Bovine Casein Research Articles

Casein network formation at oil–water interfaces is reduced by [formula omitted]-casein and increased by Ca[formula omitted]

Mixtures of bovine caseins can serve as a benchmark for understanding the functionality of microbial-based recombinant caseins at oil–water interfaces. In this work we show that, in the presence of Ca2+, the individual casein fractions form viscoelastic networks at the oil–water interface with comparable stiffness. In the absence of Ca2+, αs2- and β-casein interfacial network formation was strongly inhibited over the full deformation regime. For αs1-casein, the network stiffness was increased in the absence of Ca2+ at small deformations (<15%), but at large deformations (>50%) it was completely disrupted, to a similar stiffness as αs2- and β-casein. The interfacial structure formed by κ-casein was largely unaffected by Ca2+ due to limited phosphorylation. We hypothesize that the differences between calcium-sensitive caseins lie in the conformation they assume at the interface. Both αs2- and β-casein adsorb in a train-tail conformation with a tail extending into the aqueous bulk phase, whereas αs1-casein adsorbs in a loop-train conformation, with a loop that extends less into the bulk phase. The tail-train configuration is hypothesized to increase the inter-molecular Ca2+ bridging thereby increasing the interfacial stiffness of αs2- and β-casein.Blending the casein fractions revealed a strong negative effect of β-casein on the interfacial modulus, which was more pronounced at a higher concentration. The presence of Ca2+ remained important for interfacial network formation of a casein blend. Without Ca2+, the interfacial network was less stiff, more viscous, and behaved like a 2d polymer solution.With this work we showed that casein interfacial network formation at oil–water interfaces is mediated by Ca2+ bridging. Blending the different casein fractions decreased the interfacial viscoelastic properties through the presence of β-casein. These results indicate that future work on recombinant caseins should focus on single genetic variants, since a blend of variants will likely decrease interfacial functionality.

Analysis of cow and goat milk by spr: adulteration detection and differences in protein adsorption

The article discusses using Surface Plasmon Resonance (SPR) to detect adulteration of goat milk with cow milk. The SPR technique is based on changes in the refractive index and can be used to monitor the adsorption of proteins on the sensor surface. The SPR technique can differentiate between pure cow milk and pure goat milk by measuring the initial refractive index, which is higher for cow milk. The article then describes the changes in the refractive index that occur when goat milk is adulterated with cow milk and how adding a bovine casein film on the sensor surface can make it more selective for cow milk. The differences in the chemical compositions and molecular structures of cow and goat milk and how these differences affect the adsorption of proteins and fatty acids on the sensor surface. Finally, other proteins, such as α-lactalbumin and β-lactoglobulin, are used as indicators of adulteration.

Production of antioxidant hydrolysates from bovine caseinate and soy protein using three non-commercial bacterial proteases

The release of bioactive peptides from proteins can be achieved by enzymatic hydrolysis, whereby mainly commercial enzymes are applied, although increasing attention is focused on alternative proteases. Hence, crude proteases from Bacillus sp. CL14, CL18, and CL33A were investigated to obtain antioxidant hydrolysates from bovine sodium caseinate (NaCAS) and soy protein isolate (SPI). The proteases were able to hydrolyze both substrates. Hydrolysis improved the radical-scavenging, Fe2+-chelating, and reducing power abilities of NaCAS and SPI. Higher antioxidant activities were commonly achieved at 2–3 h of hydrolysis. Overall antioxidant activities were higher for NaCAS hydrolysates produced with CL14 > CL18 > CL33 protease, and for SPI hydrolysates obtained with CL14 > CL33A ∼ CL18 protease. Generation of antioxidant protein hydrolysates suggest applications of enzymes and hydrolysates in food/feed (bio)technology.

Urinary opioid peptides in children with autism spectrum disorders. A Pilot Study

BACKGROUND: Circulating β-casomorphins (BCM) and gluten exorphins (GE), which are exogenous opioid peptides originating from bovine milk protein casein and cereal protein gluten, respectively, have been proposed as potential trigger factor to symptoms observed in children with autism. OBJECTIVE: Given the debate surrounding the detectability of these opioid peptides in body fluids, particularly using highly sensitive and specific mass spectrometry (HPLC-MS) techniques, we aimed to investigate their presence in urine samples of autistic subjects. METHODS: We employed an HPLC-MS method for peptide detection. RESULTS: The presence of several BCMs and GEs in the urine of both autistic children (ASD) and healthy controls (HC) was documented. The detection of dietary opioid peptides even at very low concentrations underscores the sensitivity of this novel HPLC-MS method. BCM-8 was more often detected in the ASD group compared to the HC group. A higher prevalence of gastrointestinal (GI) symptoms were also observed in the ASD group. CONCLUSIONS: This pilot study supports the presence of BCMs and GEs in urine samples in subjects with autism as well as healthy controls which was the main goal of this pilot study. Prolonged exposure to bovine BCMs and GEs may play a role in the manifestation of core and GI symptoms in subgroups of autism. Further research is warranted to investigate this phenomenon thoroughly.

Fe3O4@silica-thermolysin: A robust, advantageous, and reusable microbial nanobiocatalyst for proteolysis and milk-clotting

Thermolysin (TLN) is a microbial highly-priced thermostable metallo-endoprotease with complementary substrate specificity to those of proteases widely used in science and industry for protein digestion and milk-clotting. This study is the first to immobilize TLN on aminated superparamagnetic nanoparticles (Fe3O4@silica-NH2) aiming for higher stability, recoverability, reusability, and applicability in proteolysis and as a microbial rennet-like milk-clotting enzyme. The nanobiocatalyst developed (Fe3O4@silica-TLN) displays hydrolytic activity on a synthetic TLN substrate and, apparently, was fully recovered from reaction media by magnetic decantation. More importantly, Fe3O4@silica-TLN retains TLN catalytic properties in the presence of calcium ions even after exposure to 60 °C for 48 h, storage at 4 °C for 80 days and room temperature for 42 days, use in proteolyses, and in milk-clotting for up to 11 cycles. Its proteolytic activity on bovine milk casein in 24 h furnished 84 peptides, of which 29 are potentially bioactive. Also, Fe3O4@silica-TLN catalyzed the digestion of bovine serum albumin. In conclusion, Fe3O4@silica-TLN showed to be a new, less autolytic, thermostable, non-toxic, magnetically-separable, and reusable nanobiocatalyst with highly attractive properties for both science (peptide/protein chemistry and structure, proteomic studies, and the search for new bioactive peptides) and food industry (cheese manufacture).

Release of CM-12 from A2-type casein by the cleavage of Ser-Leu-Xaa at the C-terminus using Aspergillus oryzae alkaline protease.

Aspergillus oryzae protease can release the opioid peptide β-casomorphin-10 (CM-10, YPFPGPIPNS, 60-69) from A2-type casein. However, not only is the yield of the active peptide low, but the key enzyme involved in processing has yet to be identified. A significant amount of the opioid peptide 60YPFPGPIPNSLP71 (CM-12) was produced from the A2-type casein peptide 53AQTQSLVYPFPGPIPNSLPQNIPPLTQTPV82 when the active protease in A. oryzae protease extract was fractionated with DEAE-Sepharose. The fractionated enzyme produced CM-12 from bovine A2-type casein but not from bovine A1 casein. A major protein of 34 kDa was purified and identified as an alkaline protease (Alp). Motif prediction of the Alp cleavage site using Multiple EM for Motif Elicitation analysis revealed preferable cleavage at the C-terminal end of Ser-Leu-Xaa for the release of CM-12. A2-type casein hydrolysate by Alp exhibited similar levels of opioid activity to that of synthetic CM-12 in cAMP-Glo assays with μ-opioid receptor-expressing HEK293 cells. These results suggest that CM-12 is a major opioid peptide in the casein hydrolysate. Our findings showed that Alp fractionated from A. oryzae protease extract produced the opioid peptide CM-12 from A2-type casein as a result of preferential cleavage at the C-terminal end of Ser-Leu-Xaa and the removal of coexisting enzymes. Moreover, docking predictions suggested a stable interaction between CM-12 and the 3D structure of Alp. Casein hydrolysate with Alp-containing CM-12 has the potential for use as a bioactive peptide material with opioid activity. © 2024 Society of Chemical Industry.

Changes in allergenicity characteristics of bovine casein by enzymatic hydrolysis treatment

Changes in allergenicity characteristics of bovine casein by enzymatic hydrolysis treatment

The Severity of Gastrointestinal Disorders and Autistic-Like Behaviors Could Be Associated with a Selective Humoral Response to Bovine Milk Caseins: A Case Series

The Severity of Gastrointestinal Disorders and Autistic-Like Behaviors Could Be Associated with a Selective Humoral Response to Bovine Milk Caseins: A Case Series

Comparative detection of goat's milk casein through various enzyme‐linked immunosorbent assay (ELISA) approaches using caprine alpha‐S1 casein peptides and bovine casein polyclonal antibodies

SummaryCasein causes allergic reactions in cow's milk protein allergy (CMPA) individuals. Casein can cross‐react with cows and goats. Hence, a rapid and highly specific technique is required for its detection. The study aims to evaluate and optimise multiple Enzyme‐Linked Immunosorbent Assay (ELISA) approaches for the detection of casein isolated from goat's milk. The study utilised and optimised various ELISA approaches, including direct, indirect and sandwich with various antibodies. Several parameters of ELISA including pre‐treatments of casein, blocking reagent, concentrations of antibodies and background signal were evaluated. The results showed that sandwich ELISA revealed a good signal‐to‐noise ratio at 1 mg mL−1. Indirect ELISA showed a higher signal‐to‐noise ratio without any heat treatment in comparison to the direct ELISA despite utilising an equivalent concentration (i.e., 10 μg mL−1) of goat's milk casein. The findings provide a theoretical foundation for obtaining a high signal‐to‐noise ratio for antigenicity studies of goat's milk casein to minimise its immunological response.

Extraction, catalytic study and milk-clotting properties of proteases from Brassicaoleracea

Proteases were extracted and biochemically characterized from different Brassica oleracea varieties (B. oleracea var. palmifolia (black cabbage, BC), B. oleracea var. sabellica (curly kale, K), and B. oleracea var. botrytis (white cauliflower, WC)). Calf rennet (CR) and Cynara cardunculus crude extracts (CC) were used as reference samples. The catalytic study used two different substrates: the synthetic peptide Bz-Phe-Val-Arg-pNA (BPVA-pNA), which contains para-nitroaniline (pNA), and azocasein (AzC) as a macromolecular substrate. The effect of temperature and pH on the proteolytic activity were also evaluated. Brassica proteases were further proposed as milk coagulants, and their coagulating efficiencies were evaluated. Bovine casein was incubated with the plant extracts, and the relative breakdown products were observed through electrophoresis. BC, K, and WC contain proteases which hydrolyze the macromolecular substrate (AzC) and synthetic peptides with Arg in P-1 (BPVA-pNA). Therefore, among the three B. oleracea varieties and control extracts, K leaf extracts displayed greater catalytic efficiency against AzC. Brassica extracts showed a lesser milk clotting index but visible milk curd formation. More importantly, the protease activities of BC and K extracts exhibited great catalytic activity over a broad range of pH (3.6–9.0) and temperatures (40–70 °C) using AzC as a substrate. Current pH and thermal tolerance results indicate that B. oleracea var. palmifolia and B. oleracea var. sabellica proteases could have broad applications in food technology. Many plant resources, which could represent potential sources of efficient proteases, are still unexploited.

Synthesis and dual sensing of vanadium and copper ions using protein capped selenium nanoparticles

Highly homogenous and spherical shaped Selenium nanoparticles with an average size of 20 nm were synthesized from an eco-friendly and environmentally safe approach using bovine milk protein casein as stabilizing agent (named as CasSeNPs). Protein functionalization provided excellent stability at variable temperatures (25–60 °C), pH (8–10), ionic strength (0.05–2 M), and storage period for more than five months. Synthesized CasSeNPs were extensively characterized using various spectroscopic (UV–visible, Fourier transform infrared (FTIR), circular dichroism (CD), intrinsic fluorescence spectroscopy), particle sizing (dynamic light scattering (DLS) and zeta potential), microscopic (scanning electron microscopy, energy dispersive X-ray spectroscopy (SEM/EDS), atomic force microscopy (AFM)), and X-ray diffraction (XRD) measurements. Moreover, CasSeNPs was established as highly sensitive and selective sensor for the simultaneous detection of vanadium (V+2) and copper (Cu+2) ions with significant linearity (0.5–10 and 0.1–1.0 µM, respectively), detection limits (LOD = 0.03 and 0.067 µM, respectively), and percent recoveries (94–105 %) in real environmental (tap water) and biological (human plasma and urine) samples with adequate test performance. Cumulatively, obtained results revealed that the established very simple, biocompatible and economical nanosensor has great potential for applications in analytical chemistry as well as for biological investigations.

Application of on-line sample preconcentration by large-volume dual preconcentration by isotachophoresis and stacking (LDIS) on straight-channel microchips.

In this study, large-volume dual preconcentration by isotachophoresis and stacking (LDIS) which is an on-line sample preconcentration technique coupling large-volume sample stacking with an electroosmotic flow pump (LVSEP) with transient isotachophoresis (tITP) was applied to microchip electrophoresis (MCE) for improving both detection sensitivities and peak shapes. To realize LDIS in MCE, we investigated experimental procedures for injecting a short plug of a leading electrolyte (LE) solution into a straight microchannel without any sophisticated injector apparatus. We found that a short LE plug could be injected into a sample-filled straight-channel only by making the liquid level of the LE solution in an outlet reservoir higher than that in an inlet one. By applying a reversed-polarity voltage to the microchip, anionic analytes injected throughout the microchannel were first enriched by LVSEP, followed by tITP. Through the second preconcentration effect by tITP in LDIS, sensitivity enhancement factor (SEF) and asymmetry factor for a standard dye were improved from 878 and 0.62 to 1330 and 1.14, respectively, relative to those in conventional LVSEP. It should be noted that more viscous running buffer containing sieving polymers could be employed to the LDIS analysis, which was effective for improving the SEF and the separation efficiencies, especially for bio-polymeric compounds. Finally, LDIS was applied to the oligosaccharide and protein analyses in MCE, resulting in the SEFs of 1410 and ca. 50 for maltotriose and bovine milk casein, respectively.

Effects of bovine casein hydrolysate as a dry cow therapy on prevention and cure of bovine intramammary infection,

The objectives were to investigate the efficacy of bovine casein hydrolysate (bCNH) as a dry cow therapy on: 1) preventing new intramammary infection (IMI) postpartum of all bacteria and coagulase-negative staphylococci (CNS); 2) curing existing subclinical infections, mainly of CNS; and 3) affecting on milk yield, composition and somatic cell count (SCC) during the post-calving lactation.

Enzymatic characterization and synergistic effects of protease combinations on DPP-IV inhibitory peptide release from bovine casein

This study focused on the enzymatic characteristics, with regard to primary specificity, secondary specificity, and hydrolysis ability of five proteases (Alcalase, Protamex, papain, Flavourzyme, and ProtexA), towards the release of dipeptidyl peptidase-IV (DPP-IV) inhibitory peptides from bovine casein. The secondary specificity of the five proteases, especially the release capability for Xaa-Pro-type peptide played a determining role in the release of DPP-IV inhibitory peptides from bovine casein. Moreover, protease combinations, chosen based on their Xaa-Pro-type peptide release abilities, resulted in superior DPP-IV inhibitory activity compared to other combinations. Particularly, the hydrolysate with the highest DPP-IV inhibitory activity (IC50 value of 0.45 ± 0.07 mg/mL) was obtained by the combination hydrolysis of ProtexA and papain, achieving a 74.63% ± 1.62% (peak area) release of Xaa-Pro-type peptide, and a significant improving glucose tolerance in mice (33.42% ± 1.68% reduction of blood glucose AUC compared with control mice). The mechanistic insights revealed that papain efficiently released long Xaa-Pro-type peptides in the early hydrolysis stage, and subsequently, the synergistic effect of papain and ProtexA led to an accelerated and extensive release of short Xaa-Pro-type peptides. These findings provide valuable insight into enzymatic processes for generating DPP-IV inhibiting peptides from casein.

Neonatal Gut and Immune Responses to β-Casein Enriched Formula in Piglets

Backgroundβ-casein is the main casein constituent in human milk (HM) and a source of bioactive peptides for the developing gastrointestinal tract and immune system. Infant formulas contain less β-casein than HM, but whether different concentrations of β-casein affect tolerability and gut and immune maturation in newborns is unknown. ObjectivesUsing near-term piglets as a model for newborn infants, we investigated whether increasing the β-casein fraction in bovine-based formula is clinically safe and may improve gut and immune maturation. MethodsThree groups of near-term pigs (96% gestation) were fed formula with bovine casein and whey protein (ratio 40:60): 1) standard skim milk casein (BCN-standard, 35% β-casein of total casein, n = 18); 2) β-casein enrichment to HM concentrations (BCN-medium, 65%, n = 19); and 3) high β-casein enrichment (BCN-high, 91%, n = 19). A reference group was fed 100% whey protein concentrate (WPC) as protein (WPC, n = 18). Intestinal and immune parameters were assessed before and after euthanasia on day 5. ResultsClinical variables (mortality, activity, body growth, and diarrhea) were similar among the groups, and no differences in intestinal or biochemical parameters were observed between BCN-standard and BCN-medium pigs. However, pigs receiving high amounts of β-casein (BCN-high) had lower small intestine weight and tended to have more intestinal complications (highest gut pathology score, permeability, and interleukin-8) than the other groups, particularly those receiving no casein (WPC pigs). Blood lymphocyte, thrombocyte, and reticulocyte counts were increased with higher β-casein, whereas eosinophil counts were reduced. In vitro blood immune cell responses were similar among groups. Conclusionsβ-casein enrichment of bovine-based formula to HM concentrations is clinically safe, as judged from newborn, near-term pigs, whereas no additional benefits to gut maturation were observed. However, excessive β-casein supplementation, beyond concentrations in HM, may potentially induce gut inflammation together with increased blood cell populations relative to natural β-casein concentrations or pure whey-based formula.

Isolation and characterization of a Mannheimiahaemolytica secreted serine protease that degrades sheep and bovine fibrinogen

Mannheimiahaemolytica is an opportunistic agent of the respiratory tract of bovines, a member of the Pasteurellaceae family, and the causal agent of fibrinous pleuropneumonia. This bacterium possesses different virulence factors, allowing it to colonize and infect its host. The present work describes the isolation and characterization of a serine protease secreted by M. haemolytica serotype 1. This protease was isolated from M. haemolytica cultured media by precipitation with 50 % methanol and ion exchange chromatography on DEAE-cellulose. It is a 70-kDa protease able to degrade sheep and bovine fibrinogen or porcine gelatin but not bovine IgG, hemoglobin, or casein. Mass spectrometric analysis indicates its identity with protease IV of M. haemolytica. The proteolytic activity was active between pH 5 and 9, with an optimal pH of 8. It was stable at 50 °C for 10 min but inactivated at 60 °C. The sera of bovines with chronic or acute pneumonia recognized this protease. Still, it showed no cross-reactivity with rabbit hyperimmune serum against the secreted metalloprotease from Actinobacilluspleuropneumoniae, another member of the Pasteurellaceae family. M. haemolytica secreted proteases could contribute to the pathogenesis of this bacterium through fibrinogen degradation, a characteristic of this fibrinous pleuropneumonia.

Interfacial network formation by casein blends in the presence of Ca2+ is dominated by β- and κ-casein

With the increasing interest in precision fermentation for food purposes, the opportunity arises to produce individual casein fractions or specific blends based on the requirements of an application. Therefore, the functionality of individual casein fractions has gained attention in academia and industry.This study focused on the air-water interfacial functionality of individual bovine casein fractions in casein blends, to serve as a benchmark for precision fermented casein fractions. With oscillating bubble tensiometry, we showed that the interfacial behavior of a milk-like casein blend of αs1-, αs2-, β-, &κ-casein (ratio 4:1.1:3.8:1.3) was dominated by the β- and κ-casein fractions through the formation of a disordered solid-like viscoelastic interface. The presence of Ca2+ in the buffer was essential; Without Ca2+, the casein blend formed a weak interfacial microstructure where the viscoelastic modulus was dominated by exchange between bulk and interface. The interfacial microstructure was imaged with Langmuir-Blodgett deposition followed by atomic force microscopy. Here we showed that the interfacial structure of β+κ-casein was highly similar to the milk-like casein blend. In the presence of Ca2+ in the buffer, the interfacial microstructure of the casein blend was shown to be more connected and homogeneous compared to a buffer without Ca2+. Thereby highlighting the importance of the phosphoserine residues in β-casein.These results show the opportunity to mimic the interfacial functionality of a milk-like casein blend with only a blend of β+κ-casein but that the presence of phosphoserine residues in β-casein is essential for the formation of a stiff interfacial network.

Characterization of the potential allergenicity of enzymatically hydrolyzed casein in Balb/c mouse model.

Bovine casein is a major allergen present in cow milk to induce anaphylaxis. In this study, the potential allergenicity of enzymatically hydrolyzed casein (HC) was evaluated based on in vitro and in vivo. The results showed that Alcalase and Protamex treatment (AT, PT) reduced the potential allergenicity of CN, with the greatest reductions of 68.25% and 50.75%, respectively. In addition, in vivo results showed that HC effectively alleviated allergic response symptoms of Balb/c mice; a significant tendency toward decreased serum IgG1 and mast cell tryptase levels was observed, accompanied by a decrease of Th2-associated IL-4, IL-5, and IL-13 and an increase of IFN-γ levels in spleen. Moreover, the inflammation of the lung, jejunum, and ileum was remarkably ameliorated. The findings indicated that HC induced a shift toward Th1 response and maintained the Th1/Th2 immune balance. Importantly, our results provide the basis for the production of hypoallergenic dairy products.

Formation of casein micelles from bovine caseins simulating human casein phosphorylation patterns: Micellar structure and in vitro infant gastrointestinal digestion

This study investigated the micellar structure and in vitro infant gastrointestinal digestion of formed casein micelles simulating human casein composition and phosphorylation patterns. Caseins were fractionated from bovine caseins and formulated according to human casein composition, i.e., β-, κ- and αs1-caseins with a ratio of 68:20:12. The formulated caseins were dephosphorylated for different times and were mixed to obtain mixed phosphorylation patterns (FC-MP) for β-casein, showing 0–5 phosphate groups comparable to human β-casein. The formulated caseins without dephosphorylation and with high dephosphorylation, showing 5 phosphate groups and 0–2 phosphate groups for β-casein, respectively, were categorized as high (FC-HP) and low phosphorylated (FC-LP). With the average mineral concentrations found in human milk, CaCl2, MgCl2, citrate and inorganic phosphate were added into the different formulated casein dispersions to obtain casein micelles (FM-HP, FM-MP and FM-LP). Compared to FM-HP, FM-MP was closer to human micelles in relation to particle size, micellar hydration, molar ratio of Ca:casein, morphology and internal structure, whereas FM-LP showed did not show a characteristic micellar structure and had a larger size and lower molar ratio of Ca:casein. For gastrointestinal digestion, FM-MP was closer to human micelles in terms of the gastric flocs, casein degradation rate, free amino groups, and molecular weight distribution of peptides compared to FM-HP, whereas FM-LP showed larger flocs and lower casein degradation rate. These results show the significance of post-translational phosphorylation in facilitating the assembly of caseins into micellar structures, indicating the potential to form human-like micelles for application in infant formulae.

Structure and digestibility of bovine casein complexes formed by enriching κ- and β-caseins in micellar casein concentrate together with minerals adjustment

Colloidal structure and infant in vitro gastrointestinal digestibility of casein complexes simulating caseins and minerals compositions of human casein micelles were studied. κ- and β-Caseins were fractionated from bovine micellar casein concentrate (MCC), and mixed with MCC to increase their ratios to 20% and 68%, followed by adding citrate (Cit), calcium (Ca), and inorganic phosphate (Pi) to obtain the casein complexes. The complexes enriched with κ- and β-caseins showed higher percentages of serum Ca, Pi and caseins, larger particle size, and looser internal structure. During gastric digestion, the complexes enriched with κ- and β-caseins formed smaller and looser coagula, which promoted degradation of intact caseins and formation of free amino groups and small peptides. During initial intestinal digestion, the complexes enriched with κ- and β-caseins formed more free amino groups and smaller peptides. These results offered a potential strategy to form human casein micelles analogues for use in infant formula.