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Release of β-casomorphin-7 from bovine milk of different β-casein variants after ex vivo gastrointestinal digestion
Abstract
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 βCM-7 after GI digestion.
... This enzymatic cleavage liberates a specific seven amino acid peptide known as β-casomorphine-7 (BCM-7). This peptide, though present in the sequence of β-casein in both the A1 and A2 variant, is released in higher quantities when digesting A1 milk compared to A2 milk [8]. This cleavage is conducted in three steps by pepsin, chymotrypsin, elastase and leucine aminopeptidase [9]. ...
... Since BCM-7 is generated during in vitro digestion of A1 but also of A2 milk [29] and ex vivo [8], we developed an in-house competitive ELISA using a mouse monoclonal BCM-7 antibody whose binding activity is not largely disturbed by milk components. This ELISA served as a control to confirm successful in vitro digestion and investigate whether BCM-7 was more prevalent in A1 milk compared to A2 milk after the digestion process. ...
... BCM-7, which is generated in increased amounts from A1 β-casein during digestion [8], is being discussed as a potential marker for negative effects on the immune system [16,34]. However, currently, there is a lack of data to assess the impact of BCM-7 and β-casein variants on immune cells [35]. ...
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.
... The hypothesis was that peptide bond was resistant to enzymatic cleavage in A2 instead of A1. Despite further researchers strengthened this theory, the release of BCM7 has been also observed during in vitro and ex vivo enzymatic hydrolysis of the A2 variant (Asledottir et al., 2017;Asledottir et al., 2018;De Noni, 2008;Ul Haq, Kapila & Kapila, 2015). Nonetheless, the yield of BCM7 was significantly lower than that recorded after the digestion of A1 variant (Asledottir et al., 2017;Asledottir et al., 2018;Cieślińska et al., 2007;Cieślińska et al., 2012;Duarte-Vázquez et al., 2017). ...
... Despite further researchers strengthened this theory, the release of BCM7 has been also observed during in vitro and ex vivo enzymatic hydrolysis of the A2 variant (Asledottir et al., 2017;Asledottir et al., 2018;De Noni, 2008;Ul Haq, Kapila & Kapila, 2015). Nonetheless, the yield of BCM7 was significantly lower than that recorded after the digestion of A1 variant (Asledottir et al., 2017;Asledottir et al., 2018;Cieślińska et al., 2007;Cieślińska et al., 2012;Duarte-Vázquez et al., 2017). Recently, the release of BCM7 was observed during digestion of A2-type milk using an in vitro semi-dynamic protocol for GID (Lambers, Broeren, Heck, Bragt, & Huppertz, 2021). ...
... As found in our previous studies (De Noni, 2008;De Noni & Cattaneo, 2010), the content of BCM7 was only a negligible part of the total theoretical quantity releasable from complete digestion of β-CN. Nonetheless, the amounts of BCM7 differed according to the applied GID in BCM7 formation between A1 and A2 samples appeared to be somewhat smaller than those described by Asledottir et al. (2018). These last authors reported the release of BCM7 from bovine milk containing the variants A1, A2, F or I of β-CN during ex vivo gastrointestinal digestion. ...
Beta-casomorphin-7 (BCM7) represents the fragment Val⁶⁰-Ile⁶⁶ of bovine β-casein (β-CN), and there is evidence that it is more easily released during gastrointestinal digestion (GID) of A1 β-CN variant, in comparison to the A2 variant. This study aimed at investigating the effect of type of enzymes and the protease/protein (P/S) ratio on BCM7 release during the intestinal step of in vitro static GID of bovine milk and cheeses with A1 or A2 β-CN phenotypes. BCM7 occurred in digests of both A1 and A2 samples, being the release more marked for A1 counterparts. Nonetheless, the BCM7 release depended on both the type of GID enzymes and the P/S ratio. These findings highlight the importance of GID conditions which may affect the outcomes for possible differences between A1 and A2 milk based on BCM7 release during in vitro GID.
... Based on the amino acid substitution arising from the cleavage site in the polypeptide chain, twelve to fifteen different genetic variants of β-CN exist, with variants A1 (His 67 ) and A2 (Pro 67 ) most abundant in modern cattle, which frequency depends on cow genetics (Daniloski, Cunha, et al., 2021;Nguyen, Busetti, Smolenski, Johnson, & Solah, 2021). The distinction between these two forms is a mutation in the amino acid polypeptide chain at position 67 (Asledottir et al., 2018;Nguyen, Solah, Busetti, Smolenski, & Cooney, 2020). Furthermore, the β-CN variants can be separated into two of the most prevalent families, namely A1 family, including A1, B, and F variants; and A2 family, including A2, A3, and I variants (Asledottir et al., 2018). ...
... The distinction between these two forms is a mutation in the amino acid polypeptide chain at position 67 (Asledottir et al., 2018;Nguyen, Solah, Busetti, Smolenski, & Cooney, 2020). Furthermore, the β-CN variants can be separated into two of the most prevalent families, namely A1 family, including A1, B, and F variants; and A2 family, including A2, A3, and I variants (Asledottir et al., 2018). As a result of the single nucleotide polymorphism in modern European cattle a few thousand years ago, the A1 β-CN variant (His 67 : 155.2 g/mol) started to predominate, even though, at first, all cattle originated from the A2 β-CN variant (Pro 67 : 115.1 g/mol) renowned as the oldest β-CN proteoform (Sebastiani et al., 2020). ...
... Daniloski, Cunha, et al. (2021) explained that β-casomorphin7 (BCM7) may also be released from A2 β-CN, albeit at lower levels compared to A1 β-CN and A1/A2 β-CN, and which was recently demonstrated by Lambers, Broeren, Heck, Bragt, and Huppertz (2021). Additionally, F and B variants of β-CN as part of the family of A1 β-CN, and A3 β-CN and I β-CN as part of the A2 β-CN family have also been reported to release BCMs during digestion (Asledottir et al., 2018). Therefore, upon proteolysis of β-CN from either family, short BCM opioid peptides are released (Asledottir et al., 2017). ...
Background
Complex polymorphisms in the polypeptide chain of bovine β-casein are responsible for the genetic variants that give rise to different bioactive peptides during in vitro and ex vivo digestion, or food fermentation. One specific group of bioactive peptides, known as β-casomorphins, are opioid-agonists for μ-receptors and have been suggested to assume an active role in the development of various non-communicable diseases, including diabetes mellitus, cardiovascular diseases, neurological disorders, pulmonary inflammation, to name a few. Their potential bioactivity and role in human health is dependent on their release from the latent form within the primary structure of β-casein, which can occur during the manufacture of dairy products or during gastric and intestinal digestion. Consequently, β-casomorphins can be either completely hydrolysed or absorbed in the gut or be transferred into the blood stream and internal organs in their intact form. Their biological function as opioid agonists is expressed in the gut, thus upon epithelial translocation they may affect various physiological states, such as causing gastrointestinal issues, bloating, and lactose intolerance.
Scope and approach
This review evaluated the possible disadvantages and potential beneficial effects of β-casomorphins on human health, within the scope of in vitro and ex vivo studies. Applying a systematic approach, a literature search was performed across four electronic databases (Scopus, Web of Science, PubMed, and Cochrane) to identify suitable studies.
Key findings and conclusions
The data mined from in vitro and ex vivo trials on the health impact of β-casomorphins is both inconclusive and limited to completely support the possible adverse or potential beneficial health effects of β-casomorphins. These peptides are usually further cleaved in the gut, which prevents their migration across the gut-blood-brain barrier. Nevertheless, in some individuals that are immunocompromised, their condition increases permeability of the gut barrier often referred to as a "leaky gut" condition. Thus, the absorption of β-casomorphins appears possible. This may indicate that the presence of β-casomorphins can affect gastrointestinal functions only. However, since the overall concern with β-casomorphins appears debatable and not well defined, more experimental trials are required to investigate the metabolic pathways of these identified peptides, their release during digestion, and subsequent fate after the digestion process. Consequently, repeatability of the findings under a number of other laboratory conditions is required before the data can be fully substantiated. Due to the rapidly evolving nature of the issue and emerging studies in this field, further exploration into the bioactivity of β-casomorphins is warranted.
... In subsequent studies, BCMs were identified after in vitro simulated gastroduodenal-intestinal digestion of milk (Picariello et al., 2015), as well as in human jejunal aspirates following milk ingestion (Boutrou et al., 2013). The detection of BCM7 at comparable levels after in vitro and ex vivo digestion of β-CNA1 and β-CNA2 revealed that the release of BCM7 is not exclusive to β-CNA1 (Asledottir et al., 2017;Asledottir, Poulsen, Devold, Larsen, & Vegarud, 2018). Recent investigations have confirmed the generation of BCM7 and longer BCM precursors during in vitro gastroduodenal digestion (Lambers, Broeren, Heck, Bragt, & Huppertz, 2021;Reiche et al., 2024), whereas slightly different tendencies of β-CNA1 and β-CNA2 to release BCM7 have been ascribed to fluctuations in the activity of digestive enzymes (Cattaneo, Masotti, Stuknytė, & De Noni, 2023). ...
... To address concerns related to the potential health effects of β-CNA1, many breeders have undertaken genetic selection programs that have shifted production toward milk containing exclusively β-CNA2 (Fernández-Rico et al., 2022). However, evidence of the exclusive or preferential release of BCM7 during in vivo digestion of β-CNA1 and its distribution in the human body remains weak (Asledottir et al., 2018;Lambers et al., 2021;Reiche et al., 2024). To address this issue, we aimed to detect and quantify BCMs, specifically BCM7, in the bloodstream of human volunteers who consumed bovine milk containing defined amounts of β-CN variants; the milk used in this study included β-CNA1 and β-CNA2 at proportions of 36% and 64%, respectively (Supplementary Fig. S1). ...
... On the other hand, the digestion of the other A2 variant family (A2, A3, and I) with Pro in position 67 does not produce BCM-7 because the peptide bond Ile66-Pro67 in A2 variants is more resistant to enzymatic cleavage than the Ile66-His67 bond in A1 variants (Pal et al., 2015;Cattaneo et al., 2023). Asledottir et al. (2018) revealed that A2 milk (A2 and I variants) also produces BCM-7 when digested using human GI juices, but the concentrations were much lower (297%) compared with A1 milk (A1 and F variants). The BCM-7 was also detected in the blood of dairy calves fed A1 or A2 milk, but the levels of BCM-7 were 77% lower in A2 than in A1 calves (Hohmann et al., 2021). ...
... The BCM-7 was also detected in the blood of dairy calves fed A1 or A2 milk, but the levels of BCM-7 were 77% lower in A2 than in A1 calves (Hohmann et al., 2021). Asledottir et al. (2018) observed that within each family, F (A1-like) and I (A2-like) variants release greater BCM-7 concentrations than the A1 and A2 true variants, respectively. Thus, it seems inappropriate to group variants and their behavior during digestion into "A1 family" and "A2 family" based only on which amino acid is at position 67 (His or Pro). ...
... β-casomorphin-7 (BCM7) is an opioid peptide that has effects like those of morphine and has been isolated from an enzymatic digest of β-casein (in particular, A1) and has been linked to several health concerns such as gastrointestinal disorders [2]. Minimal amounts of BCM7 are, however, released from milk that contains A2 β-casein as its main protein [3][4][5]. The difference between A1 and A2 β-casein lies in the specific amino acid at a particular position (the 67th position) in the protein sequence. ...
... The difference between A1 and A2 β-casein lies in the specific amino acid at a particular position (the 67th position) in the protein sequence. A1 β-casein contains the amino acid histidine at this position in its protein sequence, whereas A2 β-casein has the amino acid proline [4][5][6]. The pasteurization (85 • C/30 s) and UHT (140 • C/15 s) of milk inhibit the formation of BCM7 during intestinal digestion, which could be due to the protein denaturation altering the protein digestion [3,[7][8][9] or due to the formation of radicals during the Maillard reaction that could attack the protein backbone, subsequently modifying some of the peptides that are formed [10]. ...
... The presence of BCMs in "cheese" (another important term in the map) depends on the milk treatment process, pH, coagulant, and strain starter used, and the temperature and humidity employed during ripening [14]. Among BCMs, in the A2 β-casein research, BCM-7 is especially important, being more frequently released by A1 β-casein [49]. BCM-7 can influence the nervous, endocrine, and immune systems by activating the gastrointestinal tract's µ-opioid receptors [20]. ...
... The term "beta-casomorphin" (BCM) was located in the red cluster, but was also next to the green cluster, with a lot of linkages with their terms. This was probably because a high number of reviews on the A1 and A2 β-casein research field have focused on the description of the relationship between BCM-7 and possible healthrelated adverse effects, such as gastrointestinal problems [9,49], heart disease, diabetes, or autism [10,20,51,52]. However, the number of clinical trials in humans remains low [18,19], thus they cannot be considered conclusive and further studies are required to provide further insight into the possible effects of BCM-7 on human health [14,36]. ...
The protein fraction of β-casein may play a key role in the manifestation of a new intolerance: milk protein intolerance. The most common forms of β-casein among dairy cattle breeds are A1 and A2 β-casein. During gastrointestinal digestion of A1 β-casein, an opioid called peptide β-casomorphin-7 (BCM-7) is more frequently released, which can lead to adverse health outcomes. For that reason, novel products labelled as “A2 milk” or “A1-free dairy products” have appeared on the market. In this context, a bibliometric analysis on A2 β-casein research was carried out through the Web of Science (WoS) database. The main objective of this work was to provide an overview of the state of the art in the field of β-casein A2 by analyzing the number of publications per year, trends in thematic content, the most frequently used terms, and the most important institutions and countries in the field. This bibliometric study showed that a greater effort is needed to determine the possible implications of this novel product for human health and the market.
... The release of BCM7 from milk casein variant F and A1 has been reported to be 6.57 and 3.71 mg per serve of milk (mg 200 mL − 1 ) (Asledottir et al., 2018). ...
... The establishment of the A1-Free Category was based on the hypothesis that cow's milk is potentially detrimental to human health (Gerstein & VanderMeulen, 1996;Schrezenmeir & Jagla, 2000). This concept arose from the observation of differences in proteolytic digestion of milks from cows with different genotypes of b-CN, as experiments have shown that a1 b-CN releases the bioactive peptide BCM7, whereas a2 b-CN does not (Asledottir et al., 2018;Elliott et al., 1999;Kamiński et al., 2007). ...
The paper reviews analytical methods of quality control and specifications for a new food A1-Free Category consisting of dairy products produced exclusively from milk that is naturally free from the a1 form of beta-casein. Evaluation of an A1-free milk Inter-Laboratory Round Robin trial revealed that positive samples had no statistically significant differences between participants, despite reporting varying concentrations ranging from 0.04-5.07%. Following consideration of various analytical methods reported in the literature, we recommend intact a1 beta-casein protein as the most suitable molecular species for quantification, and propose potential category definitions, specifications, and laboratory methods of quality control for retail, business-to-business and global trade practices.
... Several studies with diverse outcomes have investigated the formation of BCM-7 from b-casein A1 and A2 in milk (Table 1). Milk from single cows of multiple breeds, bulk milk and model products have been tested using different digestion procedures and analytical methods to detect BCM peptide formation (Asledottir et al., 2017(Asledottir et al., , 2018Cie sli nska, Kaminski, Kostyyra, & Sienkiewicz-Szłapka, 2007;Cie sli nska et al., 2012;Duarte-V azquez, García-Ugalde, Villegas-Guti errez, García-Almend arez, & Rosado, 2017;Jinsmaa & Yoshikawa, 1999;Nguyen, Busetti, Smolenski, Johnson, & Solah, 2021;De Noni, 2008;Ul Haq, Kapila, & Kapila, 2015). However, considering today's standard for simulating gastrointestinal digestion and analytical methods, physiological relevance varies among the studies listed in Table 1. ...
... In all A1 and A2 milk samples, the amount of BCM-7 formed was only a fraction of the total amount of that could be formed from the b-casein present, although the amount of BCM-7 appeared to be slightly higher in the A1 samples compared with the A2 samples, but differences did not exceed a factor of 1.5 when comparing identically processed or control milk samples. The amounts of BCM-7 detected in the intestinal digestions, as well as the slightly higher levels from A1 milk, are in line with previous observations (Asledottir et al., 2017(Asledottir et al., , 2018, although the differences between A1 and A2 BCM-7 formation appeared to be somewhat smaller in the current study, employing a milk sample from multiple cows. A larger data set of multiple single homozygous cows (preferably from a single herd to accommodate potential effects of farm management/feed) may be required to further quantitatively elucidate possible differences in the amount of BCM-7 formed in A1 and A2 milk. ...
Variation between bovine A1-or A2-type β-caseins are suggested to affect beta-casomorphin-7 (BCM-7) formation, which may affect health. Studies assessing BCM-7 formation often use raw milk, but processing of milk is known to affect digestion. Using in vitro digestion and stable-isotope assisted peptide quantification, we reveal that BCM-7 is formed under intestinal conditions from both A1-and A2-type milk. Moreover, formation of BCM-7 was affected by industrial relevant heating in both A1 and A2 milk. Further studies with a large number of single A1-and A2-type cows are needed to elaborate if amounts of BCM-7 formed are different between A1 and A2 milk. Both longer and shorter BCM peptide sequences that may display similar activities should be taking into account as a longer BCM-7 containing peptide (BCM-7 +2) could also be detected in A2 milk intestinal digesta.
... Owing to the fact that the genetic variation between these two β-CN proteoforms has been clarified by the replacement of a single amino acid in this position, it is more likely that this mechanism results in alternation of the protein functions and functionalities, enzymatic and acidic hydrolysis and liberation of bioactive peptides (BCMs); thus, it may have an influence on further milk processing and human nutrition (Cieślińska et al., 2012;Poulsen et al., 2017). Recently, some studies have indicated that even A2 β-CN milk structurally can allow for release of β-casomorphin7 (BCM7) just less readily and less abundantly compared to A1 β-CN milk (Asledottir et al., 2017(Asledottir et al., , 2018Cieślińska et al., 2012). Notably, the other genetic variants, including F β-CN and B β-CN from the A1 β-CN family, and A3 β-CN and I β-CN from the A2 β-CN family have been found to liberate BCM7 during digestion. ...
... Notably, the other genetic variants, including F β-CN and B β-CN from the A1 β-CN family, and A3 β-CN and I β-CN from the A2 β-CN family have been found to liberate BCM7 during digestion. Therefore, upon proteolysis of A1 β-CN family or A2 β-CN family, short BCM opioid peptides are released (Asledottir et al., 2018). ...
Background
A number of randomised in vivo trials have to date investigated the health impacts of the genetic variants A1 and A2 of bovine β-casein. The primary difference between these two genetic variants is the mutation leading to an amino acid exchange at a position 67 in the peptide chain. This systematic review evaluated the effects of bovine milk, β-casein and pure β-casomorphin7, in the form of orally administered nutritional ingredients, on possible incidence and risk for chronic digestive discomfort and development of incurable conditions and diseases in human and animal randomised controlled trials.
Scope and approach
Following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) checklist, searches were performed for publications across 7 electronic databases (Scopus, Embase, Web of Science, Medline, EBSCO, PubMed, and Cochrane) up to and until July 2020, to identify randomised controlled trials. The subsequent search results were screened for relevance firstly by title, then abstract, and the chosen ones by full text, with additional screening of included articles reference lists.
Key findings and conclusions
In total 2006 peer-reviewed journal articles were identified and after applying exclusion criteria, 19 studies were deemed suitable for inclusion. Human-based and animal-based results from the clinical in vivo studies demonstrated that consumption of A2 β-casein milk can lead to improved tolerance of milk via decline in the ubiquity of gut related discomfort. However, the exact mechanism for these effects or specific individuals that may benefit from A2 β-casein milk as opposed to A1 β-casein milk is still poorly understood. Notably, consumption of A2 β-casein milk had very low to completely no effect on the other health statuses investigated, particularly non-communicable diseases, such as cardiovascular diseases, neurological disorders, and diabetes. Based on current data, there is not sufficient evidence to merit public health authority recommendations related to the consumption and health associations of A1 β-casein milk or A2 β-casein milk. Interestingly, regardless of the scientific evidence between A2 β-casein milk and health, this milk continues to gain prominence on the market, thus further functional research is required to understand the mechanisms of action of these identified peptides and gene variants and any implications A1 or A2 β-casein milk may have on human health and techno-functional properties of milks.
... In conseguenza di ciò, soprattutto in Australia, Nuova Zelanda, Stati Uniti e Cina, ma anche in Europa e in Italia, è aumentata la commercializzazione di prodotti, latte e yogurt soprattutto, a base di latte prodotto da vacche omozigoti A2A2. Dal punto di vista biologico, si ritiene che tale effetto sia legato al rilascio del peptide BCM7 (beta-casomorfina 7) che viene rilasciato durante i processi digestivi soprattutto dalla variante A1 e molto meno dalla A2 (8,(12)(13)(14). Si tratta di un oppioide naturale che può avere un effetto negativo sulla salute umana qualora venga assorbito (8). ...
... Poco si è indagato invece dei possibili effetti delle varianti della β-caseina a livello caseario (15). In realtà, alcune ricerche hanno osservato il rilascio della BCM7 anche durante la digestione del latte A2, seppur in quantità molto più modeste (12)(13)(14). ...
... Indeed, it has been demonstrated that BCM7 is released from the A1, and not from the A2 variant, during in vitro enzymatic digestion of b-Cn (Jinsmaa and Yoshikawa 1999;De Noni 2008;Raies-Ul-Haq, Kapila, and Kapila 2015) (Table 2). Nonetheless, the release of BCM7 has been also observed during in vitro or ex vivo enzymatic hydrolysis of the A2 variant (and milk products containing it), despite the yield of BCM7 was lower than that recorded from the digestion of A1 type (Cie sli nska et al. 2012;Asledottir et al. 2017;Duarte-V azquez et al. 2017;Asledottir et al. 2018) (Table 2). For instance, Asledottir et al. (2017) showed the A1 variant to release about 3-fold more BCM7/g b-Cn than the A2 type, following ex vivo digestion (Table 2). ...
... For instance, Asledottir et al. (2017) showed the A1 variant to release about 3-fold more BCM7/g b-Cn than the A2 type, following ex vivo digestion (Table 2). By applying this type of digestion protocol to milk with different b-Cn genetic variants, Asledottir et al. (2018) found BCM7 to be released in all the digested samples, although more BCM7 was found in digested A1 and F milk compared with the A2 and I samples (Table 2). Similar findings arose from the study of Duarte-V azquez et al. (2017), who found approx. ...
The genetic variant A1 of bovine β-casein (β-Cn) presents a His residue at a position 67 of the mature protein. This feature makes the Ile⁶⁶-His⁶⁷ bond more vulnerable to enzymatic cleavage, determining the release of the peptide β-Cn f(60–66), named β-casomorphin 7 (BCM7). BCM7 is an opioid-agonist for μ receptors, and it has been hypothesized to be involved in the development of different non-transmissible diseases in humans. In the last decade, studies have provided additional results on the potential health impact of β-Cn A1 and BCM7. These studies, here reviewed, highlighted a relation between the consumption of β-Cn A1 (and its derivative BCM7) and the increase of inflammatory response as well as discomfort at the gastrointestinal level. Conversely, the role of BCM7 and the effects of ingestion of β-Cn A1 on the onset or worsening of other non-transmissible diseases as caused or favored by still need proof of evidence. Overall, the reviewed literature demonstrates that the “β-Cn A1/BCM7 issue” remains an intriguing but not exhaustively explained topic in human nutrition. On this basis, policies in favor of breeding for β-Cn variants not releasing BCM7 and consumption of “A1-like” milk appear not yet sound for a healthier and safer nutrition.
... Therefore, dairy products containing A2 β-CN have a better stability than those containing A1 β-CN [6]. Other studies showed that the His67 in A1 β-CN confers reduced sensitivity to proteolytic cleavage by digestive enzymes, leading to the release of β-casomorphin-7 [7,8]. Numerous gastrointestinal conditions, including stomach pain and changes in stool consistency, have been linked to β-casomorphin-7 [9,10], and may cause other health conditions such as immune disorders [11,12]. ...
The aim of this study was to investigate the immunomodulatory effects of A2 β-casein (β-CN) in cyclophosphamide-induced immunosuppressed BALB/c mice. Experiments conducted in vitro revealed that A2 β-CN digestive products have potent immunostimulatory activities. Animal studies demonstrated that A2 β-CN improved the immunological organ index reduction trend caused by cyclophosphamide, reduced the pathological damage to the spleen tissue in immunosuppressed mice, increased the release of IL-17A, IgG, and IgA, and reduced the production of IL-4. By regulating the relative abundance of advantageous bacteria like Oscillospira, Lactobacillus, and Bifidobacteria and harmful bacteria like Coprococcus and Desulfovibrionaceae, A2 β-CN improved gut microbiota disorders in immunosuppressed mice. Moreover, A2 β-CN promoted the production of short-chain fatty acids and increased the diversity of the gut microbiota. Therefore, ingestion of A2 β-CN is beneficial to the host’s immune system and gut health. These findings provide insights for the future application of A2 β-CN-related dairy products.
... Although this difference is very slight, the bioactive peptides produced by the digestion of these two variants have significantly different effects on humans [6]. A1 β-CN, when digested, produces β-casomorphin-7 (BCM-7) [7], which can cause many diseases [8]. Conversely, BCM-7 is not generated from A2 β-CN [9]. ...
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.
... The binding of β-casomorphin-7 to its receptor in the gut was associated to an increased gastro-intestinal transit time and inflammation as well as an impairment in the gut barrier integrity [59,60]. In addition, some studies pointed out further negative effects of β-casomorphin-7 on human health, including type-2 diabetes and cardiovascular diseases [61]. However, other studies suggested positive health effects of opioid peptides on human health and in particular on the nervous system [62,63]. ...
The effect of ripening and in vitro digestion on the biological activities, peptide profiles and release of bioactive peptides in Ras cheese has been investigated. Ras cheese ripening largely influenced the extent of protein hydrolysis. The advancement in ripening resulted in an increase in total peptides (from 0.97 to 2.46 mmol leucine/g in samples at 30 and 180 days of ripening, respectively) and bioactive peptides concentration, especially angiotensin-converting enzyme (ACE)-inhibitory, dipeptidyl-peptidase-IV-(DPP-IV)-inhibitory and antioxidant peptides. In vitro gastro-intestinal digestion further promoted protein hydrolysis and the release of bioactive peptides. Digested Ras cheese at 90 and 180 days of ripening displayed the highest bioactive peptides intensity. The variations in bioactive peptides amount during ripening and in vitro digestion were correlated with the changes in ACE-inhibitory, DPP-IV-inhibitory and antioxidant activities. The highest amounts of VPP and IPP were detected in digested Ras cheese at 90 days of ripening (17.44 and 36.50 mg/kg of cheese, respectively), whereas the highest concentrations of APFPE were found in undigested and digested 180-day ripened Ras cheese (82.09 and 52.01 mg/kg of cheese, respectively). The present investigation underlined potential differences in the biological effect after the ingestion of Ras cheese at different ripening times.
... This single amino acid difference results in the alteration of protein functions and functionalities, enzymatic and acidic hydrolysis, and liberation of bioactive peptides that could have an influence on further milk processing and human nutrition [4]. Some studies have indicated that β-CN A1 digestion releases a greater quantity of beta-casomorphine-7 (BCM-7) than β-CN A2 [5][6][7][8], which is considered to be a health risk as it can potentially affect opioid receptor in the nervous, endocrine and immune system [9,10], and is associated with the development of some non-transmissible diseases [11,12]. However, the European Food Safety Authority (EFSA) did not find any clear evidence of a cause-effect relationship between BCM-7 and the development of certain non-transmissible disease, and only stated that BCMs may cause disturbances of the gastrointestinal tract, such as swelling, flatulence and abdominal pain [13]. ...
This study investigated the acid and rennet milk coagulation properties of A2 milk (β-casein (CN) A2A2 genotype), in comparison to a control milk (blend of A2A1/A1A1/A2A2 genotypes). Acid and rennet coagulation were evaluated using the Optigraph® system, measuring the coagulation time, aggregation rate, and gel density or curd firmness. The acidification kinetics were monitored using a CINAC® system, evaluating the time to reach pH 4.6, the acidification rate, the maximum acidification rate, the time required to reach it, and the latency time. The water-holding capacity of acid milk gels and the potential yield, total solids, and syneresis of enzymatic gels were also evaluated. Some variables were highly influenced by the farm factor, showing the importance of the effect of extrinsic parameters. Acid and enzymatic coagulation times were not affected in either milk. The A2 milk presented higher acid gel density and latency time than the control milk. Although the differences in water-holding capacity were not statistically significant, the A2 milk presented lower values, related with the higher gel density. The A2 milk also showed higher rennet aggregation rate and curd firmness than the control milk. Potential yield and syneresis were higher in the A2 milk, which is in accordance with the higher firmness of curd. Coagulation results and gel and curd properties indicate that it is possible to manufacture acid and rennet coagulation dairy products from A2 milk with no major differences when compared with a control milk.
... The histidine residue in A1-type β-CN allows enzymatic release of the preceding 7 amino acid residues in the gastrointestinal tract to generate the β-casomorphin-7 (BCM-7) peptide [211]. Release of this peptide is either prevented or greatly reduced from A2-type β-CN [211][212][213]. Although BCM-7 was initially associated with several adverse effects, a review by the European Food Safety Authority [214] concluded that "a cause-effect relationship between the oral intake of BCM7 or related peptides and etiology or course of any suggested non-communicable diseases cannot be established." ...
Milk is newborn’s food and an emulsion full of all necessary components for neonatal growth. Its consumption is worldwide and is the base for all dairy products. Because of the latter, many new technologies are growing, among them proteomics; in order to give new insights in milk’s compounds and to maximize the beneficial potential for consumers’ health. In this review, we aimed to gather data of proteomics studies for the majority of dairy animals and elucidate the role of milk bioactive compounds. Furthermore, special reference was made to milk fat globule membrane (MFGM) peptides and the result of thermal treatment in milk proteins. Finally, the proteomic approach regarding adulterations was included in the review.
... On the other hand, The Pro 67 of A2 milk prevents this process by not producing BCM7 and BCM5 or producing them in negligible quantities (ASLE-DOTTIR et al., 2018;DANILOSKI et al., 2021). Asledottir et al. (2018) found BCM7 in both variants, but at very low concentrations in A2 milk (0.01 mg g -1 β-CN digested) compared to conventional (1.85mg g -1 β-CN digested). ...
The objective of this study was to evaluate the potential of the A2 milk as an upgrading strategy for milk producers in Brazil. The primary data were obtained through semi-structured interviews with eight strategic actors of the A2 milk chain in September and October 2021. Five producers, two researchers and one member of the movements called the #bebamaisleite. The study was led by the theoretical framework of global value chain, focusing on the upgrading dimension. A2 milk is an upgrading opportunity for producers , following a global trend of food premiumization. However , caution is needed for producers to enter this chain because the market is incipient, the costs are significant, and the financial return is uncertain. Otherwise, there will be a reproduction of the inequalities and bottlenecks which are present in the conventional dairy chain. It was found that the A2 milk chain is not intended to replace the conventional one, but it aims to capture vegetable beverages consumers.
... On the other hand, The Pro 67 of A2 milk prevents this process by not producing BCM7 and BCM5 or producing them in negligible quantities (ASLE-DOTTIR et al., 2018;DANILOSKI et al., 2021). Asledottir et al. (2018) found BCM7 in both variants, but at very low concentrations in A2 milk (0.01 mg g -1 β-CN digested) compared to conventional (1.85mg g -1 β-CN digested). ...
The objective of this study was to evaluate the potential of the
A2 milk as an upgrading strategy for milk producers in Brazil. The
primary data were obtained through semi-structured interviews
with eight strategic actors of the A2 milk chain in September and
October 2021. Five producers, two researchers and one member
of the movements called the #bebamaisleite. The study was led by
the theoretical framework of global value chain, focusing on the
upgrading dimension. A2 milk is an upgrading opportunity for pro�ducers, following a global trend of food premiumization. Howe�ver, caution is needed for producers to enter this chain because
the market is incipient, the costs are significant, and the financial
return is uncertain. Otherwise, there will be a reproduction of the
inequalities and bottlenecks which are present in the conventional
dairy chain. It was found that the A2 milk chain is not intended
to replace the conventional one, but it aims to capture vegetable
beverages consumers.
... The mutation resulted in an amino acid change: proline on the A2 β-casein was replaced by histidine on the A1 β-casein (Bell et al., 2006). It has been reported that the A1 and A2 β-caseins are digested differently in the gut because of a structural dissimilarity (Asledottir et al., 2018). During digestion, the A1 β-casein releases the bioactive peptide β-casomorphin-7 (BCM-7) whilst the A2 β-casein does not. ...
Bovine milk mainly contains two types of β-casein: A1 and A2 variants. In recent years, a new variety of cows’ milk has emerged in dairy sector called “A2 milk”. This novel product is characterised by the absence of A1 β-casein, which has been associated with possible gastrointestinal discomfort due to β-casomorphin-7 (BCM-7) release during gastrointestinal digestion. In this context, methods to verify A1 allele absence in A2 milk are required as a quality control in the A2 milk commercialisation. Therefore, the aim of the present study was to develop a locked nucleic acid (LNA) probe-based duplex real-time PCR (qPCR) assay for A1 allele detection in A2 milk samples. Firstly, four DNA isolation methods from milk somatic cells were optimised and evaluated. Results suggests that the commercial kit NucleoSpin Tissue was the most suitable method in terms of DNA quality and amplificability for downstream applications. Then, optimisation and validation of the qPCR assay were carried out. For both A1 and A2 alleles, the absolute limits of detection of this qPCR assay were 7.3 DNA copies/reaction (2 x 10⁻⁵ ng DNA) and 30.4 DNA copies/reaction (0.1 ng DNA) at a 95% confidence level with synthetic reference DNA samples and heterozygous genotyped DNA sample, respectively. The relative limits of detection were 2% (15 copies) and 5% (152 copies) for A1 allele in A2 samples at a 95% confidence with synthetic reference and genotyped DNA samples, respectively. The qPCR assay was robust, with intra- and inter-assay variability below 4.3%, and specific, differentiating between A1 and A2 alleles with 100% genotyping accuracy. In conclusion, this cost-effective and fast method could be used to discriminate A1 allele in A2 samples and, consequently, to verify A1 allele absence in “A2 milk” by screening commercial product on the market.
... The stomach discomfort discussed resembles the discomfort experienced by individuals with lactose intolerance, but recent studies may indicate that some of these individuals may benefit from drinking A 2 milk instead of milk containing A 1 , even when the lactose has been hydrolysed (He et al., 2017). The release of the BCM-7 peptide by digestion of either purified β-CN or of β-CN in milk from homozygous cows has been studied in two recent studies (Asledottir et al., 2017(Asledottir et al., , 2018. Using mass spectrometry for BCM-7 detection, it was found that BCM-7 was released from the A 1 family milk as expected, but also from the A 2 family milk, though at lower levels (around 4-5 times lower). ...
This chapter reviews the genetic factors affecting the composition and quality of cow's milk. It starts by discussing how different breeds of cattle can affect the composition of milk. The chapter then goes on to examine milk proteins, genetic variants and post-translational modifications. It then discusses milk coagulation and other functional properties, before highlighting the genetic influence of fatty acids on minor milk components. The chapter also discusses using mid-infrared spectroscopy for genetic parameter estimation, before concluding with a section on the possibilities for genetic improvement in relation to dairy milk.
... On the other hand, The Pro 67 of A2 milk prevents this process by not producing BCM7 and BCM5 or producing them in negligible quantities (ASLE-DOTTIR et al., 2018;DANILOSKI et al., 2021). Asledottir et al. (2018) found BCM7 in both variants, but at very low concentrations in A2 milk (0.01 mg g -1 β-CN digested) compared to conventional (1.85mg g -1 β-CN digested). ...
The study aimed to assess the potential of A2 milk as na upgrading strategy for milk producers in Brazil. The primary data were obtained through semi-structured interviews with eight strategic actors of the A2 milk chain in September and October 2021. Five producers were interviewed, two researchers (one from nutrition and the other from bovine genetic improvement) and one from the movement called #drinkmilk. The theoretical framework of the Global Value Chain, focusing on the upgrading dimension, guided the study. A2 milk is an upgrading opportunity for producers, following a global trend towards food premiumization. However, it is necessary to be cautious for producers to enter this chain as the market is incipient, the costs are signicant and the financial return is uncertain. Otherwise, there will be a reproduction of the inequalities and bottlenecks present in the conventional dairy chain. It was found that the A2 milk chain is not intended to replace the conventional one, but to capture consumers of vegetable drinks.
... β-casmorphin-7 (BCM-7) is released through the digestion of the A 1 and B variants, by cleavage driven by elastase of the bond between peptides 66 (isoleucine) and 67 (histidine), it contains residues 60-66 of β-CN A 1 [140,141], as a part of γ 1 -CN, whereas no hydrolysis by elastase happens for the A 2 variant, which has a proline at position 67 [142]. However, in more recent studies, BCM-7 has been found to be released in A 2 milk as well, but at a lower level [143,144]. This peptide has been controversially reported to be associated with milk intolerance symptom [145], cardiovascular disease [146], type I diabetes [146], autism [147], the aggravation of schizophrenia [13] and sudden infant death syndrome (SIDS) [148]. ...
Milk protein comprises caseins (CNs) and whey proteins, each of which has different genetic variants. Several studies have reported the frequencies of these genetic variants and the effects of variants on milk physicochemical properties and functionality. For example, the C variant and the BC haplotype of αS1-casein (αS1-CN), β-casein (β-CN) B and A1 variants, and κ-casein (κ-CN) B variant, are favourable for rennet coagulation, as well as the B variant of β-lactoglobulin (β-lg). κ-CN is reported to be the only protein influencing acid gel formation, with the AA variant contributing to a firmer acid curd. For heat stability, κ-CN B variant improves the heat resistance of milk at natural pH, and the order of heat stability between phenotypes is BB > AB > AA. The A2 variant of β-CN is more efficient in emulsion formation, but the emulsion stability is lower than the A1 and B variants. Foaming properties of milk with β-lg variant B are better than A, but the differences between β-CN A1 and A2 variants are controversial. Genetic variants of milk proteins also influence milk yield, composition, quality and processability; thus, study of such relationships offers guidance for the selection of targeted genetic variants.
... Mihaela Claudia SPATARU , C. SPATARU , 4) (β-CN), alpha-s2-casein (αs2-CN), and kappa-casein (κ-CN), while the gamma-casein (γ-CN) is considered to be a product of β-CN degradation. The share of the four caseins in the total caseins' content of milk is 39-46%,25-35%,8-11%, and 8-15%, respectively. ...
The paper reviews literature data with respect to the risk of beta-casein A1 milk variant (A1 β-CN) consumption in humans and etiopathogenic mechanisms involved. This allele of β-CN particularly came into notice by the possibility of its cleavage during the digestion process, with the release at the level of the digestive system of a strong opioid named beta-casomorphin-7. This opioid is associated with pathological conditions in humans by affecting the digestive, circulatory, and nervous systems (for instance, diabetes mellitus type I, ischemic heart disease, autism, and schizophrenia). The debated topic is important for public health, but also for animal science, completing the multitude of studies focused on associations of different allelic variants within each type of casein or whey proteins with various technological properties of milk and even with certain qualitative and quantitative characteristics. This time, a potential alarm signal was debated for a more efficient selection of dairy cows for the benefit of consumer health and not only in terms of the profitability of their breeding. Although there are evidences of negative effects on human health of liberated bioactive beta-casomorphin-7, these evidences cannot be generalized and are mostly dependent on the individual reactivity.
... The digestion of beta-casein encoded by the A1 allele produces a bioactive peptide called beta-casomorphin 7, which causes intestinal discomfort because of its opioid characteristics. 4,5 Hence, there is a market interested in A2 milk obtained from cows whose betacasein genotype is A2A2. New techniques are even being developed to meet growing market demands. ...
A2 milk is an easily digestible product since it has only A2 beta-casein. In cattle, the A1 and A2 alleles are found in the population and the A2 milk is produced from A2A2 animals. Little is known about these alleles in other domestic dairy species. The present study aims to analyze sequence of genetic material available on public databases and quantify the animals genotyped. Eight domestic species were analyzed. There is strong evidence that domestic non-bovine species only carry A2 beta-casein. The data reported here for goats already confirm it due to the large number of animals genotyped as well as buffaloes. It means that they naturally produce A2 milk and no selection must be done. Thus, the fact that A2 milk is easier to digest can be used to add value to dairy product of these species. It helps to conquer new markets. It also improves people’s health and breeder profitability.
... Multiple or selected reaction monitoring (MRM or SRM) has been applied to the quantification of lactosylated milk proteins, mostly at relative concentration (Le et al., 2013). Nevertheless, there have been a number of studies published on absolute quantification of either milk bioactive peptides (Asledottir et al., 2017;Asledottir et al., 2018), human milk proteins (Chen et al., 2016) or cow's milk proteins in processed dairy products (Lutter et al., 2011). Recently, the application of MRM for the absolute and simultaneous quantification of twenty bovine milk proteins was reported (B€ ar et al., 2019). ...
High-value milk proteins, which can be obtained by optimized fractionation procedures, are ideal ingredients in many food applications. Thus, a simple and robust analytical method is required for the identification and quantification of these individual milk proteins. Here, we present a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using multiple reaction monitoring (MRM) to simultaneously detect and measure target peptides of two major milk proteins, α-lactalbumin (α-LA) and β-casein (β-CN), in raw milk samples from 662 Danish Holstein cows. The MRM quantification of α-LA and β-CN was achieved with limit of detection (LOD) of 0.14 and 0.16 g/L, respectively and reproducibility of the assay <15%. By this newly established MRM-based method, the concentration of α-LA and β-CN in an individual cow's milk ranged from 0.5 to 1.9 (average 1.1) g/L, and from 7.5 to 23.4 (average 15) g/L, respectively. There was no significant effect of parity, whereas significantly increasing concentrations of α-LA and β-CN were observed through lactation (P < 0.001). This shows a considerable biological variation of these two ingredient milk proteins, providing potential varying outputs of fractionation in the dairy streams.
... The different release of bBCM7 in digestates of BF and PF was likely the result of the diverse hydrolyzed CN content of the two fortifiers, but it also could be affected by the ratio among the different genetic variants of β-CN present in the same samples. Indeed, it has been demonstrated that lower (or unquantifiable) amount of bBCM7 releases during enzymatic hydrolysis of the A2 genetic variant of β-CN in comparison to the A1 type (Asledottir et al., 2017(Asledottir et al., , 2018De Noni, 2008). The A1 and A2 are the most widespread β-CN variants among Holstein Friesian cows and, likely, they characterized the starting milk used for the manufacturing of the studied fortifiers. ...
Pasteurized donor human milk (PDHM) for preterm infant nutrition is fortified with hydrolyzates of cow's milk proteins, which have been poorly investigated in relation to heat-damage and occurrence of the bioactive peptides β-casomorphins (BCMs). Therefore, thermal protein modifications of three commercial fortifiers were assessed by measuring well-recognized indexes of heat load. The fortifiers did not contain pyrraline, whereas furosine and lysinoalanine levels roughly overlapped the lowest values reported for liquid formulas addressed to term infant nutrition. Bovine BCMs 3 to 7 and human BCMs 3 to 9 were searched. Bovine BCMs 3, 4, 6 and 7 were found in the undigested fortifiers. Following in vitro digestion simulating the digestive conditions of premature infant, bovine BCMs still occurred in fortified PDHM; the human BCMs 3, 7, 8 and 9 formed. Overall, these results better address the nutritional features of protein fortifiers and fortified PDHM intended for nutrition of preterm infants.
... The histidine residue in A1-type β-CN allows enzymatic release of the preceding 7 amino acid residues in the gastrointestinal tract to generate the β-casomorphin-7 (BCM-7) peptide (Jinsmaa and Yoshikawa, 1999). Release of this peptide is either prevented or greatly reduced from A2-type β-CN (Jinsmaa and Yoshikawa, 1999;Noni, 2008;Asledottir et al., 2018). Although BCM-7 was initially associated with several adverse effects, a review by the European Food Safety Authority (2009) concluded that "a cause-effect relationship between the oral intake of BCM7 or related peptides and etiology or course of any suggested noncommunicable diseases cannot be established." ...
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.
Objectives
Acute respiratory infections (ARIs) and diarrhea are common in toddlers. Milk free of A1 β‐casein (A1PF milk) may support the immune system, but few studies have investigated A1PF milk in toddler formula and any potential effects on ARI/diarrhea. This study's objective was to investigate the incidence of ARI and diarrhea with two toddler formulas, A1PF formula (A1PF) or conventional formula (CON), which differed in milk base and nutrient composition.
Methods
This randomized, open‐label, multicenter study (19 December 2022 to 17 May 2023) evaluated the occurrence of ARI and/or diarrhea in toddlers (aged 2–3 years) who consumed A1PF or CON over 90 days.
Results
A total of 200 toddlers were enrolled, and 180 completed the study. The relative risk of ARI or diarrhea in the A1PF group versus the CON group did not differ significantly, but the median (interquartile range) ARI duration was significantly shorter in the A1PF group (3 [2–4] days vs. 5 [3–6] days, p = 0.012). At Day 90, toddlers consuming A1PF had significantly less severe bloating, gassiness, and fewer regurgitation events (all p < 0.05). Both formulas were well tolerated, and no serious adverse events were reported.
Conclusion
Toddlers who consumed A1PF had a reduced duration of ARI and improved diarrhea outcomes, reducing the burden on their families compared with toddlers who consumed CON. Although this is consistent with other studies, further research is required to determine whether these effects are solely attributable to the A1PF milk base or other differences between the formulas.
Cow's milk has a complex structure and includes all the necessary nutrients to support human life. The content of milk protein ranges from 2.6 to 5.3 %. The main protein fractions are β-casein, κ-casein and whey proteins (immunoglobulins, α-lactalbumin, β-lactoglobulin, lactoferrin and transferrin). The biological value of milk proteins is determined by the fact that they contain a full range of essential amino acids. Thus, all cow's milk proteins are a rich source of lysine, and casein is also characterized by a high content of proline. The main source of energy in milk is milk fat, the content of which reaches 4.5 %. In the vast majority, it is represented by glycerides, lipoids (phosphatides and sterols) and free fatty acids, among which saturated fatty acids predominate (up to 65 %). Carbohydrates in milk are represented by lactose, which, like fat, is used by the body as an energy source. It is the only source of galactose, which is part of the coenzymes involved in the synthesis of proteins, fats, carbohydrates, vitamins, enzymes, and is important for metabolic processes in the human body. Minerals in cow's milk are salts of inorganic and organic acids, which are contained in an easily digestible form, which is an important factor for the normal growth and development of the body. In total, up to 80 elements were found in milk, of which calcium, potassium, phosphorus, sodium, magnesium and iron predominate. Almost all fat-soluble (A, D, E, K) and water-soluble vitamins (groups B, C, H, etc.) are found in cow's milk.
By-products generated in the winemaking industry contain compounds with health-promoting properties, which can be reintroduced into the food production chain. This study evaluated the use of a by-product from the industrial processing of grapes as an ingredient in the manufacture of Petit Suisse cheese, made with A2A2 milk and the addition of the probiotic Bifidobacterium animalis subsp. lactis HN019. Two Petit Suisse formulations were made in three independent batches: a control formulation without the addition of the by-product (F0) and a formulation containing 10% of the by-product (F1). The proximate composition of the cheeses was characterized on the first day after manufacturing them. The addition of the by-product led to an increase in ash, lipids, and carbohydrates and a reduction in moisture and protein contents. The physicochemical characterization and the texture profile analysis showed no changes throughout the product’s shelf life. The probiotic counts remained abundant (~eight log CFU/g) in both formulations with no changes seen throughout the shelf life period. Scanning electron microscopy images showed the added bacteria had typical structures. No differences were observed in the fatty acid profiles of the formulations, and both exhibited a total of 18 fatty acids, including saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs), and polyunsaturated fatty acids (PUFAs). Additionally, the by-product conferred antioxidant activity to the F1 formulation. The addition of the by-product in fresh cheese may be an interesting approach in regards to the processing technology used, its microbiological safety, and its nutritional value. The use of A2A2 milk and a probiotic culture thus enhanced the Petit Suisse cheese, resulting in a healthier product.
The six major bovine milk proteins are encoded by highly polymorphic chromosomes. Genetic variation has become a topic of interest and controversy in recent years due to supposed adverse health effects, beginning with the A2 variant of β‐casein. The functional implications of genetic variants have been well characterised, specifically heat stability and coagulation (i.e. rennet and acid) properties of milk, which have found that the B variant of β‐casein, κ‐casein and β‐lactoglobulin are the most beneficial. Genetic variants of milk proteins provide considerable potential to create new dairy products to add value to milk.
Utilization of human gastrointestinal juices in model digestion presents a promising approach for simulating human digestion of food matrixes in a highly relevant and physiological manner. This approach offers advantages in terms of gastrointestinal enzyme specificity, that more closely mimics human physiology. In this protocol, we outline the process of aspirating gastrointestinal juices, conducting enzyme assays, and performing ex vivo model digestions. Additionally, we supply examples of foods and matrixes where the use of human gastric and duodenal juices has significantly enhanced the authenticity and utility of digestion studies.
β-Casomorphin-7 (BCM-7) is a peptide released through the proteolysis of β-casein (β-CN), which is considered a bioactive peptide displaying evidence of promoting the binding and activation of the μ-opioid receptor located in various body parts, such as the gastrointestinal tract, the immune system and potentially the central nervous system. The possible effects of BCM-7 on health are a theme rising in popularity due to evidence found in several studies on the modulation of gastrointestinal proinflammatory responses that can trigger digestive symptoms, such as abdominal discomfort. With the advancement of studies, the hypothesis that there is a correlation of the possible effects of BCM-7 with the microbiota–gut–brain axis has been established. However, some studies have suggested the possibility that these adverse effects are restricted to a portion of the population, and the topic is controversial due to the small number of in vivo studies, which makes it difficult to obtain more conclusive results. In addition, a threshold of exposure to BCM-7 has not yet been established to clarify the potential of this peptide to trigger physiological responses at gastrointestinal and systemic levels. The proportion of the population that can be considered more susceptible to the effects of BCM-7 are evidenced in the literature review. The challenges of establishing the adverse effects of BCM-7 are discussed, including the importance of quantifying the BCM-7 release in the different β-CN genotypes. In summary, the reviewed literature provides plausible indications of the hypothesis of a relationship between β-CN A1/BCM-7 and adverse health effects; however, there is need for further, especially in vivo studies, to better understand and confirm the physiological effects of this peptide.
The purpose of this study was to compare the structural and physical properties between normal bovine milk and A2 bovine milk fermented using commercial fermentation bacteria mixing with/without Lactiplantibacillus plantarum MWLp-12 and Limosilactobacillus fermentum MWLf-4 screening from human milk. Scanning Electron Microscopy (SEM), SDS-PAGE and inverted fluorescence microscope were used for analyzing the structural differences. Titratable acidity and pH, water holding capacity, rheology, texture performances, and chromaticity value were determined for evaluating differences in the physical properties. We found that A2 fermented milk had better viscosity characteristics and stable structure than normal fermented milk. Importantly, the significant differences in structure and physical properties would affirm that differences structures were existing in casein micelle between normal bovine milk and A2 bovine milk. In addition, the addition of MWLp-12 and MWLf-4 strains could enhance the proteolysis and bring in more pilotaxitic texture leading to the fermented milk possessing a greater texture performance. Therefore, this study will be an interest to the dairy industry for developing novel dairy products, and the two strains will be the potential probiotics to be applied on fermented milk preparation.
Mass fingerprinting is a fast and straightforward approach for analyzing the chemical composition of biological materials. This chapter details how to generate mass fingerprints from diverse food sources, from the extraction of metabolites to data analysis, using different mass spectrometry (MS) methods. We cover four high-throughput ionization techniques for MS: Direct liquid-introduction electrospray ionization (DLI-ESI), low-temperature plasma (LTP) ionization, matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF), and leaf spray (LS). Further, we describe data visualization with clustering methods and data mining.
Ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS/MS) enables simultaneous identification and quantification of beta-casomorphine 5 (β-CM5) and beta-casomorphin 7 (β-CM7) at the level of ng/mL in milk. This analytical technique uses stable isotope-labeled beta-casomorphin 5 (β-CM5-d10) and beta-casomorphin 7 (β-CM7-d10) to achieve accurate quantitation of target peptides. In addition, solid-phase extraction (SPE) can be used for concentration and purification of milk extracts to improve the limits of detection and quantitation of the method.
Whilst there are only four genes that encode caseins found in bovine milk, there is a vast array of naturally occurring variants of each protein arising from both genetic polymorphisms and post-translational modification. Effects of the different casein genetic variants on production traits, composition and functional properties of milk have been discovered over the last two decades, and their potential use as an active ingredient in products for improved health outcomes has formed the basis for consumer marketing. Research focused on casein post-translational modifications is an emerging area, and the influence of phosphorylation and glycosylation modifications on the compositional and functional properties of milk is not yet fully elucidated. This review article outlines the influence of casein genetic variants, phosphorylation and glycosylation on the structure of casein and how the properties of certain casein variants associate with human nutrition and the physicochemical and sensory characteristics of dairy products.
Bovine A1-or A2-type β-caseins have attracted a growing interest due to their variation in beta-casomorphin-7 (BCM-7) formation, which may affect health. In the present work, identification and quantification of A1 and A2 types of β-casein proteins at the peptide level was achieved for the first time. An automated and online immobilized trypsin digestion system was employed for high throughput digesting of proteins into peptides. Tryptic peptides were separated and analyzed subsequently by liquid chromatography coupled to mass spectrometry platform. Two specific peptides ranging from the position of 49 to 97 in the peptide chain were selected for the identification and quantification of A1 and A2 β-casein, which covered the different amino acids between them. Synthetic isotopically labeled winged peptides were used for absolute quantification. Compared with traditional in-solution digestion, online digestion shortens digestion times from 2 to 24 h to 4 min. The limits of quantification (LOQ) of A1 and A2 β-casein in pasteurized milk are 0.8 and 2.4 µg/g, respectively. To further demonstrate the applicability of the proposed method, commercial pasteurized milk tests were performed with satisfactory results.
Supplementary information:
The online version contains supplementary material available at 10.1007/s13197-022-05376-6.
Background
Dairy products from a range of species, including caprine products, have been labelled as A2. Yet these simple terms do not accurately capture the complexity of casein protein sequences or properties, particularly when used to describe non-bovine proteins.
Scope and approach
This review examines the current state of knowledge of the beta casein (β-CN) proteins in caprine and bovine milk. It explores differences in the naming, sequence and function of caprine and bovine β-CN proteins and questions whether caprine milk can be considered ‘A2’ or ‘A2-like’.
Key findings and conclusions
None of the twelve caprine β-CN alleles or proteins have been scientifically named ‘A2’ to date and the caprine β-CN silent allele A1 differs in sequence to bovine β-CN A1. While the bovine β-CN proteins A1 and A2 differ by one amino acid at position 67 (histidine in A1 and proline in A2), all caprine β-CN proteins have proline at this position. These caprine proteins could be considered ‘A2-like’ based on this criterion alone but there are several other differences in sequence within and outside the β-casomorphin-7 (BCM-7) region that differentiate caprine β-CN proteins from bovine β-CN proteins. These sequence differences in caprine β-CN protein sequence may affect functional properties or the physiological effects of β-casomorphins (BCMs) potentially generated during consumption although there is little information from studies performed to date. Given the current state of understanding of caprine β-CN proteins, it appears inappropriate to label caprine dairy products as ‘A2-like’. These knowledge gaps offer promising areas for future research that could provide new insights into the differences between caprine and bovine animals and their milk and dairy products including the bioactivity, functionality and digestibility of different proteins.
Gastrointestinal (GI) digestion of bovine milk promotes release of biologically active peptides, including β-casomorphins (BCMs). Previous studies demonstrate release of BCM-7 by digestion of A¹ β-casein (β-CN), but the conditions required to increase the amount of BCM-7 released during digestion are unclear. In this study, a simple in vitro digestion method employing pepsin, pancreatin and leucine aminopeptidase (LAP) enzymes was used to digest CN isolated from regular and A2 milk. LC-MS analysis revealed that CN isolated from regular milk digested with pepsin-pancreatin released BCM-7 and Val-BCM-7 over time. Additionally, LAP, which cleaves the N-terminal valine, increased the yield of BCM-7. In contrast, significantly less BCM-7 and Val-BCM-7 were released by digestion of CN isolated from A2 milk. This simple method for digesting bovine casein can now be used to provide a platform for future work aimed at better understanding the reported gut health benefits of A2 compared to A1 milk.
Food protein-derived bioactive peptides are recognized as valuable ingredients of functional foods and/or nutraceuticals to promote health and reduce the risk of chronic diseases. However, although peptides have been demonstrated to exert multiple benefits by biochemical assays, cell culture, and animal models, the ability to translate the new findings into practical or commercial uses remains delayed. This fact is mainly due to the lack of correlation of in vitro findings with in vivo functions of peptides because of their low bioavailability. Once ingested, peptides need to resist the action of digestive enzymes during their transit through the gastrointestinal tract and cross the intestinal epithelial barrier to reach the target organs in an intact and active form to exert their health-promoting properties. Thus, for a better understanding of the in vivo physiological effects of food bioactive peptides, extensive research studies on their gastrointestinal stability and transport are needed. This review summarizes the most current evidence on those factors affecting the digestive and absorptive processes of food bioactive peptides, the recently designed models mimicking the gastrointestinal environment, as well as the novel strategies developed and currently applied to enhance the absorption and bioavailability of peptides.
Background
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.
Objective
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.
Methods
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, partial) which are potential sources of β-casomorphin-7.
Results
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 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.
Conclusions
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.
Highlights
New methods are presented for the analysis of total and A1-type β-casein in milk and milk-derived ingredients. Mass fraction of A1-type β-casein in commercial β-casein standard was determined to enable its use as calibrant.
This study assessed the impact of heat treatment on beta-casomorphin 5 (β-CM5) and beta-casomorphin 7 (β-CM7) after in-vitro gastrointestinal digestion of milk representing beta-casein (β-CN) A1A1, A2A2 and A2I phenotype. After heat treatment at 73 °C/20 s, 85 °C/5 min and 121 °C/12 min, milk samples were subjected to in-vitro gastrointestinal digestion. β-CM5/7 were analysed using ultra high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and further confirmed using ultra high-performance liquid chromatography-high resolution accurate mass Orbitrap™ mass spectrometry (UHPL-HRMS). β-CM5 was not released after in-vitro gastrointestinal digestion of all heated milk. Similarly, β-CM7 was not released in all milk with β-CN A2A2 phenotype. However, this peptide level ranged from 127.25 to 198.10 ng/mL (4.94–7.70 ng/mg protein) in heated milk with β-CN A1A1 phenotype, whereas it was released at much lower levels ranging from 19.35 to 24.50 ng/mL (0.71–0.91 ng/mg protein) in heated milk with β-CN A2I phenotype.
The influence of ripening and in vitro digestion on the peptidomic profile of Parmigiano-Reggiano (PR) cheeses was investigated. Ripening and in vitro digestion thoroughly modified the peptidomic profile of the three cheeses. Twenty-six bioactive peptides were identified in undigested PR. Some peptides were degraded and others released during ripening. After digestion, 52 bioactive peptides were identified. Semi-quantitative data suggested that bioactive peptides released after digestion can be clustered in 5 groups according to the ripening time. VPP and IPP peptide levels in undigested samples were in the range of 4.52–11.34 and 0.66–4.24 mg kg⁻¹, with the highest amounts found in 18-month ripened PR. YPFPGPI peptide was absent in undigested PRs but was released after digestion, especially in the 12-month-old sample (20.18 mg kg⁻¹). The present study suggests possible differences in bioactive peptide levels after digestion as a function of the duration of ripening of PR cheese.
Background
Cows’ milk generally contains two types of β-casein, A1 and A2 types. Digestion of A1 type can yield the peptide β-casomorphin-7, which is implicated in adverse gastrointestinal effects of milk consumption, some of which resemble those in lactose intolerance. This study aimed to compare the effects of milk containing A1 β-casein with those of milk containing only A2 β-casein on inflammation, symptoms of post-dairy digestive discomfort (PD3), and cognitive processing in subjects with self-reported lactose intolerance. Methods
Forty-five Han Chinese subjects participated in this double-blind, randomized, 2 × 2 crossover trial and consumed milk containing both β-casein types or milk containing only A2 β-casein. Each treatment period was 14 days with a 14-day washout period at baseline and between treatment periods. Outcomes included PD3, gastrointestinal function (measured by smart pill), Subtle Cognitive Impairment Test (SCIT), serum/fecal laboratory biomarkers, and adverse events. ResultsCompared with milk containing only A2 β-casein, the consumption of milk containing both β-casein types was associated with significantly greater PD3 symptoms; higher concentrations of inflammation-related biomarkers and β-casomorphin-7; longer gastrointestinal transit times and lower levels of short-chain fatty acids; and increased response time and error rate on the SCIT. Consumption of milk containing both β-casein types was associated with worsening of PD3 symptoms relative to baseline in lactose tolerant and lactose intolerant subjects. Consumption of milk containing only A2 β-casein did not aggravate PD3 symptoms relative to baseline (i.e., after washout of dairy products) in lactose tolerant and intolerant subjects. Conclusions
Consumption of milk containing A1 β-casein was associated with increased gastrointestinal inflammation, worsening of PD3 symptoms, delayed transit, and decreased cognitive processing speed and accuracy. Because elimination of A1 β-casein attenuated these effects, some symptoms of lactose intolerance may stem from inflammation it triggers, and can be avoided by consuming milk containing only the A2 type of beta casein. Trial registrationClinicalTrials.gov/NCT02406469
True lactose intolerance (symptoms stemming from lactose malabsorption) is less common than is widely perceived, and should be viewed as just one potential cause of cows' milk intolerance. There is increasing evidence that A1 beta-casein, a protein produced by a major proportion of European-origin cattle but not purebred Asian or African cattle, is also associated with cows' milk intolerance. In humans, digestion of bovine A1 beta-casein, but not the alternative A2 beta-casein, releases beta-casomorphin-7, which activates μ-opioid receptors expressed throughout the gastrointestinal tract and body. Studies in rodents show that milk containing A1 beta-casein significantly increases gastrointestinal transit time, production of dipeptidyl peptidase-4 and the inflammatory marker myeloperoxidase compared with milk containing A2 beta-casein. Co-administration of the opioid receptor antagonist naloxone blocks the myeloperoxidase and gastrointestinal motility effects, indicating opioid signaling pathway involvement. In humans, a double-blind, randomized cross-over study showed that participants consuming A1 beta-casein type cows' milk experienced statistically significantly higher Bristol stool values compared with those receiving A2 beta-casein milk. Additionally, a statistically significant positive association between abdominal pain and stool consistency was observed when participants consumed the A1 but not the A2 diet. Further studies of the role of A1 beta-casein in milk intolerance are needed.
Within each milk protein there are many individual protein variants and marked alterations to milk functionality can occur depending on the genetic variants of each protein present. Bovine A(1) and A(2) β-casein (β-CN) are 2 variants that contribute to differences in the gelation performance of milk. The A(1) and A(2) β-CN variants differ by a single AA, the substitution of histidine for proline at position 67. β-Casein not only participates in formation of the casein micelle but also forms an oligomeric micelle itself and functions as a molecular chaperone to prevent the aggregation of a wide range of proteins, including the other caseins. Micelle assembly of A(1) and A(2) β-CN was investigated using dynamic light scattering and small-angle X-ray scattering, whereas protein functionality was assessed using fluorescence techniques and molecular chaperone assays. The A(2) β-CN variant formed smaller micelles than A(1) β-CN, with the monomer-micelle equilibrium of A(2) β-CN being shifted toward the monomer. This shift most likely arose from structural differences between the 2 β-CN variants associated with the adoption of greater polyproline-II helix in A(2) β-CN and most likely led to enhanced chaperone activity of A(2) β-CN compared with A(1) β-CN. The difference in micelle assembly, and hence chaperone activity, may provide explain differences in the functionality of homozygous A(1) and A(2) milk. The results of this study highlight that substitution of even a single AA can significantly alter the properties of an intrinsically unstructured protein such as β-CN and, in this case, may have an effect on the functionality of milk.
Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.
Simulated gastro-intestinal digestion is widely employed in many fields of food and nutritional sciences, as conducting human trials are often costly, resource intensive, and ethically disputable. As a consequence, in vitro alternatives allowing for the determination of a variety of endpoints such as bioaccessibility of nutrients and non-nutrients, or digestibility of macronutrients such as lipids, proteins and carbohydrates, are used for screening and building new hypotheses. Various digestion models have been proposed, often impeding the possibility to compare results across research teams. For example, a large variety of enzymes from different sources such as of porcine, rabbit or human origin have been used, differing in their activity and characterization. Differences in pH, mineral type, ionic strength and digestion time, which alter enzyme activity and other phenomena, may also considerably alter results. Other parameters such as the presence of phospholipids, individual enzymes such as gastric lipase and digestive emulsifiers vs. their mixtures (e.g. pancreatin and bile salts), and the ratio of food bolus to digestive fluids, have also been discussed at length. In the present consensus paper, within the COST Infogest network, we propose a general standardised and practical static digestion method based on physiologically relevant conditions that can be applied for various endpoints, which may be amended to accommodate further specific requirements. A frameset of parameters including the oral, gastric and small intestinal digestion are outlined and their relevance discussed in relation to available in vivo data and enzymes. This consensus paper will give a detailed protocol and a line-by-line, guidance, recommendations and justifications but also limitation of the proposed model. This harmonised static, in vitro digestion method for food should aid the production of more comparable data in the future.
Abstract We compared the gastrointestinal effects of milk-based diets in which the β-casein component was either the A1 or A2 type in male Wistar rats fed the experimental diets for 36 or 84 h. Gastrointestinal transit time was significantly greater in the A1 group, as measured by titanium dioxide recovery in the last 24 h of feeding. Co-administration of naloxone decreased gastrointestinal transit time in the A1 diet group but not in the A2 diet group. Colonic myeloperoxidase and jejunal dipeptidyl peptidase (DPP)-4 activities were greater in the A1 group than in the A2 group. Naloxone attenuated the increase in myeloperoxidase activity but not that in DPP-4 activity in the A1 group. Naloxone did not affect myeloperoxidase activity or DPP-4 activity in the A2 group. These results confirm that A1 β-casein consumption has direct effects on gastrointestinal function via opioid-dependent (gastrointestinal transit and myeloperoxidase activity) and opioid-independent (DPP-4 activity) pathways.
Elevated concentrations of circulating casomorphins (CM), the exogenous opioid peptides from milk casein, may contribute to the pathogenesis of autism in children. Because several mass spectrometry studies failed to detect casomorphins in autistic children, it was questioned whether these peptides can be detected in body fluids by mass spec. Here we demonstrated, using a novel high sensitivity ELISA method, that autistic children have significantly higher levels of urine CM-7 than control children. The severity of autistic symptoms correlated with concentrations of CM-7 in the urine. Because CMs interact with opioid and serotonin receptors, the known modulators of synaptogenesis, we suggest that chronic exposure to elevated levels of bovine CMs may impair early child development, setting the stage for autistic disorders.
The objective of this study was to examine variation in overall milk, protein, and mineral composition of bovine milk in relation to rennet-induced coagulation, with the aim of elucidating the underlying causes of milk with impaired coagulation abilities. On the basis of an initial screening of 892 milk samples from 42 herds with Danish Jersey and Holstein-Friesian cows, a subset of 102 samples was selected to represent milk with good, poor, or noncoagulating properties (i.e., samples that within each breed represented the most extremes in regard to coagulation properties). Milk with good coagulation characteristics was defined as milk forming a strong coagulum based on oscillatory rheology, as indicated by high values for maximum coagulum strength (G'(max)) and curd firming rate (CFR) and a short rennet coagulation time. Poorly coagulating milk formed a weak coagulum, with a low G'(max) and CFR and a long rennet coagulation time. Noncoagulating milk was defined as milk that failed to form a coagulum, having G'(max) and CFR values of zero at measurements taken within 1 h after addition of rennet. For both breeds, a lower content of total protein, total casein (CN) and κ-CN, and lower levels of minerals (Ca, P, Mg) were identified in poorly coagulating and noncoagulating milk in comparison with milk with good coagulation properties. Liquid chromatography/electrospray ionization-mass spectrometry revealed the presence of a great variety of genetic variants of the major milk proteins, namely, α(S1)-CN (variants B and C), α(S2)-CN (A), β-CN (A(1), A(2), B, I, and F), κ-CN (A, B, and E), α-lactalbumin (B), and β-lactoglobulin (A, B, and C). In poorly coagulating and noncoagulating milk samples of both breeds, the predominant composite genotype of α(S1)-, β-, and κ-CN was BB-A(2)A(2)-AA, which confirmed a genetic contribution to impaired milk coagulation. Interestingly, subtle variations in posttranslational modification of CN were observed between the coagulation classes in both breeds. Poorly coagulating and noncoagulating milk contained a lower fraction of the least phosphorylated α(S1)-CN form, α(S1)-CN 8P, relative to total α(S1)-CN, along with a lower fraction of glycosylated κ-CN relative to total κ-CN. Thus, apparent variation was observed in the milk and protein composition, in the genetic makeup of the major milk proteins, and in the posttranslational modification level of CN between milk samples with either good or impaired coagulation ability, whereas the composition of poorly coagulating and noncoagulating milk was similar.
The aim of this study was to characterise the individual human gastric and duodenal juices to be used in in vitro model digestion and to examine the storage stability of the enzymes. Gastroduodenal juices were aspirated, and individual variations in enzymatic activities as well as total volumes, pH, bile acids, protein and bilirubin concentrations were recorded. Individual pepsin activity in the gastric juice varied by a factor of 10, while individual total proteolytic activity in the duodenal juice varied by a factor of 5. The duodenal amylase activity varied from 0 to 52.6 U/ml, and the bile acid concentration varied from 0.9 to 4.5 mM. Pooled gastric and duodenal juices from 18 volunteers were characterised according to pepsin activity (26.7 U/ml), total proteolytic activity (14.8 U/ml), lipase activity (951.0 U/ml), amylase activity (26.8 U/ml) and bile acids (4.5 mM). Stability of the main enzymes in two frozen batches of either gastric or duodenal juice was studied for 6 months. Pepsin activity decreased rapidly and adjusting the pH of gastric juice to 4 did not protect the pepsin from degradation. Lipase activity remained stable for 4 months, however decreased rapidly thereafter even after the addition of protease inhibitors. Glycerol only marginally stabilised the survival of the enzymatic activities. These results of compositional variations in the individual gastrointestinal juices and the effect of storage conditions on enzyme activities are useful for the design of in vitro models enabling human digestive juices to simulate physiological digestion.
The aim of this study was to compare the digestion of milk proteins from different species using an in vitro gastrointestinal model. Raw and heated milks from bovine, caprine, human and equine species were digested by human digestive enzymes. Digestion was performed in two 30-min sequential steps by digestive juices from the stomach (pH 2.5/37 °C) and from the duodenum (pH 8.0/37 °C). The degradation patterns of the milk proteins were visualized by SDS-PAGE and quantified using the ImageQuant program. Caseins in the equine milk were rapidly digested by the gastric juice in contrast to the caseins from the other species. During the subsequent digestion by the duodenal juice most of the caseins from all species were degraded within 5 min, and within 30 min only traces of caseins were detected. The mean casein micellar size varied between species in the range of 146.0–311.5 nm (equine > caprine > bovine > human). The α-lactalbumin from all species appeared to be very resistant to both gastric and duodenal digestions. A similar trend was shown for β-lactoglobulin from bovine and caprine milks, of which ∼ 60% intact protein remained, while only 25% remained intact in equine milk after total digestion. Equine milk contained a high amount of lysozyme, of which 60% remained intact in the present study. In heated milks from all species, only α-lactalbumin degradation increased approximately 12–20% in comparison to the raw milk. This study shows that equine milk with fast digestible proteins could be considered as a replacement for bovine milk in the diet of people with special needs, such as infants and the elderly.
The 6 main milk proteins in cattle are encoded by highly polymorphic genes characterized by several nonsynonymous and synonymous mutations, with up to 47 protein variants identified. Such an extensive variation was used for linkage analysis with the description of the casein cluster more than 30 yr ago and has been applied to animal breeding for several years. Casein haplotype effects on productive traits have been investigated considering information on the whole casein complex. Moreover, mutations within the noncoding sequences have been shown to affect the specific protein expression and, as a consequence, milk composition and cheesemaking. Milk protein variants are also a useful tool for breed characterization, diversity, and phylogenetic studies. In addition, they are involved in various aspects of human nutrition. First, the occurrence of alleles associated with a reduced content of different caseins might be exploited for the production of milk with particular nutritional qualities; that is, hypoallergenic milk. On the other hand, the frequency of these alleles can be decreased by selection of sires using simple DNA tests, thereby increasing the casein content in milk used for cheesemaking. Furthermore, the biological activity of peptides released from milk protein digestion can be affected by amino acid exchanges or deletions resulting from gene mutations. Finally, the gene-culture coevolution between cattle milk protein genes and human lactase genes, which has been recently highlighted, is impressive proof of the nonrandom occurrence of milk protein genetic variation over the centuries.
This report of the American Dairy Science Association Committee on the Nomenclature, Classification, and Methodology of Milk Proteins reviews changes in the nomenclature of milk proteins necessitated by recent advances of our knowledge of milk proteins. Identification of major caseins and whey proteins continues to be based upon their primary structures. Nomenclature of the immunoglobulins consistent with new international standards has been developed, and all bovine immunoglobulins have been characterized at the molecular level. Other significant findings related to nomenclature and protein methodology are elucidation of several new genetic variants of the major milk proteins, establishment by sequencing techniques and sequence alignment of the bovine caseins and whey proteins as the reference point for the nomenclature of all homologous milk proteins, completion of crystallographic studies for major whey proteins, and advances in the study of lactoferrin, allowing it to be added to the list of fully characterized milk proteins.
A proteomic approach was applied to characterize genetic variation and relative protein content of β-casein (β-CN) in milk from two minor native dairy breeds in Denmark; Red Danish 1970 and Jutland cows in comparison to the major dairy breeds; Danish Holstein and Danish Jersey. Genetic β-CN variants were related to the relative concentration of β-CN. Furthermore, a very high frequency of variant F in the native breeds was found. This was remarkable as this variant is only found in a very low frequency in the major Danish breeds. Novel associations were found between β-CN variant F and milk coagulation properties in the native breeds, which is an important finding for understanding the genetic background for non- and poor coagulating milk and potential exploitation of β-CN variants in selective breeding programs. Furthermore, in Red Danish 1970 a very high prevalence of non-coagulating milk was found (18%).
This study investigated whether different genetic variants of 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 BCM7 does not seem to be linked solely to variant A1, as earlier suggested by relevant published literature on in vitro digestion.
The effect on rennet-induced milk coagulation of three novel genetic haplotypes in close proximity to CSN3 encoding κ-casein was evaluated. Milk samples were collected from 71 Danish Holstein cows homozygous for the three novel haplotypes named according to which genetic variants of CSN3 they were characterised by: AE, A and B, respectively. The results documented that haplotype AE had significantly longer rennet coagulation time and lower curd firming rate compared with haplotypes A and B. Haplotype AE milk was further characterised by larger casein micelles and lower relative content of κ-casein, whereas the total protein contents did not differ among haplotypes. These findings indicate that the genetic κ-casein A variant can be divided into two groups with poor and good milk coagulation properties. Furthermore, three milk samples were identified as non-coagulating. These were all associated with the haplotype AE.
Milk from 94 Ethiopian goats (four breeds) was assessed for casein level, micellar size, coagulation properties and whey syneresis to evaluate cheese-making potential. Casein content (g kg−1) ranged from 6.84 to 8.94 (αs1); 4.16 to 4.98 (αs2); 12.51 to 15.37 (β) and: 6.89 - 8.93 (κ). Arsi-Bale goats had the highest αs1-casein (8.94 g/kg) followed by the Somali goats (7.90 g/kg); and were higher than Boer (7.05 g/kg) and Cross (6.84 g/kg) (P < 0.001). Milk from Arsi-Bale and Somali goats had significantly (P < 0.05) smaller micelles; 207.47 and 209.29 nm respectively than Boer (230.30 nm) and Cross (228.71 nm). Significantly stronger gels (P < 0.05) were obtained from milk samples with higher αs1-CN level and smaller micelles (Arsi-Bale 37.99 mm and Somali 36.19 mm) than samples with lower αs1-CN level and larger micelles (Boer 31.13 mm and Cross 33.51 mm). Syneresis was significantly higher (P < 0.001) in milk with good coagulation properties. Curd firmness was negatively correlated with coagulation rate (R, -0.832) and micellar size (R, -0.647). Milk from Arsi-Bale and Somali goats had significantly higher αs1-CN level, smaller micelle and better cheese making property than the cross and Boer goats. This study shows that there exists high potential among indigenous goats for cheese production.
Substantial variation in milk coagulation properties has been observed among dairy cows. Consequently, raw milk from individual cows and breeds exhibits distinct coagulation capacities that potentially affect the technological properties and milk processing into cheese. This variation is largely influenced by protein composition, which is in turn affected by underlying genetic polymorphisms in the major milk proteins. In this study, we conducted a large screening on 3 major Scandinavian breeds to resolve the variation in milk coagulation traits and the frequency of milk with impaired coagulation properties (noncoagulation). In total, individual coagulation properties were measured on morning milk collected from 1,299 Danish Holstein (DH), Danish Jersey (DJ), and Swedish Red (SR) cows. The 3 breeds demonstrated notable interbreed differences in coagulation properties, with DJ cows exhibiting superior coagulation compared with the other 2 breeds. In addition, milk samples from 2% of DH and 16% of SR cows were classified as noncoagulating. Furthermore, the cows were genotyped for major genetic variants in the αS1- (CSN1S1), β- (CSN2), and κ-casein (CSN3) genes, revealing distinct differences in variant frequencies among breeds. Allele I of CSN2, which had not formerly been screened in such a high number of cows in these Scandinavian breeds, showed a frequency around 7% in DH and DJ, but was not detected in SR. Genetic polymorphisms were significantly associated with curd firming rate and rennet coagulation time. Thus, CSN1S1C, CSN2B, and CSN3B positively affected milk coagulation, whereas CSN2A(2), in particular, had a negative effect. In addition to the influence of individual casein genes, the effects of CSN1S1-CSN2-CSN3 composite genotypes were also examined, and revealed strong associations in all breeds, which more or less reflected the single gene results. Overall, our results strongly suggest that milk coagulation is under the influence of additive genetic variation. Optimal milk for future cheese production can be ensured by monitoring the frequency of unfavorable variants and thus preventing an increase in the number of cows producing milk with impaired coagulation. Selective breeding for variants associated with superior milk coagulation can potentially increase raw milk quality and cheese yield in all 3 Scandinavian breeds.
The A1 variant protein of the β-casein family has been implicated in various disease states although much evidence is weak or contradictory. The primary objective was to measure, for the first time, the proportions of the key β-casein variant proteins in UK retail milk over the course of one year. In total, 55 samples of semi-skimmed milk were purchased from five supermarkets over the course of a year and the proportions of the A1, A2, B and C casein variant proteins were measured, using high resolution HPLC-MS. The results showed that β-casein in UK retail milk comprises approximately 0.58, 0.31, 0.07 and 0.03 A2, A1, B and C protein variants, respectively. The proportion of A2 is higher than some early studies would predict although the reasons for this and any implications for health are unclear.
Capillary zone electrophoresis (CZE) was tested for reproducibility and was shown to be a suitable method for estimating the relative concentration of α-lactalbumin, β-lactoglobulin, αS2-casein (αS2-CN), αS1-CN, κ-CN, and β-CN in milk. This study showed how the method can also be used to determine the relative concentration of two different phosphorylation states of αS1-CN (αS1-CN-8P and αS1-CN-9P) and three of αS2-CN (αS2-CN-10P, αS2-CN-11P and αS2-CN-12P). Furthermore, with CZE it was possible to determine most common genetic variants including the κ-CN E variant that has not been identified by CZE before. Analyses of milk samples of 1948 Dutch Holstein-Friesian cows showed large variation in both the relative protein concentration and the relative concentration of different phosphorylation states for all milk proteins studied. Correlations between different phosphorylation states of αS1- and αS2-CN indicated that the relative concentration of specific phosphorylation states of αS1- and αS2-CN were related to each other.
The aim of the present investigation was to study the underlying causes of noncoagulating (NC) milk. Based on an initial screening in a herd of 53 Danish Holstein-Friesians, 20 individual Holstein-Friesian cows were selected for good and poor chymosin-induced coagulation properties; that is, the 10 cows producing milk with the poorest and best coagulating properties, respectively. These 20 selected cows were followed and resampled on several occasions to evaluate possible changes in coagulation properties. In the follow-up study, we found that among the 10 cows with the poorest coagulating properties, 4 cows consistently produced poorly coagulating (PC) or NC milk, corresponding to a frequency of 7%. Noncoagulating milk was defined as milk that failed to form a coagulum, defined as increase in the storage modulus (G') in oscillatory rheometry, within 45min after addition of chymosin. Poorly coagulating milk was characterized by forming a weak coagulum of low G'. Milk proteomic profiling and contents of different casein variants, ionic contents of Ca, P and Mg, κ-casein (CN) genotypes, casein micelle size, and coagulation properties of the 4 NC or PC samples were compared with milk samples of 4 cows producing milk with good coagulation properties. The studies included determination of production of caseinomacropeptide to ascertain whether noncoagulation could be ascribed to the first or second phase of chymosin-induced coagulation. Caseinomacropeptide was formed in all 8 milk samples after addition of chymosin, indicating that the first step (cleavage of κ-CN) was not the cause of inability to coagulate. Furthermore, the effect of mixing noncoagulating and well-coagulating milk was studied. By gradually blending NC with well-coagulating milk, the coagulation properties of the well-coagulating samples were compromised in a manner similar to titration. Milk samples from cows that consistently produced NC milk were further studied at the udder quarter level. The coagulation properties of the quarter milk samples were not significantly different from those of the composite milk sample, showing that poor coagulation traits and noncoagulation traits of the composite milk were not caused by the milk quality of a single quarter. The milk samples exhibiting PC or NC properties were all of the κ-CN variant AA genotype, and contained casein micelles with a larger mean diameter and a lower fraction of κ-CN relative to total CN than milk with good coagulation properties. Interestingly, the relative proportions of different phosphorylation forms of α-CN differed between well-coagulating milk and PC or NC milk samples. The PC and NC milk samples contained a lower proportion of the 2 less-phosphorylated variants of α-CN (α(S1)-CN-8P and α(S2)-CN-11P) compared with samples of milk that coagulated well.
This review outlines a hypothesis that A1 one of the common variants of beta-casein, a major protein in cows milk could facilitate the immunological processes that lead to type I diabetes (DM-I). It was subsequently suggested that A1 beta-casein may also be a risk factor for coronary heart disease (CHD), based on between-country correlations of CHD mortality with estimated national consumption of A1 beta-casein in a selected number of developed countries. A company, A2 Corporation was set up in New Zealand in the late 1990s to test cows and market milk in several countries with only the A2 variant of beta-casein, which appeared not to have the disadvantages of A1 beta-casein. The second part of this review is a critique of the A1/A2 hypothesis. For both DM-I and CHD, the between-country correlation method is shown to be unreliable and negated by recalculation with more countries and by prospective studies in individuals. The animal experiments with diabetes-prone rodents that supported the hypothesis about diabetes were not confirmed by larger, better standardised multicentre experiments. The single animal experiment supporting an A1 beta-casein and CHD link was small, short, in an unsuitable animal model and had other design weaknesses. The A1/A2 milk hypothesis was ingenious. If the scientific evidence had worked out it would have required huge adjustments in the world's dairy industries. This review concludes, however, that there is no convincing or even probable evidence that the A1 beta-casein of cow milk has any adverse effect in humans. This review has been independent of examination of evidence related to A1 and A2 milk by the Australian and New Zealand food standard and food safety authorities, which have not published the evidence they have examined and the analysis of it. They stated in 2003 that no relationship has been established between A1 or A2 milk and diabetes, CHD or other diseases.
Beta-kasein i NRF-mjølk
- A G Larsgard
Larsgard, A. G. (2017). Beta-kasein i NRF-mjølk. Buskap, 7, 20e21. Retrieved from:
http://viewer.zmags.com/publication/1f00a22d#/1f00a22d/20.