Article

Occurrence of β-casomorphins 5 and 7 in commercial dairy products and in their digests following in vitro simulated gastro-intestinal digestion

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Abstract

The occurrence of β-casomorphin-5 (BCM5) and β-casomorphin-7 (BCM7) was investigated in commercial dairy products and in their digests, following in vitro simulated gastro-intestinal digestion (SGID), by means of HPLC–MS. The analysed dairy products were as follows: 10 cheeses (Gorgonzola, Caprino, Brie, Taleggio, Gouda, Fontina, Cheddar and Grana Padano 10-, 15- or 25-m ripened); 4 samples of drinking milk (unprocessed, pasteurised, UHT and in bottle-sterilised); 2 yoghurts and 4 fermented milks containing probiotics; 7 infant formulas; and 4 dried milk-derivatives (skim milk powder, calcium caseinate and milk protein concentrates). β-Casomorphin-5 was not detected in dairy products, either prior to or after SGID. β-Casomorphin-7 was detected only in cheeses with the exception of Taleggio, Caprino and Grana Padano samples. Peptide amount was in the range 0.01–0.15 mg kg−1 the highest level being recovered in Brie sample. Following SGID, BCM7 formed in all dairy samples or increased up to 21.77 mg kg−1 in digests of cheeses. The peptide level ranged from 0.29 to 1.23 mg kg−1in fermented milks and from 3.46 to 22.18 mg kg−1 in dried milk-derivatives. Digests of commercial infant formulas contained BCM7 at concentrations of 0.04–0.21 mg l−1. For the first time, this work reports quantitative values for BCM5 and BCM7 in a range of dairy products providing evidence that, during processing, only proteolytic systems involved in manufacturing and ripening of cheese can potentially hydrolyse β-CN to BCM7. Nevertheless, formation or further release of BCM7 is mainly promoted by the action of gastrointestinal proteinases during in vitro digestion irrespective of the type of dairy product.

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... Destaca-se que este peptídeo bioativo além de apresentar efeitos locais no trato digestório, poderá ser absorvido e atingir o sistema nervoso central, sendo esta absorção possivelmente maior em estados de hiperpermeabilidade intestinal. A βcasomorphina-7 também pode ser encontrada em derivados do leite A1, tais como como iogurte e queijos [18,19] , embora seja proposto que certos micro-organismos presentes nestes produtos teriam a capacidade de hidrolisar o BCM-7 até peptídeos menores ou mesmo em aminoácidos [18] . Ainda não foi demostrado se tais micro-organismos também apresentam similar influência dentro do trato gastrintestinal [20] . ...
Article
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... Evidence on the release of BCM7 by SGID has been derived primarily from enzymatic digestions including an initial digestion of BC with pepsin at pH 2.0. However, recent research is now considering that physiological pH values of infant's stomach are quite different from the optimum for pepsin [31,47,48]. In the present investigation, SGID was performed with an initial peptic attack at pH 4 followed by proteolytic enzymes hydrolysis including several amino-and carboxi-peptidases in addition to trypsin and α-chymotrypsin. ...
... In this work, the amount of BCM7 released from Holstein Friesian raw cow's milk was significantly higher (2.11 ± 0.19 µg/100 mL) than BC A2 milk (0.74 ± 0.008 µg/100 mL) and IF 1 (0.86 ± 0.01 µg /100 mL). Similar observations on BCM7 release from cow's milk have been reported by other authors [31,32,47]. Raies et al. [32] have shown that SGID of BC fraction from cow's milk previously genotyped as CSN2 A2A1 results in the release of BCM7. ...
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Beta-casein (BC) is frequently expressed as BC A2 and BC A1 in cow's milk. Gastrointestinal digestion of BC A1 results in the release of the opioid peptide beta-casomorphin 7 (BCM7) which is less likely to occur from BC A2. This work was aimed to produce milk containing BC A2 with no BC A1 (BC A2 milk) using genetically selected CSN2 A2A2 Jersey cows. Additionally, we aimed to develop an infant formula (IF) suitable for healthy full-term infants during the first six months of life based on BC A2 milk. The concentration of BCM7 released from BC A2 IF, from commercially available IFs as well as from human milk and raw cow's milk was evaluated after simulated gastrointestinal digestion (SGID). BC A2 IF presented the lowest mean relative abundance of BC A1 (IF 1 = 0.136 ± 0.010), compared with three commercially available IFs (IF 2 = 0.597 ± 0.020; IF 3 = 0.441 ± 0.014; IF 4 = 0.503 ± 0.011). Accordingly, SGID of whole casein fraction from BC A2 IF resulted in a significantly lower release of BCM7 (IF 1 = 0.860 ± 0.014 µg/100 mL) compared to commercially available IFs (IF 2 = 2.625 ± 0.042 µg/100 mL; IF 3 = 1.693 ± 0.012 µg/100 mL; IF 4 = 1.962 ± 0.067 µg/100 mL). Nevertheless, BCM7 levels from BC A2 IF were significantly higher than those found in SGID hydrolysates of BC A2 raw milk (0.742 ± 0.008 µg/100 mL). Interestingly, results showed that BCM7 was also present in human milk in significantly lower amounts (0.697 ± 0.007 µg/100 mL) than those observed in IF 1 and BC A2 milk. This work demonstrates that using BC A2 milk in IF formulation significantly reduces BCM7 formation during SGID. Clinical implications of BC A2 IF on early infant health and development need further investigations.
... If it has A2A2 genotype, it can be used in mating program in selection. Secondly, in case there are stages to be applied according to the genotypes of the female offspring to be obtained in selection, Based on the research results showing that BCM-7 is ineffective in the production process until the production of A2 milk from A1 milk obtained Ö. ŞAHIN, S. BOZTEPE from female animals with A1A1 and A1A2 genotype, these milks can be used for yoghurt and some cheeses (De Noni and Cattaneo, 2010;Nguyen et al., 2015). When the transition phase is completed, Animals with A1A1 genotypes cannot be used for selection in the next generation that can be sent to slaughter at the slaughter age. ...
Article
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... Overall, the existing data report a BCM7 amount in the order of mg per liter of liquid or reconstituted IFs submitted to SGID. Only De Noni and Cattaneo (2010) reported the BCM7 content in some powdered milk derivatives submitted to SGID (with pepsin and Corolase PP): SMP 3.46 mg/kg; sodium caseinate 17.68 mg/kg; MPC 16.99-22.18 mg/kg. ...
Article
Infant biscuits (IBs) are commonly used during the complementary feeding of infants from the 6th month of life. They contain wheat flour and dairy ingredients, which can release the opioid-acting peptides β-casomorphins (BCMs) and gluten exorphins (GEs) after gastrointestinal digestion. In the present study, five model IBs were prepared with or without gluten and different powdered milk derivatives in the formulations. IBs were digested simulating an in vitro static gastrointestinal digestion for infants aged 6–12 months. BCMs and GEs were identified and quantified by UPLC/HR-MS. The amounts of BCM7 and the GE A5 were related to the β-CN and gluten content of the formulations. To date, levels of BCMs and GEs in digests of IBs have not been reported in literature. This work represents an in vitro investigation regarding the release of opioid-acting peptides in IBs. It could add additional knowledge on complementary foods for infant health.
... Bioactive peptides vary due to the genetic polymorphism for β-casein protein. β-Casomorphin7 (BCM7), having opioid like properties, can be released easily from A1 type β-casein [24,25]. BCM7 has potential negative effect on the opioid receptors of nervous system, endocrine system, and immune system of the human body. ...
... Other dairy products such as cheese and yogurt are also reported as sources of BCMs that parallel their A1/A2 milk lineages (De Noni and Cattaneo 2010). ...
Article
Beyond being a source of key nutrients, bovine milk influences physiological functions by synthesising bioactive peptides during the process of digestion. Some of the claimed negative health outcomes associated with milk consumption, such as cardiovascular diseases and type 1 diabetes may be attributed to an opioid peptide, beta-casomorphin-7 (BCM-7), derived from A1 beta-casein. BCM-7 exerts its function by binding to the μ-opioid receptors in the body. It is hypothesised that activation of the μ-opioid receptors in the gut can alter gut microbial composition, impair gut barrier integrity and bile acid metabolism, in addition to increasing gastrointestinal transit time and gut inflammation. Further, it is hypothesised that BCM-7 may influence fractures and obesity via μ-opioid receptor pathways. In conclusion, it appears that BCM-7 might have multiple functions pertinent to human health; however, the evidence is limited and warrants further pre-clinical and clinical studies for hypothesis confirmation.
... The controversy surrounding A1 versus A2 relates to the digestion of β-casein. When proteins are digested, bioactive peptides can be released [31]. A peptide is simply a small chain of amino acids. ...
Article
Full-text available
Milk proteins are a heterogeneous group of polymeric compounds that have a wide range of different molecular structures and properties. They occur as caseins, whey proteins, enzymes, minor proteins and nitrogen compounds. Caseins constitute about 80% of the total proteins of cow’s milk. β-casein is an important part of the caseins, which makes up about 37% of the total caseins. Within β-casein, there are a number of variants which are genetically determined. However, there are thirteen genetic variants of β-casein found in cow´s milk. A1 and A2 are the most common variants, which are called A1 β-casein (A1-milk) and A2 β-casein (A2-milk). The only difference between A1- and A2-milk is a difference in the 67th amino acid in the chain. At this position, A2-milk has a proline amino acid, while A1-milk has histidine amino acid. Several studies have reported that cow’s milk with a dominant or singular A2-milk may be healthier than A1-milk. These studies are based on digestion of A1milk which lead to release β-casomorphin-7 (BCM-7). Subsequently increase inflammation, Type-1-diabetes, heart disease, autism, gastrointestinal discomfort and other disease in the consumer. For this reason, there is a growing global interest in A2-milk. In conclusion, the effects of A1-milk compared to A2-milk on human health show mixed results. On the basis of the available results, we cannot conclusively assess the health effects of A1milk and A2-milk. Therefore, further investigations are needed.
... However, A2 β-casein not been linked to any of such health issues ( Kaminski et al., 2007). A1, B and C β-casein have a histidine residue at position 67 that allows an enzymatic cleavage to occur during digestion, releasing the seven amino acid peptide, β-casomorphin 7 (De Noni and Cattaneo, 2010). ...
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Milk is a complete food as it contains all essential micro-nutrients needed for growth and development of human health as well as for neonate animals. However, milk derivative peptides may cause deleterious effect on human health by elevating risk for type I diabetes (DM-1), coronary heart disease (CHD), schizophrenia and autism. The higher occurrences of these diseases have relationship with consumption of variants A1, B and C beta-casein from cow's milk. The production of BCM-7 is more in A1 milk than A2 milk and the difference is basically at 67th position of the beta casein chain. Due to presence of histidine at amino acid at 67th position, digestion of A1 β-casein milk releases a 7 amino acid bioactive peptide called β-casomorphin 7 (BCM-7) in small intestine, while proline in A2 milk at 67th position prevents splitting at this particular site. So propensity is now towards consumption of A2 milk. It is a matter of great concern for the health of people in India.
... The casein and whey proteins found in milk are a good source of the bioactive peptides that have a positive impact on body functions. These peptides are inactive within the sequence of precursor proteins but can be released in vivo or in vitro by enzymatic hydrolysis [10] or during fermentation with lactic acid bacteria [11]. Other methods have also been confirmed for releasing bioactive peptides, such as chemical synthesis [12], extraction from natural foods [13], and DNA recombinant technology [14]. ...
Article
Full-text available
Angiotensin I-converting enzyme (ACE) peptides are bioactive peptides that have important value in terms of research and application in the prevention and treatment of hypertension. While widespread literature is concentrated on casein or whey protein for production of ACE-inhibitory peptides, relatively little information is available on selecting the proper proteases to hydrolyze the protein. In this study, skimmed cow and goat milk were hydrolyzed by four commercial proteases, including alkaline protease, trypsin, bromelain, and papain. Angiotensin I-converting enzyme-inhibitory peptides and degree of hydrolysis (DH) of hydrolysates were measured. Moreover, we compared the difference in ACE-inhibitory activity between cow and goat milk. The results indicated that the DH increased with the increase in hydrolysis time. The alkaline protease-treated hydrolysates exhibited the highest DH value and ACE-inhibitory activity. Additionally, the ACE-inhibitory activity of hydrolysates from goat milk was higher than that of cow milk-derived hydrolysates. Therefore, goat milk is a good source to obtain bioactive peptides with ACE-inhibitory activity, as compared with cow milk. A proper enzyme to produce ACE-inhibitory peptides is important for the development of functional milk products and will provide the theoretical basis for industrial production.
... mg/kg have recently been found in bread and pasta following the simulation of in vitro GI digestion (using pepsin-trypsin-chymotrypsin) [137]. Similarly, casomorphin releases were analyzed in milk and its products after simulated GI digestion by different enzymes [138]. ...
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World Health Organization data suggest that stress, depression, and anxiety have a noticeable prevalence and are becoming some of the most common causes of disability in the Western world. Stress-related disorders are considered to be a challenge for the healthcare system with their great economic and social impact. The knowledge on these conditions is not very clear among many people, as a high proportion of patients do not respond to the currently available medications for targeting the monoaminergic system. In addition, the use of clinical drugs is also associated with various side effects such as vomiting, dizziness, sedation, nausea, constipation, and many more, which prevents their effective use. Therefore, opioid peptides derived from food sources are becoming one of the safe and natural alternatives because of their production from natural sources such as animals and plant proteins. The requirement for screening and considering dietary proteins as a source of bioactive peptides is highlighted to understand their potential roles in stress-related disorders as a part of a diet or as a drug complementing therapeutic prescription. In this review, we discussed current knowledge on opioid endogenous and exogenous peptides concentrating on their production, purification, and related studies. To fully understand their potential in stress-related conditions, either as a drug or as a therapeutic part of a diet prescription, the need to screen more dietary proteins as a source of novel opioid peptides is emphasized.
... Although some cattle breeds maintain the A2 β-casein variant, a single nucleotide polymorphism in modern western cattle breeds means that they exhibit mixed A1 and A2 β-casein variants [3][4][5]. Digestive enzymes act on A1 β-casein and hydrolyze it, releasing beta-casomorphin-7 (BCM-7) [6][7][8][9][10]. The histidine residue in A1 β-casein allows cleavage to form BCM-7, whereas the proline residue in A2 β-casein limits such cleavage and BCM-7 formation [11]. ...
Article
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Acute-feeding and multiple-day studies have demonstrated that milk containing A2 β-casein only causes fewer symptoms of lactose intolerance (LI) than milk containing both A1 and A2 β-caseins. We conducted a single-meal study to evaluate the gastrointestinal (GI) tolerance of milk containing different concentrations of A1 and A2 β-casein proteins. This was a randomized, double-blind, crossover trial in 25 LI subjects with maldigestion and an additional eight lactose maldigesters who did not meet the QLCSS criteria. Subjects received each of four types of milk (milk containing A2 β-casein protein only, Jersey milk, conventional milk, and lactose-free milk) after overnight fasting. Symptoms of GI intolerance and breath hydrogen concentrations were analyzed for 6 h after ingestion of each type of milk. In an analysis of the 25 LI subjects, total symptom score for abdominal pain was lower following consumption of milk containing A2 β-casein only, compared with conventional milk (p = 0.004). Post hoc analysis with lactose maldigesters revealed statistically significantly improved symptom scores (p = 0.04) and lower hydrogen production (p = 0.04) following consumption of milk containing A2 β-casein only compared with conventional milk. Consumption of milk containing A2 β-casein only is associated with fewer GI symptoms than consumption of conventional milk in lactose maldigesters.
... BCM is one of the β-casomorphins (BCMs) in the opioid peptide group and is generated from the digestion of β-casein (De Noni & Cattaneo, 2010). Several studies have reported the formation of BCM in cheese and raw milk. ...
Article
Food-derived opioid peptides that are released from proteins by digestion, fermentation, or food production processes lead to several health problems. The opioids are generally resistant to hydrolyze by proteases, except the dipeptidyl peptidase IV (DPPIV, EC 3.4.14.5) enzyme, because of proline amino acid. β-casomorphin (BCM) from milk casein, gluteomorphin (GM) from wheat gluten, and soymorphin (SM) from the soybean β-conglycinin β-subunit are natural substrates of DPPIV because of their amino acid sequences and proline location. In the present study, DPPIV from Lactococcus lactis spp. lactis was purified and characterized by mass spectrometry. Purified DPPIV was added to standard BCM, GM, and SM, and hydrolysis percentages of morphins were measured by HPLC analysis. The results indicated that DPPIV enzyme hydrolyzed food-derived opioids (from 0.1 mM to 2 mM), BCM (33.42% for 2 mM), SM (83.81% for 2 mM), and GM (45.73% for 2 mM) in vitro. Hydrolysis percentages of SM were considerably higher than the same concentrations with BCM and GM. For dietary supplements to be promising for reducing the adverse effects of food derived opioids, this must be supported by in vivo studies of DPPIV use in the human body.
... An example of a peptide that interacts with the CNS is β-casomorphine that exert analgesic effects by acting like opioids (132). Studies have also demonstrated peptides' gut interference via enhanced mucin production, thereby inhibiting the pathogens adherence while enhancing the GI retention which has been linked to a better weight management, through regulated food intake (133). ...
Article
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Coronavirus disease (COVID-19) is a global health challenge, caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) triggers a plethora of respiratory disturbances and even multiple organs failure that can be fatal. Nutritional intervention is one of the key components towards to a proper management of COVID-19 patients, especially in those requiring medication, and should thus be considered the first-line treatment. Immuno-modulation and -stimulation are currently being explored in COVID-19 management and are gaining interest by food and pharmaceutical industries. Various dietary combinations, bioactive components, nutrients and fortified foods have been reported to modulate inflammation during disease progression. Dietary combinations of dairy-derived products and eggs are gaining an increasing attention given the huge immunomodulatory and anti-inflammatory properties attributed to some of their chemical constituents. Eggs are complex dietary components containing many essential nutrients and bioactive compounds as well as a high-quality proteins. Similarly, yogurts can replenish beneficial bacteria and contains macronutrients capable of stimulating immunity by enhancing cell immunity, reducing oxidative stress, neutralizing inflammation and regulating the intestinal barriers and gut microbiome. Thus, this review highlights the impact of nutritional intervention on COVID-19 management, focusing on the immunomodulatory and inflammatory effects of immune-enhancing nutrients.
... Histidine present at position 67 in A1 β-casein results in the cleavage of the preceding seven amino acid residues, generating the peptide β-casomorphin-7 (βCM-7) (Jinsmaa & Yoshikawa, 1999). Compared to A2, the CSN2 A1 variant is more easily hydrolysed by digestive enzymes present in the human gastrointestinal tract, due to weaker binding between the isoleucine and histidine, releasing the peptide β-casomorphine-7 (βCM-7) (De Noni & Cattaneo, 2010;De Noni, 2008;Nguyen, Johnson, Busetti, & Solah, 2015). βCM-7 obtained after A1 milk digestion may be associated with increased risk of diseases, such as Type 1 diabetes mellitus (Chia et al., 2017), autism (Jarmołowska et al., 2019;Whiteley et al., 2010), schizophrenia (Severance et al., 2010) and increased gastrointestinal inflammation (Jianqin et al., 2016). ...
Article
Cows' milk may contain two types of β-casein: A1 and A2. A1 digestion is associated with the release of β-casomorphine-7 peptide, which can cause adverse gastrointestinal effects. Two methods - high-resolution melting (HRM) and rhAmp® SNP genotyping - were developed to identify the β-casein gene (CSN2) A1 and A2 alleles directly in milk. DNA milk samples from 45 animals were examined and 10 samples were also sequenced to confirm the accuracy of the assays. The analytical sensitivities of both strategies for A1 allele identification were evaluated by testing decreasing dilutions of A1 allele DNA copies (500 - 5 copies) in the A2 sample. The limits of detection for A1 in A2 samples were 10% (100 copies) and 2% (10 copies) for HRM and rhAmp, respectively. Both techniques were specific, differentiating between A1 and A2 alleles. However, we recommend rhAmp genotyping testing over HRM because of its enhanced sensitivity for A1.
... In addition, the B variant differs from the A1 variant in a substitution of arginine for serine at position 122. Due to the existence of a histidine in the beta casein protein sequence at position 67 that has the potential to result in cleavage occurring upon digestion and a bioactive peptide, â casomorphin being librated (Kamiñski et al., 2007;Caroli et al., 2009;De Noni and Cattaneo, 2010). ...
Article
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The beta casein gene (CSNS2) has 12 genetic variants divided into two groups: the first group (A1, B, and G) which differ from the second group (A2, A3 C, D, E, F, H, H2 and I) where A base replaces C base, this leads to potential liberation of a bioactive peptide, b-casomorphin, upon digestion where a histidine replaces a proline at position 67. The allele specific polymerase chain reaction (AS-PCR) was evaluated to distinguish between the beta casomorphin releasing variants (A1 and B) and the non-releasing variants. The sequence analysis was used to determine these variants and confirm it in goat, sheep and cattle. The results showed that cattle carrying allele A1 either homozygous or heterozygous more than sheep and goats. The allele frequency of A1 and A2 is 0.44, 0.56 in goats, 0.43, 0.57 in sheep and 0.54, 0.46 in cattle, respectively. The sequence results reported changing of C base to A base in goat, sheep and cattle. Therefore, this study reported that goat and sheep milk was more safe than cattle milk.
... One research group has consistently reported small amounts of BCM-7 in fresh milk and in hydrolyzed milk from both A1/A1 and A2/A2 cows after an intensive 24-h acidic (pH 2.0) pepsin digestion (22,23). The presence of BCM-7 in fresh milk is not confirmed elsewhere in the literature, and it has alternatively been proposed that this finding may be explained by proteolysis of caseins by enzymes associated with somatic cells in the milk, which are normally associated with clinical or subclinical mastitis (2,24). However, it could be more likely that small amounts of BCM-7 detected by this group after pepsin digestion were the result of extended acidic hydrolysis rather than enzymatic action and are not reflective of human multienzyme gastrointestinal digestion in which gastric digestion may occur for <1 h and is unlikely to exceed 6 h (25). ...
Article
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This is the first systematic review, to our knowledge, of published studies investigating the gastrointestinal effects of A1-type bovine β-casein (A1) compared with A2-type bovine β-casein (A2). The review is relevant to nutrition practice given the increasing availability and promotion in a range of countries of dairy products free of A1 for both infant and adult nutrition. In vitro and in vivo studies (all species) were included. In vivo studies were limited to oral consumption. Inclusion criteria encompassed all English-language primary research studies, but not reviews, involving milk, fresh-milk products, β-casein, and β-casomorphins published through 12 April 2017. Studies involving cheese and fermented milk products were excluded. Only studies with a specific gastrointestinal focus were included. However, inclusion was not delimited by specific gastrointestinal outcome nor by a specific mechanism. Inclusion criteria were satisfied by 39 studies. In vivo consumption of A1 relative to A2 delays intestinal transit in rodents via an opioid-mediated mechanism. Rodent models also link consumption of A1 to the initiation of inflammatory response markers plus enhanced Toll-like receptor expression relative to both A2 and nonmilk controls. Although most rodent responses are confirmed as opioid-mediated, there is evidence that dipeptidyl peptidase 4 stimulation in the jejunum of rodents is via a nonopioid mechanism. In humans, there is evidence from a limited number of studies that A1 consumption is also associated with delayed intestinal transit (1 clinical study) and looser stool consistency (2 clinical studies). In addition, digestive discomfort is correlated with inflammatory markers in humans for A1 but not A2. Further research is required in humans to investigate the digestive function effects of A1 relative to A2 in different populations and dietary settings.
... Casomorphins are detected in human colostrum as BCM5 and BCM7 up to 27 μg/mL (Jarmołowska et al., 2007) and in baby formula at 0.2-0.9 μg/mL (Cattaneo et al., 2017;De Noni & Cattaneo, 2010). Out of eight casomorphins present in dairy, BCM7 was proposed as one of the most likely agents responsible for the adverse health effects of regular milk. ...
Article
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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.
... Few previous studies performed in vitro digestion of PR, but mainly focused on quantification of 385 specific peptides (Bordoni et al., 2011;De Noni and Cattaneo, 2010;Basiricò et al., 2015predictable and specific cleavage sites could be found in peptide sequences, consistently with the fact that the 391 proteolytic activity of pancreatin is due to trypsin, chymotrypsin, but also elastase, carboxypeptidase and 392 other minor proteases, and suggesting that many peptides are generated by the action of an exopeptidase or 393 ...
Article
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Parmigiano Reggiano (PR) is a raw-milk, hard cooked, long-ripened cheese of high quality and nutritional value. Long ripening times allow for extensive proteolysis of milk proteins to yield a number of peptides, some of which have potential healthy bioactive properties. This study aimed to: i) determine the peptide profile of PR cheese subjected to simulated gastrointestinal transit; ii) evaluate in vitro whether the peptides could support growth of beneficial microbial groups of the gut microbiota. PR samples were subjected to in vitro digestion, simulating oral, gastric, and duodenal transit. Liquid chromatography coupled with tandem mass spectrometry revealed that digestion caused the disappearance of the serum proteins and most of the original peptides, while 71 new peptides were found, all ranging from 2 to 24 residues. The digests were given as sole nitrogen source to pure cultures of Bifidobacterium (27 strains) and Lactobacillus (30 strains), and to bioreactor batch cultures of human gut microbiota. Most of bifidobacteria and lactobacilli grew more abundantly on PR digests than on the control peptone, and exhibited strain- or species-specific peptide preferences, as evidenced by principal component analysis. Bifidobacteria generally consumed a greater amount of peptides than lactobacilli, in terms of both the mean peptide consumption and the number of peptides consumed. For bifidobacteria, peptide preferences were very diverse, but a core of 10 peptides with 4 or 5 residues were consumed by all the strains. Lactobacilli behaved more homogenously and consumed nearly only the same 6 peptides, mostly dipeptides. The peptide preferences of the different groups of bifidobacteria and lactobacilli could not be ascribed to features such as the length of the peptide or the abundance of residues with peculiar properties (hydrophobicity, polarity, charge) and likely depend on specific proteases and/or peptide transporters preferentially recognizing specific sequence motifs. The cultures of human colonic microbiota confirmed that PR digest promoted the growth of commensal bifidobacteria. This study demonstrated that peptides derived from simulated gastrointestinal digestion of PR supported the growth of most lactobacilli and bifidobacteria.
... [34]. Впоследствии появилась целая серия работ, которые подтверждали данный факт [35,36,37]. ...
Article
The literature review is devoted to biologically active metabolites of casein — the results of its hydrolysis — oligopeptides casomorphins. These peptides with a chain length of 4 to 11 amino acids are derived from milk β-casein and are released during digestion, both in vivo and in vitro. Caseomorphins exhibit opioid and pharmacological activity due to binding to μ-receptors located in the central nervous system, gastrointestinal tract and some immune cells. Understanding the biological role of caseomorphins in the milk of mammals, including humans, and their effect on organs and systems, will bring specialists closer to deciphering the etiology of a whole group of diseases.
... These processes can significantly affect the proteolysis of the cheese, subsequently releasing peptides during processing including BCMs. De Noni and Cattaneo (2010) observed the presence of BCM7 and BCM5 in several commercial cheeses, however, these cheeses were not produced from milk of homozy- gous variants, rather batched milk (as it occurs in the industries) containing the variants A1, A2 and B. ...
Article
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.
... Regarding the potential enzymatic degradation of the β-CN sequence in the region 60-66, in vitro simulated gastrointestinal digestion of dairy products released bioactive peptide β-casomorphin-7 (BCM-7) and other related β-casomorphins (BCMs) (De Noni, 2008;De Noni & Cattaneo, 2010;Haq, Kapila, & Kapila, 2015). BCMs and peptide precursors encrypting BCM-7 have been detected in human intestinal effluents (Boutrou et al., 2013;Boutrou, Henry, & Sanchez-Rivera, 2015;Sanchón et al., 2018). ...
Article
A dedicated two-step purification procedure prior to nanoliquid chromatography-electrospray-tandem mass spectrometry analysis enabled the identification of bovine milk-derived peptides absorbed and circulating in the plasma of three healthy volunteers who received 250 mL of pasteurized milk after a 10-days washout. The appearance and clearance of milk peptides in plasma were monitored at various time points. Overall, 758, 273 and 212 unique peptides derived from 15, 15 and 18 bovine milk proteins, respectively, were identified in the plasma of these volunteers, evidencing a substantial inter-individual variability. Peptides encrypting possible bioactive and/or immunogenic molecules originating from caseins, β-lactoglobulin and minor milk proteins were detected. Peptide representation data revealed the combined action of endoproteases involved in primary hydrolysis during gastroduodenal digestion and exopeptidases that hydrolyse peptides in the small intestine. It remains to be established whether the half-life and concentration ranges of circulating milk-derived peptides may have any impacts on human health.
... It is degraded by DPP4 found on the surface of the absorptive cells and the blood [33,58]. In addition to fresh milk, it can also be identified in different milk products including cheese and butter [28]. ...
Article
This article aims to review the effects of cow milk containing A1 or A2 or mixed variants of β-casein on human health. The information based on the health effects of cow milk was collected from the most authentic scientific database including Scopus, PubMed, and Google Scholar by searching specific keywords like “cow milk”, “A1A2 beta-casein”, “beta-casomorphins”, “A2 cow milk”, and “A2 milk”. The search hits a total of 197 articles including patents in which about 70 most relevant articles were critically reviewed. The literature revealed that the most abundant category of cow milk found worldwide is mixed A1/A2 in which both A1 and A2 variants of β-casein are found in equal ratio. Among the major three categories, A2 cow milk received much attention both from the scientific community and the wider public due to its possible health benefits over A1 milk mainly in diabetes and heart-related problems. On the other hand, milk containing the A1 variant of β-casein is supposed to be harmful due to the formation of β-casomorphin-7 (BCM-7) peptide, although the scientific community is not unanimous for this claim. The claim for the harmful effects of the A1 variant should be further validated with more scientific studies.
... These observations are largely thought to be due to the action of β-casomorphin-7 (BCM-7), a bioactive peptide with opioid-like effects released upon gastrointestinal digestion and processing of milk products (58,59). However, BCM-7 is degraded during the fermentation process and is found in lower amounts in yogurt and cheese when compared with milk, which may in part explain the observed findings (60,61). ...
Article
Background Despite the putative health benefits of fermented dairy products, evidence on the association between fermented dairy and non-fermented dairy intake and depression incidence is limited. Objectives This study examined cross-sectional and prospective associations between total dairy, fermented dairy, and non-fermented dairy intake with 1) the presence of elevated depressive symptoms and 2) the risk of a future hospital discharge or outpatient diagnosis of depression. Methods Data from 2603 Finnish men (aged 42–60 years), recruited as part of the Kuopio Ischaemic Heart Disease Risk Factor Study, were included. Multivariable logistic regression models were used to examine odds ratios (ORs) and 95% confidence intervals (CIs) for elevated depressive symptoms (Human Population Laboratory Scale ≥ 5 points) at baseline. Cox proportional hazard's regression models were used to estimate hazards ratios (HRs) and 95% CIs between dairy categories and risk of depression diagnoses. Results In cross-sectional analyses, fermented dairy intake in the highest (vs lowest) tertile was associated with lower odds for having elevated depressive symptoms (adjusted-OR 0.70, 95% CI 0.52–0.96). Each 100g increase in non-fermented dairy intake was associated with higher odds for having elevated depressive symptoms (adjusted-OR 1.06, 1.01–1.10). During a mean follow-up time of 24 years, 113 males received a diagnosis of depression. After excluding cheese intake, higher fermented dairy intake was associated with a lower risk of depression diagnosis (adjusted-HR 0.62, 0.38–1.03), which was strengthened after excluding those with elevated depressive symptoms at baseline (adjusted-HR 0.55, 0.31–0.99). Whereas non-fermented dairy intake in the highest tertile was associated with a two-fold higher risk of depression (adjusted-HR 2.02, 1.20–3.42). Conclusions Fermented dairy and non-fermented dairy intake were differentially associated with depression outcomes when examined cross-sectionally and over a mean period of 24 years. These findings suggest that dairy fermentation status may influence the association between dairy intake and depression in Finnish men.
... certain peptides, such as b-casomorphins have opioid-like actions, functioning similar to an analgesic and tranquilizer affecting the central nervous system (19). Experimental studies have also demonstrated that some peptides interfere with the gastrointestinal tract, favoring the production of mucin, thus preventing the adhesion of the pathogen to the intestinal surface, exerting effects on intestinal motility that may justify a possible role in weight control through the regulation of food intake (20). ...
Article
The present study was conducted on milk samples from 30 Italian Mediterranean buffaloes, and as many goats, sheep and cattle. Milk samples were subjected to chemical-nutritional analysis and compared with commercially available milk samples. In the experiments, the management conditions could have influenced this parameter, determining the observed values. The higher fat content in sheep's milk differed significantly from that in the milk of the other animals analyzed. A higher lactose content was found in the milk of buffaloes and sheep, than in cows and goats. The highest cholesterol content was observed in cow and buffalo milk. These differences were statistically significant. To avoid excessive cholesterol intake, it is necessary to pay attention to both the quantity and quality of fats contained in food, bearing in mind that the introduction of large quantities of dairy products in the diet can cause a significant intake in cholesterol levels. Furthermore, when selecting the type of milk to be consumed, it is necessary to consider that the amount of total cholesterol is dependent on both the animal of origin and on the integrity of the lipid fraction. To complete the study, a chemical-nutritional analysis of milk samples normally marketed, both of vegetable and animal origin, was also included. Observing the results, some differences appear evident in the chemical-nutritional composition of goat and cow milk compared to the raw milk analyzed. In particular, goat's milk has a higher percentage of lipids. However, the differences observed herein were not significant and could be explained by the manufacturing process the samples were subjected to from the stable to the packaging industry. From the results obtained in the present study in the compositional analyses performed, and as also obtained from previous studies, it was found that sheep and goat milk, in particular, may be a valid substitute for cow's milk.
... For instance, Nguyen et al. (2014Nguyen et al. ( , 2015Nguyen et al. ( , 2019 determined the concentration of BCM7 in (raw or pasteurized) cow milk and yoghurt ( Table 2). The research group of De Noni (De Noni 2008;De Noni and Cattaneo 2010;De Noni, Stuknyt_ e, and Cattaneo 2015;Cattaneo et al. 2017) studied BCM7 occurrence in diverse dairy products (fermented milk, infant formula and cheeses) prior to and after in vitro simulated gastrointestinal digestion (SGID) ( Table 2). Very different amounts of BCM7 were found among undigested cheeses and the related digestates, suggesting that the manufacturing and ripening processes exert a pivotal role in modulating the release of this BAP in matured cheeses and during subsequent SGID. ...
Article
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.
Thesis
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This thesis describes possible physiological consequences of genetic variation in bovine beta- and kappa-casein. These caseins constitute approximately 40% of the protein in bovine milk, and are known sources of a number of bioactive peptides, which are protein fragments that are believed to exert health-promoting effects. The studies revealed that the fragmentation pattern of caseins during simulated gastrointestinal digestion is affected by amino acid substitutions, and that peptides differing in bioactivity are generated as a consequence. These findings may be of relevance for the differentiation of raw materials in the dairy industry.
Article
In recent years there has been an increasing interest in pure casein fractions, particularly beta-casein due to its physiochemical properties as well as its bio- and techno-functional properties. A range of methods has been developed for the fractionation of casein into its individual proteins. The present review deals with the current technologies for the isolation and purification of beta-casein, especially on a technical scale. Furthermore, the main techno-functional properties of beta-casein as well as examples for functional food applications are presented.
Chapter
Casomorphins are bovine and human milk-derived opioid peptides. These are released from casein milk proteins. Among casomorphins, β-casomorphins (BCMs) are the most abundant group of exorphins released from the β-casein of human and bovine milk. Casomorphins differ from each other on the source of milk (bovine/human) and the number and sequence of amino acids. The commonly studied casomorphins include bovine BCM-4, 5, 6, 7, 8 and neocasomorphin-6 and human BCM-4, 5, 7, 8 and α-casomorphin. Among all casomorphins, BCM-7 is the most significant exorphins studied, because it correlates with the incidence of few human illnesses. BCMs have selectivity for all three types of opioid receptors including μ, δ, and κ. These exorphins (except α-casomorphin) act as agonists for all these opioid receptors. Casomorphins are released from the milk casein during gastrointestinal digestion (in vivo), simulated gastrointestinal digestion (in vitro), or fermentation. Reports have established the production of these peptides from various types of milk, cheese samples, infant formulas, and yogurt. The A1/A2 milk hypothesis establishes that BCM-7 is released from only A1 milk of bovines due to the presence of histidine at position 67 of the β-casein. The presence of proline at this position in A2 milk resists such cleavage and hence this exorphin is not produced from this variant of β-casein.
Article
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.
Article
Fermented milk products are valued by consumers and the food industry for their nutritional properties, pleasant taste, and texture. To rapidly discriminate the peptide and small molecular fingerprints of fermented milk with different flavour characteristics, we assessed the suitability of high-throughput protocols for the screening of these samples using rapid evaporative ionisation mass spectrometry (REIMS) and matrix-assisted laser desorption ionisation–time-of-flight mass spectrometry (MALDI-TOF MS). With minimal sample preparation, we can distinguish between different fermented milk produced from different bacterial strains. These techniques can aid in quick and cost-effective fingerprinting to screen for new and interesting fermented products and bacterial cultures. Furthermore, we have demonstrated for the first time the suitability of REIMS for the measurement of liquid dairy samples.
Chapter
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.
Article
Bioactive peptides (BPs) derived from animal and plant proteins are important food functional ingredients with many promising health-promoting properties. In the food industry, enzymatic hydrolysis is the most common technique employed for the liberation of BPs from proteins in which conventional heat treatment is used as pre-treatment to enhance hydrolytic action. In recent years, application of non-thermal food processing technologies such as ultrasound (US), high-pressure processing (HPP), and pulsed electric field (PEF) as pre-treatment methods has gained considerable research attention owing to the enhancement in yield and bioactivity of resulting peptides. This review provides an overview of bioactivities of peptides obtained from animal and plant proteins and an insight into the impact of US, HPP, and PEF as non-thermal treatment prior to enzymolysis on the generation of food-derived BPs and resulting bioactivities. US, HPP, and PEF were reported to improve antioxidant, angiotensin-converting enzyme (ACE)-inhibitory, antimicrobial, and antidiabetic properties of the food-derived BPs. The primary modes of action are due to conformational changes of food proteins caused by US, HPP, and PEF, improving the susceptibility of proteins to protease cleavage and subsequent proteolysis. However, the use of other non-thermal techniques such as cold plasma, radiofrequency electric field, dense phase carbon dioxide, and oscillating magnetic fields has not been examined in the generation of BPs from food proteins
Article
The aim of this study is to reveal β-casein polymorphism of some cattle breeds and also the potential to produce A2 milk from existing animals and to develop strategies in this area. Therefore, a total of 400 cattle, 100 animals from each breed of Holstein, Brown Swiss, Jersey and Simmental raised commonly in Turkey, were obtained, and C > A polymorphism in 67th amino acid in the 7th exons of β-casein gene was determined by TaqI enzyme with PCR-ACRS method. Blood samples were collected from dairy cattle farms raising Holstein, Brown Swiss and Jersey breeds from Konya province and Simmental breed from Kütahya province in Turkey. A1 and A2 allele frequencies in Holstein, Brown Swiss, Jersey and Simmental cattle breeds were determined as 0.475/0.525, 0.370/0.630, 0.215/0.785 and 0.440/0.560, respectively. The highest A2 allele frequency (0.785) was found in Jersey breed. A1A1 genotypes in Holstein, Brown Swiss, Jersey and Simmental breeds were 0.240, 0.150, 0.030 and 0.160, respectively; A1A2 genotypes were 0.470, 0.440, 0.370 and 0.560, respectively; A2A2 genotypes were determined as 0.290, 0.410, 0.600 and 0.280, respectively. In these breeds, the highest A2A2 genotype frequency was found in Jersey (0.600), the lowest A1A1 genotype frequency (0.030) was found in Jersey and the highest A1A2 genotype frequency (0.560) was found in Simmental. Holstein, Brown Swiss, Simmental and Jersey populations were at the level of Hardy-Weinberg in terms of β-casein gene (p > 0.05). The average Ho, He and PIC values were calculated as 0.460, 0.469 and 0.605, respectively. In conclusion, the frequency of commonly reared cattles in Turkey especially Brown Swiss, and Jersey breeds in A2A2 genotype are satisfactory, but it can be said that the use of animals with A2 allele in selection is extremely important for increasing A2 milk producing potential in the future.
Chapter
The present chapter shows an overview of the production of bioactive peptides (BAPs) obtained from food matrices, using fermentation processes. It shows that it is possible to obtain BAPs from milk, meat, and vegetable proteins and emphasizes scientific production and the proven benefits that milk protein-derived BAPs provide to health. It also emphasizes a promising outlook in BAP production by fully using meat and vegetable proteins using food industry by-products, which also helps to mitigate waste environmental issue. For viable and safe BAPs industrial production, advances about in vivo research and adaptations of biotechnological processes for this scale of production are required.
Chapter
Fermented food and beverages constitute a significant part of the human diet (5%–40%) worldwide. Fermentation has been used for preservation and to augment the flavor, texture, and nutritional qualities of the food, since antiquity. During fermentation, the bioavailability of vitamins, minerals, and other constituents increases due to the microorganisms’ metabolic activities. Besides enhancing nutritional quality, fermented foods contain live organisms reported to prevent/treat many health disorders. Types of the fermentation process are also classified based on these microorganisms. In developing countries, fermented foods were usually prepared using traditional methods without any standardized techniques. Considering the beneficial effects of fermented foods, industrial-level production requires consistent specific microorganisms, fermentation methods, evaluation of nutritional compositions, and food safety testing. This chapter discusses the fermented foods and associated organisms, different sources available for the consumption of fermented foods, and food component’s effect on microorganism’s efficacy.
Article
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Cow’s milk-intolerance is a digestive problem on people who not able to digest milk. This problem may relate to the variant of (3-casein (CSN2), especially A1, suggested due to (3-casomorphins (BCM-7) formation during enzymatic digestion, for that selecting cattle free BCM-7 become a concern to produce digestive friendly milk. This study aimed to differentiate A1 and A2 allele variant of CSN2 gene in selected population of Indonesian Holstein cattle. In total 70 cows DNA were collected, and fragment of CSN2 exon 7 which contain Single Nucleotide Polymorphism (SNP) rs43703011 and rs43703013 were amplified. Variant analysis was done by mutation site analysis using PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) with Mspl restriction enzyme and DNA sequence for confirmation. Result shows 2 allele variants of (3-casein that are B type, representing A1 family variant, and A2 in mutation site rs43703013C>G. We found A2 allele in the studied population is superior in frequency than A1 (0.916 vs. 0.084). Of that, 8.6% cattle were heterozygotes that is BA2 and 91.4% were homozygotes A2A2. Cattle which carry A1 allele variant should be excluded from dairy cattle breeding program for further milk production free of BCM-7.
Article
Milk proteins are recognized as the main source of biologically active peptides. Casein's primary structure contains several bioactive amino acid sequences on its latent inactive form. These potential active sequences can be released during cheese manufacture and ripening given rise to peptides with biological activities such as antihypertensive, antidiabetic, antioxidant, immunomodulatory, and mineral binding property. However, the presence of biopeptides in cheese does not imply the actual biological activity in vivo since these peptides can be further hydrolyzed during gastrointestinal transit. This paper reviews the recent advances in biopeptides formation in ripened cheeses production, focusing on the influence of technological parameters affecting proteolysis and the consequent release of peptides. In addition, the main discoveries on cheese peptides digestion through recent in vivo and in vitro model studies are also reviewed. This article is protected by copyright. All rights reserved.
Chapter
The generated ecological data, human case-control findings, in vivo and in vitro studies followed by biochemical and pharmacological mechanisms are strong enough to establish a correlation between A1 “like” milk consumption and increased risk for various health disorders. BCM-7 released from A1 milk is actually considered as a proposed risk element. However, the counter arguments furnished by EFSA, American Nutritionists and Truswell can’t be ignored. The present status of the hypothesis isn’t in a position to make consumer health recommendations. More mechanistic studies involving well-designed animal and human trials with explored cellular, molecular and immunological mechanisms are needed to reach at final conclusions.
Article
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.
Article
β-Casomorphin-7 (BCM-7) is a heptapeptide dietary molecule derived from the digestion of the β-casein of dairy and dairy products. In this review, we have covered the extensive details about BCM and its derived peptides out of the gastrointestinal and enzymatic digestion of milk and milk products, its structure and properties, and its immunological aspects related to human health among infants and adults of both genders. We have left judgment about BCM’s pros and cons to the reader by describing the details in a cyclopedic perspective. In addition, a section on the possible ways to detect BCMs from their sources using proteomics, genome-based techniques, such as PCR and aptamers, and other analytical techniques equip the reader to get an idea about the details of the diagnostics available and possible applications in future. Overall, this review will provide information to the end-users of milk and milk products to enable them to make their own decisions about BCMs.
Chapter
Food-derived exorphins are usually 4–20 amino acid peptides released from food proteins like caseins, whey, gluten, RuBisCo, β-conglycinin, and albumin. They are generated through gastrointestinal digestion, simulated gastrointestinal digestion (SGID), or fermentation. They show structural features that enable them to bind opioid receptors (μ, δ, and κ). These opioid receptors are distributed widely in the central and peripheral nervous system, gastrointestinal tract, some immune cells, and other tissues. These exorphins may act as agonists or antagonists for these opioid receptors. They play various physiological roles in gastrointestinal motility, analgesia, anxiolysis, emotional and behavior development, memory consolidation, blood pressure regulation, prolactin secretion, food and fat intake, hormone release, appetite, and mucous formation. Moreover, these exorphins are correlated with the development of various physiological complications. The conventional opioids show numerous side effects and thereby limit the clinical effectiveness. Food-derived exorphins are safe alternatives for the pharmaceutical and food industry. The merits include oral consumption and bioavailability, safety due to a lack of side effects, and activation at the endogenous opioid receptors. Therefore, these peptides have enough scope in the food and pharmaceutical industry for the development of functional foods and nutraceuticals. Moreover, deeper research with explored signal cascade mechanisms at the cellular and molecular level is needed to explore food exorphins as therapeutic mediators, functional foods, or nutraceuticals for human health promotion.
Chapter
Bioactive peptides (BAPs) are biologically active protein fragments. BAPs are released from the intact food proteins during gastrointestinal digestion, fermentation, bacterial cultures, or a combination of these processes. Food-derived exorphins are BAPs released from foods that show opioid activity. These include casomorphins, lactorphins, lactoferroxins, and casoxins (milk), gluten exorphins (wheat), rubiscolins (spinach), soymorphins (soybean), and oryzatensin (rice). They have selectivity for the endogenous opioid receptors including mu (μ), delta (δ), and kappa (κ). These opioid receptors are distributed in central and peripheral nervous systems, gastrointestinal tract, immune cells, and some other tissues. These exorphins possess all the structural features that are critical for binding these opioid receptors and in turn manifestation of opioid activity. They show different physiological activities like analgesic property, anxiolysis, hormone secretion, gastrointestinal transit, ACE inhibition, lymphocyte proliferation, food and fat intake, memory consolidation, and behavioral and pharmacological effects. Moreover, the consumption of a few exorphins is correlated with some human illnesses. Overall, these peptides have enough scope in the food and pharmaceutical industry for the development of functional foods and nutraceuticals.
Article
Genetic variations of the beta casein gene hold importance because of their probable association with human health. Comparative sequence analysis of β-casein gene across Indian native, crossbred and exotic breeds in India revealed 15 SNPs and 4 INDELs corresponding to 14 haplotypes. The frequency of A2 type haplotype was maximum (0.941) across all Indian native breeds. Among the 15 variants reported for taurine breeds, only three (A1, A2 and B) were observed in analysed populations. Allelic profiling of A1/A2 β-casein variants in ~ 4000 animals belonging to three cattle types and breeding bulls also revealed the predominance of A2 allele (0.95) in Indian cattle. The high proportion of A2 allele/haplotype indicates that Indian native cattle are the best suited to meet the demands for A2 milk globally. However, a higher percentage of heterozygous genotype (A1A2) in breeding bulls warrants the need to screen sire lines so as to drift the herd towards A2. Supplementary information: The online version contains supplementary material available at 10.1007/s13205-022-03232-0.
Article
In this study, an ultrasensitive electrochemical aptasensor for the determination of the β-casomorphin 7 (BCM-7) as a promising biomarker of autism disorder is introduced. According to the proposed strategy, a glassy carbon electrode (GCE) was modified by thiourea capped-ZnS QDs (ZnS-QDs) and gold nanoparticles (AuNPs) to further immobilization the amino-aptamer (NH2-Apt) on its surface. The NH2-Apt as a receptor of the BCM-7 was attached to the embedded surface via a formation of the Au–N bonds between the AuNPs and amino groups of the Apt. The evaluation of aptasensor by various electrochemical techniques exhibited successful sensing of the BCM-7 under two broad linear concentration ranges from 1 fM to 0.6 μM with a limit of detection (LOD) down to 350 aM. Also, the performance of the aptasensor in BCM-7 detection in real human urine samples was satisfactorily investigated. These findings may facilitate distinguish of this challenging disorder and help human health.
Article
In recent years there has been an increasing interest in pure casein fractions, particularly β-casein due to its physiochemical properties as well as its bio- and techno-functional properties. A range of methods has been developed for the fractionation of casein into its individual proteins. The present review deals with the current technologies for the isolation and purification of β-casein, especially on a technical scale. Furthermore, the main techno-functional properties of β-casein as well as examples for functional food applications are presented.
Article
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In addition to its nutritional value, β-casein is a source of bioactive peptides called β-casomorphins, which are produced during digestion of raw or processed milk. It has been shown that β-casomorphin 7 should originate exclusively from β-casein genetic variants A1 or B and may be a serious risk factor in the etiology of human diseases. This study measured the level of this peptide in milk produced by cows of different β-casein genotypes. Ten Polish Holstein-Friesian cows were included in the experiment, 5 carrying the A1/A1 genotype, 5 carrying the A2/A2 genotype. The genotypes of the β-casein locus were determined on the basis of DNA by PCR-Allele Created Restriction Site (ACRS) method. Caseins were extracted and then hydrolyzed by pepsin for a 24 h incubation followed by HPLC isolation of β-casomorphin 7. In hydrolyzed milk from A1/A1 cows, the content of β-casomorphin 7 was 4 times higher than in milk produced by A2/A2 cows. In fresh (not hydrolyzed) milk, traces of β-casomorphin 7 were found. The results confirm the hypothesis that β-casein A1 milk during hydrolysis produces significant amounts of β-casomorphin 7 and, therefore, A1 milk may be considered an undesirable factor influencing human health.
Article
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Twenty-one Lactobacillus strains isolated from three types of Balkan homemade yogurts were grown on sodium caseinate, β-casein or whey proteins, and the proteolysis was followed by electrophoresis and reversed-phase high-performance liquid chromatography. The best conditions allowing obtaining proteolysis without casein precipitation are 0.8% casein in 50-mM phosphate buffer. The strains tested showed a relatively high proteolytic activity despite the limited conditions for bacterial growth. Within 72 and 96 h of incubation, 80–90% of β-casein was consumed. They showed also a proteolytic activity toward α-lactalbumin (ALA), being able to reduce its concentration between 5 and 55%, depending on the strains used. The capacity of the strains to hydrolyze β-lactoglobulin was lower as compared with hydrolysis of ALA. Hydrolysis of casein by all strains produced peptides with an antibacterial effect against Escherichia coli. Consequently, to obtain a maximal hydrolysis of the dairy proteins seconded by appearance of antimicrobial peptides, a combination of strains with different beneficial properties to be used as starters was proposed.
Article
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The peptides released from beta-casein by the action of PI-type proteinase (PrtP) from Lactococcus lactis subsp. cremoris Wg2 have been identified by on-line coupling of liquid chromatography to mass spectrometry. After 24 h of incubation of beta-casein with purified PrtP, a stable mixture of peptides was obtained. The trifluoroacetic acid-soluble peptides of this beta-casein hydrolysate were fractionated by high-performance liquid chromatography and introduced into the liquid chromatography-ion spray mass spectrometry interface. Multiply charged ions were generated from trifluoroacetic acid-soluble peptides under low nozzle voltage conditions, yielding the MH+ mass of each eluted peptide. All peptides corresponding to each of the MH+ calculated masses were determined. In those cases in which different peptides were possible, further identification was achieved by collision-induced dissociation under higher nozzle voltage conditions. Hydrolysis of beta-casein by PrtP was observed to proceed much further than reported previously. More than 40% of the peptide bonds are cleaved by PrtP, resulting in the formation of more than 100 different oligopeptides. With the exception of Phe, significant release of amino acids or di- and tripeptides could not be observed. Interestingly, one-fifth of the identified oligopeptides are small enough to be taken up by the oligopeptide transport system. Uptake of these peptides could supply L. lactis with all amino acids, including the essential ones, indicating that growth of L. lactis might be possible on peptides released from beta-casein by proteinase only.
Article
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The water-soluble extract of Cheddar cheese was fractionated by diafiltration using 10 kDa cut-off membranes. Peptides were isolated from the diafiltrate retentate by chromatography on DEAE-cellulose with a linear NaCl gradient in 50 mM-Tris-HCl. pH 8.6, and reversed-phase HPLC or electroblotting from urea-PAGE gels. Peptides were identified by determining N-terminal amino acid sequences and mass spectrometry. Most (45) of the total 51 peptides identified in the diafiltrate retentate originated from beta-casein, especially from a short region in the N-terminal half of the molecule. Only six peptides originated from alpha s1-casein; beta-lactoglobulin was also identified in the retentate. The origin of most of these peptides could be explained on the basis of known specificities of lactococcal cell envelope proteinases.
Article
Full-text available
Several peptides were isolated from the diafiltration retentate, prepared using 10 kDa membranes, of the water-soluble extract from a commercial mature Cheddar cheese and identified by amino acid sequencing and mass spectrometry. Most of the peptides were from the N-terminal half of the beta-casein, but peptides from alpha s1- and alpha s2-caseins were also identified; the extract also contained alpha-lactalbumin. Identified peptides showed the important role played by lactococcal cell envelope proteinases in the degradation of primary proteolytic products from alpha s1- and beta-caseins, produced by chymosin and plasmin respectively. Plasmin seemed to be involved in the hydrolysation of alpha s2-casein. Several phosphopeptides were identified and the action of phosphatase on these peptides was evident.
Article
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Conditions for the release of beta-casomorphin-7 from bovine beta-casein by gastrointestinal proteases in vitro were investigated. beta-Casomorphin-7 was released only from a genetic variant of beta-casein containing a His residue at the 67th position of the peptide chain. Elastase cleaved the peptide bond between Ile66 and His67, releasing the carboxyl terminus of beta-casomorphin-7. Pepsin and leucine aminopeptidase were required to release the amino terminus of this peptide. beta-Casomorphin-9, -13, and -21 also were isolated, and their opioid activities were measured. In this study, we also isolated a novel opioid peptide neocasomorphin-6 (Tyr-Pro-Val-Glu-Pro-Phe), which was released by action of trypsin or pepsin and chymotrypsin.
Article
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To test the correlation of per capita A1 beta-casein (A1/capita) and milk protein with: 1) ischaemic heart disease (IHD) mortality; 2) Type 1 (insulin-dependent) diabetes mellitus (DM-1) incidence. A1/capita was estimated as the product of per capita cow milk and cream supply and its A1 beta-casein content (A1/beta) (calculated from herd tests and breed distribution, or from tests of commercial milk), then tested for correlation with: 1) IHD five years later in 1980, 1985, 1990 and 1995, in 20 countries which spent at least US $1000 (purchasing power parities) per capita in 1995 on healthcare; 2) DM-1 at age 0-14 years in 1990-4 (51 were surveyed by WHO DiaMond Project; 19 had A1 data). For comparison, we also correlated 77 food, and 110 nutritive supply FAO (Food and Agriculture Organization)-based measures, against IHD and DM-1. For IHD, cow milk proteins (A1/capita, r = 0.76, p <0.001; A1/capita including cheese, r = 0.66; milk protein r = 0.60, p = 0.005) had stronger positive correlations with IHD five years later, than fat supply variables, such as the atherogenic index (r = 0.50), and myristic, the 14-carbon saturated fat (r = 0.48, p <0.05). The Hegsted scores for estimating serum cholesterol (r = 0.42); saturated fat (r = 0.37); and total dairy fat (r = 0.31) were not significant for IHD in 1995. Across the 20 countries, a 1% change in A1/capita in 1990 was associated with a 0.57% change in IHD in 1995. A1/capita correlations were stronger for male than female mortality. On multiple regression of A1/capita and other food supply variables in 1990, only A1/capita was significantly correlated with IHD in 1995. DM-1 was correlated with supply of: A1/capita in milk and cream (r = 0.92, p <0.00001); milk and cream protein excluding cheese (r = 0.68, p <0.0001); and with A1/beta in milk and cream (r = 0.47, p <0.05). Correlations were not significant for A2, B or C variants of milk beta-casein. DM-1 incidence at 0-4, 5-9 and 10-14 years was equally correlated (r = 0.80, 0.81, 0.81 respectively) with milk protein supply. A 1% change in A1/capita was associated with a 1.3% change in DM-1 in the same direction. Cow A1 beta-casein per capita supply in milk and cream (A1/capita) was significantly and positively correlated with IHD in 20 affluent countries five years later over a 20-year period--providing an alternative hypothesis to explain the high IHD mortality rates in northern compared to southern Europe. For DM-1, this study confirms Elliott's 1999 correlation on 10 countries for A1/capita,1 but not for B beta-casein/capita. Surveys of A1 beta-casein consumption in two-year-old Nordic children, and some casein animal feeding experiments, confirm the A1/capita and milk protein/capita correlations. They raise the possibility that intensive dairy cattle breeding may have emphasised a genetic variant in milk with adverse effects in humans. Further animal research and clinical trials would be needed to compare disease risks of A1-free versus 'ordinary' milk.
Article
This study was conducted to investigate the rate of proteolysis and peptide profiles of different Finnish fermented milk products. The highest rate of proteolysis was observed in Biokefir, while the greatest change in the rate of proteolysis was observed in Gefilus®. Differences in starters and manufacturing processes reflected on the peptide profiles of the products. Most of the identified peptides originated from either the N- or C-terminal region of β-casein or from the N-terminal region of αs1-casein.
Article
Increasing attention is being focused on physiologically active peptides derived from food proteins. A great number of peptide sequences derived from food proteins with specific bioactivities and the conditions for their release have been determined. A variety of naturally formed bioactive peptides have been found in fermented products. Industrial-scale technologies suitable for the commercial production of bioactive milk peptides have been developed and launched recently. Furthermore, there are a few commercial dairy products supplemented with milk protein - derived bioactive peptides whose health benefits have been documented in clinical human studies. It is envisaged that this trend will expand as more knowledge is gained about the multifunctional properties and physiological functions of food derived peptides.
Article
Milk proteins contain a variety of biologically active peptides, released in proteolysis induced by digestive enzymes and lactic acid bacteria. In this study, release of bioactive peptide fractions derived from Lactobacillus GG fermented UHT milk following hydrolysis by pepsin and trypsin was investigated. The molecular weight of the protein hydrolysate obtained was studied using fast atom bombardment mass spectrometry (FAB-MS). The hydrolysate was further fractionated using fast protein liquid chromatography (FPLC). Several molecular weight related ions were observed in the FAB-MS spectra of the fractions. Also weak molecular ions corresponding to immunostimulating and opioid peptides referred to in the literature were detected in the subfractions. The results indicate that in vitro hydrolysis of bovine milk proteins with the probiotic L. GG strain and digestive enzymes releases bioactive peptides. Their potential role in vivo (in the human body) is discussed.
Article
In this study, the in vitro angiotensin-converting enzyme (ACE)-inhibitory (ACE-I) activity of peptide fractions from different yoghurt batches was assessed. Inhibition of ACE activity resulted in an overall antihypertensive effect. Yoghurts were prepared either using a sole yoghurt culture including Lactobacillus delbrueckii ssp. bulgaricus Lb1466 and Streptococcus thermophilus St1342, or L. acidophilus L10, L. casei L26 and Bifidobacterium lactis B94 in addition to yoghurt culture. ACE-I activity was determined at weekly intervals during 28 days of cold storage. Peptide fractions showing high ACE-I activity were further purified using multiple-steps of RP-HPLC. All probiotic yoghurts showed appreciable ACE-I activity during initial stages of storage compared with the control yoghurt, with a significant (po0.05) decrease afterwards. The ACE-I activity ranged from IC 50 of 103.30–27.79 mg mL À1 with the greatest ACE inhibition achieved during first and third week of storage. The in vitro ACE-I activity could be related to the peptide liberation via degradation of caseins. In total, 8 ACE-I peptides were characterized originating from a s2 -casein (1), k-casein (2) and b-casein, of which two well-known ACE-inhibiting peptides, namely Val–Pro–Pro (VPP) and Ile–Pro–Pro (IPP), were identified. These peptides are already used in commercial products.
Article
The K-i values for the inhibition of the 70 kDa intracellular, o-phenanthroline-sensitive endopeptidase from Lactococcus lactis ssp. lactis MG1363 by bovine beta-casein (CN) f58-72, beta-CN f58-70, beta-CN f60-68, beta-CN f60-70, and beta-CN f60-66 were 0.018, 0.01, 0.045, 0.150 and 0.28 mM, respectively. The 95 kDa aminopeptidase, which is also, sensitive to o-phenanthroline, from the cytoplasm of the same microorganism was inhibited by the above peptides but the corresponding K-i values were 3-10 times higher. The X-prolyl-dipeptidyl aminopeptidase from the same source, which is insensitive to o-phenanthroline, was not inhibited by any of the above peptides and in fact hydrolyzed beta-CN f60-66 and beta-CN f60-70. beta-Casein f58-72 did not inhibit thermolysin and was degraded by it. The results demonstrate that cheese ripening may be influenced by inhibitory peptides originating from beta-casein.
Article
Detection of a casomorphin and a new casomorphin-like peptide from a casein enzymatic hydrolysate was achieved using second-order derivative spectra and UV-spectra comparison. In this method, synthetic casomorphins were used as standards. The β 1–3 casomorphin and α 90–94 casomorphin-like, were detected and rapidly purified with RP-HPLC coupled with photodiode array detector. This technique simplifies the identification and the purification of bioactive peptides containing aromatic amino acids from complex hydrolysates. It could be applied to follow up the evolution of these peptides during casein hydrolysis.
Article
The isolation and identification of low molecular mass peptides formed during the ripening of Parmigiano-Reggiano cheese is described. A strategy was used based on the fractionation of nitrogenous material using chemical methods followed by HPLC to isolate peptides and fast atom bombardment-mass spectrometry to identify them. It was found that the majority of cheese oligopeptides arose from the proteolysis of β-casein. Several phosphopeptides and oligopeptides known in vivo to be biologically active have also been identified during the ripening of cheese.(Received July 18 1991)(Accepted February 26 1992)
Article
Peptide extract from Brie cheese was examined. The opioid peptide was isolated from the extract. Its amino acid composition and N- and C-terminal amino acids were determined. Based on these analyses, the isolated peptide was assumed to correspond to β-casomorphin-7 (Tyr-Pro-Phe-Pro-Gly-Pro-IIe). The opioid activity was measured by examining the effects of peptide extract and isolated opioid peptide on the motor activity of isolated rabbit intestine.© 1999 Society of Chemical Industry
Article
The immunomodulation potential of four pressed-curd cheeses was studied in vitro on human T lymphocytes. T lymphocyte proliferation and metabolic activity were evaluated with optimized 5-bromo-2′-deoxyuridine and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide assays, respectively. The up to 4.6-fold stimulation of T-lymphocyte proliferation and metabolic activity by Abondance and Tomme de Savoie water-soluble extracts (WSEs) at the lag phase suggested the presence of immunomodulating compounds in these cheeses. Since peptides released from milk proteins during cheesemaking are potential immunomodulatory compounds, the high-performance liquid chromatography peptide profiles of the cheese WSEs were determined. While no correlation between peptide composition and in vitro immunomodulation of T-lymphocyte cells could be established, peptide quantity, size and hydrophobicity were related to cheesemaking technological parameters such as cheese age and cooking temperature.
Article
Recent investigations have suggested that novel food should be evaluated not only from the nutritional but also from the physiological point of view. Opioid peptides derived from milk belong to compounds whose physiological importance has not yet been fully recognized. The commercial Humana formula for newborns sold on the Polish market was assayed in the study. Four opioid peptides with agonistic and antagonistic activity were found in a peptide extract from the Humana formula and its pepsin–trypsin hydrolysate: β-casomorphin-5, casoxin C and 6 and lactoferroxin A. The opioid activity of the peptide extracts was determined by examining their influence on the motor activity of isolated rabbit intestine. In conclusion, it was suggested that the opioid activity of the formulas could be an additional indicator of their diversity. Copyright © 2007 Society of Chemical Industry
Article
 Skimmed milk was inoculated with a mixed culture of Streptococcus salivarius ssp. thermophilus and Lactobacillus delbrueckii ssp. bulgaricus and fermented at 44  °C. Peptides were isolated by treatment with a Dowex 50 WX 2 cation exchanger and then fractionated by ultrafiltration using membranes with cut-off sizes of 10, 5 and 1 kDa. Final separation of the oligopeptides was carried out by reversed-phase HPLC using gradient elution with an aqueous trifluoroacetic acid/acetonitrile mixture. Structure determination was performed by automated Edman degradation. Several peptides were additionally characterized by electrospray ionization-mass spectrometry. Peptides were evaluated for their hydrophobicity according to Ney's rule. Among the 30 peptides characterized, several precursors of bioactive peptides were detected.
Article
The release of β-casomorphin-5 (BCM5) and β-casomorphin-7 (BCM7) was investigated during simulated gastro-intestinal digestion (SGID) of bovine β-casein variants (n=3), commercial milk-based infant formulas (n=6) and experimental infant formulas (n=3). SGID included pepsin digestion at pH 2.0, 3.0 and 4.0 and further hydrolysis with Corolase PP™. β-Casein (β-CN) variants were extracted from raw milks coming from cows of Holstein-Friesian and Jersey breeds. Genomic DNA was isolated from milk and the β-CN genotype was determined by a PCR-based method. Phenotype at protein level was determined by capillary zone electrophoresis in order to ascertain the level of gene expression. Recognition and quantification of BCMs involved HPLC coupled to tandem MS. Regardless of the pH, BCM7 generated from variants A1 and B of β-CN (5-176mmol/mol casein) the highest amount being released during SGID of form B. As expected, the peptide was not released from variant A2 at any steps of SGID. BCM5 was not formed in hydrolysates irrespective of either the genetic variant or the pH value during SGID. Variants A1, A2 and B of β-CN were present in all the commercial infant formulae (IFs) submitted to SGID. Accordingly, 16-297nmol BCM7 were released from 800ml IF, i.e. the daily recommended intake for infant. Industrial indirect-UHT treatments (156°C×6-9s) did not modify release of BCM7 and, during SGID, comparable peptide amounts formed in raw formulation and final heat-treated IFs. Copyright © 2008 Elsevier Ltd. All rights reserved.
Article
Storage of UHT milk results in physico-chemical changes, which can sometimes lead to aggregation or sedimentation of milk. In this study, close attention was paid to the reactions occurring in semi-skimmed UHT milk during 6 months of storage at 4, 20 and 40 °C. Overall milk characterization revealed the development of the Maillard reaction and proteolysis which led to acidification of the milk. One hundred eighty-one peptides were identified by mass spectrometry for the freshly processed UHT milk and the milks stored 6 months at 4, 20 and 40 °C. The cleavage sites gave information concerning the possible actors of proteolysis. Plasmin, cathepsins B, D and G, elastase and proteases from Pseudomonas fluorescens B52 were thus found to be potential contributors to enzymatic proteolysis. Non-enzymatic proteolysis induced by heat-treatment and storage was also observed. Despite these modifications, milk particles (casein micelles and homogenized fat globules) did not exhibit many changes in zeta-potential, except for the last storage time at 40 °C where a decrease of the absolute value of about -3 mV was observed. The decrease in size in milks stored at 20 and 40 °C was only about 20 nm after 6 months. Similarly, storage of UHT milk showed that the higher the storage temperature the lower the heat stability and the higher the phosphate stability. Storage leads to physico-chemical changes in milk and, although some reactions such as acidification and proteolysis are known to be destabilizing, some of them are probably stabilizing to counterbalance the negative effects.
Article
The presence of the lysosomal cysteine proteinase cathepsin B in milk has been demonstrated recently. The potential significance of this enzyme to proteolysis in milk and dairy products was evaluated by the study of its proteolytic specificity towards the caseins. Bovine cathepsin B (1.4 units mL−1) was added separately to β-casein or αs1-casein (5 mg mL–1, in 100 mm Na acetate buffer, pH 6.0, containing 1.5 mm dithiothreitol). Samples were taken over a 24 h period and analysed by urea polyacrylamide gel electrophoresis and RP-HPLC; peptides were identified by N-terminal sequencing and mass spectrometry. Cathepsin B cleaved β-casein at 32 sites and αs1-casein at 35 sites, extensively hydrolysing both proteins. Thus, cathepsin B has a very broad specificity against the caseins, with an apparent preference for bonds incorporating the amino acids Leu, Val, Gln, Pro and Ser. Cathepsin B cleaved some bonds in common with chymosin (including the Phe23–Phe24 bond of αs1-casein), plasmin and the cell envelope-associated proteinases of Lactococcus, suggesting that it could be involved in proteolysis in dairy products, particularly if manufactured from high somatic cell count milk.
Article
Milk proteins exert a wide range of nutritional, functional and biological activities. Many milk proteins possess specific biological properties that make these components potential ingredients of health-promoting foods. Increasing attention is being focused on physiologically active peptides derived from milk proteins. These peptides are inactive within the sequence of the parent protein molecule and can be liberated by (1) gastrointestinal digestion of milk, (2) fermentation of milk with proteolytic starter cultures or (3) hydrolysis by proteolytic enzymes. Milk protein derived peptides have been shown in vivo to exert various activities affecting, e.g., the digestive, cardiovascular, immune and nervous systems. Studies have identified a great number of peptide sequences with specific bioactivities in the major milk proteins and also the conditions for their release have been determined. Industrial-scale technologies suitable for the commercial production of bioactive milk peptides have been developed and launched recently. These technologies are based on novel membrane separation and ion exchange chromatographic methods being employed by the emerging dairy ingredient industry. A variety of naturally formed bioactive peptides have been found in fermented dairy products, such as yoghurt, sour milk and cheese. The health benefits attributed to peptides in these traditional products have, so far, not been established, however. On the other hand, there are already a few commercial dairy products supplemented with milk protein-derived bioactive peptides whose health benefits have been documented in clinical human studies. It is envisaged that this trend will expand as more knowledge is gained about the multifunctional properties and physiological functions of milk peptides.
Article
The angiotensin converting enzyme (ACE)-inhibitory activity of several infant formulas was evaluated. Most of these products showed moderate inhibitory activity, but two exceptions that corresponded to an extensively hydrolysed whey formula and an extensively hydrolysed casein formula were detected. Two products (a non-hydrolysed milk protein-based formula and an extensively hydrolysed whey formula) were subjected to a two-stage in vitro enzymatic procedure, which simulates physiological digestion, in order to study the impact of digestion on ACE-inhibitory activity. The ACE-inhibitory activity of the non-hydrolysed formula increased during simulated gastrointestinal digestion, while no significant change was observed in the activity of the hydrolysed whey formula prior to and after, digestion. The peptides generated from these two products during simulated physiological digestion were sequenced by tandem spectrometry. At the end of the digestion, most peptides found in the non-hydrolysed milk protein-based formula were formed during incubation with the pancreatic extract, but, in the hydrolysed whey formula, many peptides present in the undigested product survived simulated digestion. The potential ACE-inhibitory activity of these peptides is discussed with regard to their amino-acid sequences.
Article
Yoghurt: Science and Technology is a standard work in its field for both industry professionals and those involved in applied research. Because manufacture is still, essentially, a natural biological process, it remains difficult to control the quality of the final product. Such control depends on a thorough understanding of the nature of yoghurt and both the biochemical changes and process technologies involved in production. Yoghurt: Science and Technology provides just such an understanding. Since the last edition, the industry has been transformed by the introduction of mild-tasting "bio-yoghurts", changing both consumer markets and manufacturing practices. This new edition has been comprehensively revised to take on board this and another major developments in the industry. Thus, today, millions of gallons of yoghurt are produced each year, yet manufacture is still, in essence, a natural biological process in which success can never be taken for granted. It is this capricious nature of the fermentation that leaves the system prone to variation. So, though some aspects of production of yoghurt have become fairly standard, there are so many areas of potential difficulty. This book offers preliminary guidance on the intricacies of production and distribution of yoghurt so as to minimize product failure.
Article
A material which displayed opioid activity in the guinea pig ileum longitudinal muscle-myenteric plexus preparation was extracted from an enzymatic casein digest into chloroform/methanol. The extract was roughly purified by adsorption/desorption procedures using charcoal and Amberlite XAD-2 resin as adsorbents. A high degree of purity was achieved by high-pressure liquid chromatography of the material on muBondapak C18 and mu-Porasil columns and finally by gel filtration chromatography on a Bio-Gel P-2 column. Several pronase-resistant compounds with opioid activity were obtained.
Article
The assessment of proteolysis levels is often achieved by global quantification of the peptides soluble at different TCA concentrations, but little information is available on the features of this precipitation mechanism. Peptic, tryptic and chymotryptic digests of alpha s1, beta, and kappa caseins have been prepared and fractionated by RP-HPLC and each isolated peptide was identified. Each digest was precipitated by adding TCA to different final concentrations (2, 4, 8, and 12%). The soluble fraction was analysed by RP-HPLC. Relationships have been searched between the properties of 75 peptides obtained in this way, and their solubilities in TCA. The best correlation was found with the peptide retention time in RP-HPLC, which can be regarded as the experimental measure of peptide hydrophobicity. We concluded that TCA, by interacting with peptides, induces an increase of the hydrophobicity of peptides which can lead to aggregation through hydrophobic interactions.
Article
Commercial cheese products were surveyed for beta-casomorphin peptides. Two extraction methods were compared: 1) water and 2) chloroform and methanol. Peptide profiles were determined using reverse-phase HPLC and multiple wavelength detection. beta-Casomorphin standards were used for comparison with cheese peptide profiles. Results indicated that peptides were present in cheeses with HPLC elution times that were similar to those for beta-casomorphins. However, comparison of absorbancies of the peaks at multiple wavelengths did not indicate peptides similar to beta-casomorphins. Therefore, beta-casomorphins were absent, or concentrations were below the HPLC detection threshold for beta-casomorphin of 2 micrograms/ml of cheese extract. The susceptibility of beta-casomorphins to the proteolytic system of a commercial strain of Lactococcus lactis ssp. cremoris was investigated. beta-Casomorphin standards were incubated at 4 degrees C with bacterial cell lysate at pH 5.0, 5.2, 5.4, and 5.7. Salt concentrations varied among 0, 1.5, and 5%. The concentration of added beta-casomorphins and the degradation products were monitored over 15 wk using HPLC. Enzymatic degradation of beta-casomorphins was influenced by the combination of pH and salt concentrations at cheese ripening temperatures. Therefore, if formed in cheese, beta-casomorphins may be degraded under conditions common for Cheddar cheese.
Article
Previously published Type I (insulin-dependent) diabetes mellitus incidence in 0 to 14-year-old children from 10 countries or areas was compared with the national annual cow milk protein consumption. Countries which were selected for study had appropriate milk protein polymorphism studies, herd breed composition information and low dairy imports from other countries. Total protein consumption did not correlate with diabetes incidence (r = +0.402), but consumption of the beta-casein A1 variant did (r = +0.726). Even more pronounced was the relation between beta-casein (A1+B) consumption and diabetes (r = +0.982). These latter two cow caseins yield a bioactive peptide beta-casomorphin-7 after in vitro digestion with intestinal enzymes whereas the common A2 variant or the corresponding human or goat caseins do not. beta-casomorphin-7 has opioid properties including immunosuppression, which could account for the specificity of the relation between the consumption of some but not all beta-casein variants and diabetes incidence.
Article
Seven kinds of ripened cheeses (8-mo-aged and 24-mo-aged Gouda, Emmental, Blue, Camembert, Edam, and Havarti) were homogenized with distilled water, and water-soluble peptides were prepared by C-18 hydrophobic chromatography. The inhibitory activity to angiotensin I-converting enzyme and decrease in the systolic blood pressure in spontaneously hypertensive rats were measured before and after oral administration of each peptide sample. The strongest depressive effect in the systolic blood pressure (-24.7 mm Hg) and intensive inhibitory activity to angiotensin I-converting enzyme (75.7%) were detected in the peptides from 8-mo-aged Gouda cheese. Four peptides were isolated by HPLC with reverse-phase and gel filtration modes. Their chemical structures and origins, clarified by combination analyses of protein sequencing, amino acid composition, and mass spectrometry, were as follows: peptide A, Arg-Pro-Lys-His-Pro-Ile-Lys-His-Gln [alpha(s1)-casein (CN), B-8P; f 1-9]; peptide B, Arg-Pro-Lys-His-Pro-Ile-Lys-His-Gln-Gly-Leu-Pro-Gln (alpha(s1)-CN, B-8P; f 1-13); peptide F, Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro-Asn (beta-CN, A2-5P; f 60-68); and peptide G, Met-Pro-Phe-Pro-Lys-Tyr-Pro-Val-Gln-Pro-Phe (beta-CN, A2-5P; f 109-119). Peptides A and F, which were chemically synthesized, showed potent angiotensin I-converting enzyme inhibitory activity with little antihypertensive effects.
Article
The risk factors identified with cardiovascular disease studied in the WHO MONICA project have been shown to have a limited relationship with the coronary heart disease mortality rates between centres, and in mirroring the historical rise and decline in deaths from the disease. Here we show that correlation of the calculated consumption of the milk protein, beta-casein A1 (excluding milk protein in cheese) against ischaemic heart disease (IHD) mortality has a r2 = 0.86. In the states of the former West Germany, where the breed composition of regional cattle herds has remained virtually constant since the 1950s, IHD mortality by state correlates with the estimated consumption of beta-casein A1. Information on other recognized dietary risk factors does not indicate any significant regional difference. Similarly, the populations of Toulouse in France and Belfast in Northern Ireland have almost identical collective 'traditional' risk factors for heart disease, yet the respective mortality rates vary more than threefold. People from Northern Ireland are estimated to consume 3.23 times more beta-casein A1, excluding cheese, than the French. The remarkable agreement between mortality and the consumption of this allele suggests that this factor is worthy of serious consideration as a potential source of cardiovascular disease when taken in conjunction with regional variations in the traditional risk factors. beta-casein A1 consumption also correlates strongly with type 1 diabetes incidence in 0-14-year-olds, suggesting that IHD and diabetes may share at least one causative risk factor.
Article
A simple in vitro protocol simulating gastrointestinal digestion of proteins and peptides to investigate the effect of digestive enzymes on the biological activity of peptides present in dairy products was developed. This protocol consisted in a 30 min incubation with pepsin followed by a 4 h incubation with trypsin or pancreatin. It was applied to an Emmental cheese water-soluble extract (WSE) and to a casein solution (as a control). Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) allowed to monitor the digestion of proteins. Reversed-phase high-performance liquid chromatography (RP-HPLC) allowed to monitor the conversion of proteins and peptides into peptides and amino acids: it is proposed to use the mean retention time corresponding to the overall retention time distribution of molecules to assess the effect of digestive enzymes. The biological activity focused in this study was the angiotensin I converting enzyme (ACE) inhibitory activity. Digestion of Emmental WSE induced an increase of the ACE inhibition as compared to undigested WSE while a 10 kDa ultrafiltered WSE lost a part of its ACE inhibitory activity after digestion process. These results strongly suggest that digestive enzymes diminished the ACE inhibition by the peptides present in Emmental cheese WSE, while the digestion of peptides of high molecular weight would generate new ACE inhibitory peptides.