to A2 milk challenge. However, day-to-day symptoms, hydrogen, serum inflammatory markers, and antioxidant concentrations were not different after A1/A2 and A2 milk consumption adaptation periods. Adaptation over two weeks did not improve lactose digestion or tolerance of A1/A2 milk to match that of A2 milk.

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.

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.

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.

A new trend in cow\'s milk has emerged in the market called type A1 and A2 milk. These products have piqued the interest of both consumers and researchers. Recent studies suggest that A2 milk may have potential health benefits beyond that of A1 milk, which is why researchers are investigating this product further. It is interesting to note that the A1 and A2 milk types have area-specific characteristics compared to breed-specific characteristics. Extensive research has focused on milk derivatives obtained from cow\'s milk, primarily through in vitro and animal studies. However, few clinical studies have been conducted in humans, and the results have been unsatisfactory. New molecular techniques for identifying A1 and A2 milk may help researchers develop new studies that can clarify certain controversies surrounding A1 milk. It is essential to exercise extreme caution when interpreting the updated literature. It has the potential to spread panic worldwide and have negative economic implications. Therefore, this study aims to investigate the differences between A1 and A2 milk in various research areas and clarify some aspects regarding these two types of milk.

Milk is a widely consumed nutrient-rich food containing protein variants such as casein A2 and A1. A1 differs from A2 in an amino acid at position 67 (Pro67 to His67). The breakdown of β-casein yields β-casomorphins (BCM), among which BCM-7 is extensively studied for its effects on the human body. Animal studies have shown that A1 β-casein milk increases digestive transit time and enhances myeloperoxidase activity. Individuals with lactose intolerance prefer A2 milk to conventional A1 milk, as BCM-7 in A1 milk can lead to inflammation and discomfort in sensitive individuals. A2 milk, which contains A2 β-casein, is believed to be more easily digestible than A1 β-casein. Its popularity has grown owing to reports linking A1 casein to diseases such as type 1 diabetes, heart disease, and autism. A2 milk has gained popularity as an alternative to A1 milk, primarily because of its potential benefits for individuals with certain diseases. This review aims to provide an updated understanding of A2 milk consumption and its health benefits. This review aims to provide an updated understanding of A2 milk consumption and its health benefits.

The A2 milk marker is gaining popularity worldwide; thus, many farms plan to convert their dairy cattle herds to the A2A2 genotype. Variation in beta-casein genotypes needs to be monitored in large dairy cattle populations. Therefore we aimed to evaluate the genotypic distributions, population genetics, and diversity parameters in Holstein-Friesian cows. A total of 1200 cattle were genotyped using the Affymetrix® Axiom® array system. We performed an association analysis regarding the CSN2 genotypes and phenotypic traits, including lactation and test-day milk yield. We next evaluated the effects of the genotypes considering the genetic merit of the animals. Animals were grouped based on their PTAs for milk production, fat, protein, and daughter pregnancy rate. Thus, we tested the genotype × genetic merit interaction for significance. The A2 allele frequency is remarkably high (0.68), and the heterozygous genotype is predominant (46.25%). The marker showed intermediate variability and diversity levels, indicating a considerable frequency of the A1A1 genotype (9.33%) remains in the population. ANOVA results showed no significant association between the CSN2 genotypes and milk yield traits. A similar finding is valid for the genotype × genetic merit regarding the genomic test results. The data presented here may be helpful for further investigations and applications on A2 milk production.

The possible adverse effect of consuming bovine milk with A1 β-casein (but not with A2 β-casein) on health aspects due to the release of β-casomorphin-7 (BCM-7) is currently under debate. The aim of this study was to perform a bibliometric analysis of studies extracted from Scopus to explore the relationship between BCM-7, A1 or A2 bovine milk with different aspects of health. Over time, several research groups were formed that are no longer active and although some authors have returned to the field of study, they have focused their efforts mainly on conducting reviews that show the same imprecise conclusions due to the few original articles. Research is concentrated in Europe and Asia, where New Zealand, China and Germany are the countries with the most publications, records and citations on the subject, respectively. On the other hand, no country in Africa or South America has scientific production, which opens the possibility of building collaborations between countries and exploring areas that lack scientific studies. Based on conflicting information from primarily in vitro and animal studies, and limited clinical trials with poor designs, A1 milk presents pro-inflammatory and oxidative activity, but the evidence is insufficient to associate its consumption with negative health effects. However, A2 milk may be better tolerated by the digestive system of some individuals, suggesting its possible modulating role in the intestinal microbiota. Stronger scientific evidence is needed to reach a consensus on whether the presence of β-casein A1 can significantly negatively affect health. The information shown will allow a better understanding of the subject and consumers will be able to make their own decisions regarding A1 or A2 milk.

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.

We previously developed a genotyping method to detect the A1 and A2 alleles of the bovine β-casein gene. This method required DNA extraction from hair samples. Recently, demand for A2 milk (milk from cows homozygous for the A2 allele) has increased, and dairy farms are required to have certification to produce A2 milk. Here, we describe the development of a new, simple, and sensitive genotyping method for the β-casein gene that does not require DNA extraction. This method uses the CycleavePCR technique and can amplify the β-casein gene directly from raw milk samples. Genotypes obtained from the milk samples (n = 27) were completely coincident with those obtained from genomic DNA. In addition, this method could quantify the A1 allele in the milk samples. The limit of detection for the A1 allele in A2 milk was 2%. The copy numbers of the A1 allele corresponding to the 2% detection limit were estimated to be 30.5 ± 24.3 molecules/μL. These findings indicate that this new genotyping method is simple and fast for detecting the A1 allele in milk samples and can therefore be potentially used to certify A2 milk.