Biodegradable methacrylated casein for cardiac tissue engineering applications.

Casein is a naturally derived amino group (-NH2) rich protein, that enables surface functionalization leading to hydrophilicity, which in turn facilitates better cell adhesion. Casein obtained from either commercial ß-casein rich skim milk (A2 milk) or dissolved air flotation (DAF) technology was tested for its potential for tissue engineering applications in a comparative study. A novel biodegradable biomaterial was synthesized from casein by chemically modifying with methacrylic anhydride (MA) and combined with polyvinyl alcohol (PVA) and polyvinyl pyrrolidone (PVP) blend. The resulting methacrylated casein (CasMA) with the two polymers was processed into porous scaffolds with low and high MA concentrations to demonstrate CasMA's ease of modification and reproducibility. Fourier Transform Infrared Microscopy (FTIR) and Proton Nuclear Magnetic Resonance (1H NMR) revealed the presence of all the components and the successful modification of casein. The rheological and morphological analysis presented viscous behaviour and columnar hollow tube-like microstructures in agreement with the biomaterials' swelling and biodegradation behaviour. The live/dead in vitro assay showed high cell viability that agreed with the cell proliferation (MTT) assay in vitro, which indicated increased proliferation upon casein modification at appropriate biomaterial concentrations and volumes. This study not only showed a possible mechanism of casein methacrylation but also presented the potential use of waste materials like DAF-casein as a value-added product for tissue engineering applications.

Synthesis and characterization of alkylated caseinate, and its structure-curcumin loading property relationship in water.

Alkylated caseinates (Cn-caseinates) containing selected C8-C16 alkyl groups were successfully synthesized through a two-step reaction between the protein and fatty acids. The substitution degree (SD) of the alkyl groups was 5.2-72.9%, which depended on the feed molar ratio of reagents and fatty acid chain length. The SD value was positively associated with the surface hydrophobicity index (S0) of the Cn-caseinate. Among the tested Cn-caseinates (n = 0, 8, 12, 14 and 16), C16-caseinate showed the best self-assembly and curcumin-loading properties in water. With increased palmitoyl group SD, the critical micelle concentration (CMC) of C16-caseinate was decreased from 5.15 to 3.77 mg/L and the encapsulation efficiency of curcumin-loaded C16-caseinate self-assemblies was increased from 31.16% to 69.87%. Transmission electron microscopy analysis demonstrated a desirable sphere assembly. In addition, the C16-caseinate self-assemblies had good re-dispersibility and storage stability at 4 °C for 6 weeks.

Anti-Inflammatory and Antioxidant Properties of Casein Hydrolysate Produced Using High Hydrostatic Pressure Combined with Proteolytic Enzymes.

Casein-derived peptides are shown to possess radical scavenging and metal chelating properties. The objective of this study was to evaluate novel anti-inflammatory properties of casein hydrolysates (CH) produced by an eco-friendly process that combines high hydrostatic pressure with enzymatic hydrolysis (HHP-EH). Casein was hydrolysed by different proteases, including flavourzyme (Fla), savinase (Sav), thermolysin (Ther), trypsin (Try), and elastase (Ela) at 0.1, 50, 100, and 200 MPa pressure levels under various enzyme-to-substrate ratios and incubation times. Casein hydrolysates were evaluated for the degree of hydrolysis (DH), molecular weight distribution patterns, and anti-inflammatory properties in chemical and cellular models. Hydrolysates produced using HHP-EH exhibited higher DH values and proportions of smaller peptides compared to atmospheric pressure-enzymatic hydrolysis (AP-EH). Among five enzymes, Fla-digested HHP-EH-CH (HHP-Fla-CH) showed significantly higher antioxidant properties than AP-Fla-CH. The anti-inflammatory properties of HHP-Fla-CH were also observed by significantly reduced nitric oxide and by the suppression of the synthesis of pro-inflammatory cytokines in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) revealed that 59% of the amino acids of the peptides in HHP-Fla-CH were composed of proline, valine, and leucine, indicating the potential anti-inflammatory properties. In conclusion, the HHP-EH method provides a promising technology to produce bioactive peptides from casein in an eco-friendly process.

Fabrication of epigallocatechin-3-gallate nanocarrier based on glycosylated casein: stability and interaction mechanism.

Polyphenols normally have strong binding affinity with proteins, which may lead to protein precipitation. Glycosylation of protein via Maillard reaction in mild condition may inhibit the precipitation. This study prepared nanocomplexes of epigallocatechin-3-gallate (EGCG) and protein, and their stability against environmental stress was investigated. Subsequently, these findings were correlated with the interactions between EGCG and casein. Results showed that glycosylated casein displayed strong encapsulating and retaining capacity to EGCG, and no obvious aggregation or fusion appeared in the concentration range of 0.25-5.00 mg/mL during storage. The in vitro release demonstrated that casein, especially glycosylated casein, could effectively protect EGCG from degradation in alkaline pH and displayed a slow and sustained release in intestinal fluid, which may be attributed to the inhibiting effects of casein binding on the motion freedom of EGCG. Fluorescence quenching spectra demonstrated that the steric hindrance induced by dextran could inhibit the interaction between casein and EGCG. These findings demonstrated that glycosylated casein could be used as a promising and effective nanocarrier for EGCG.

The design and characterization of a novel beta-casein nano-vehicle loaded with platinum anticancer drug for drug delivery.

We developed a drug-delivery system comprising a novel platinum drug (Pt(II) complex) entrapped within ß-Casein (ß-CN) nanoparticles referred to as nano-vehicles. Fluorescence spectroscopy, UV-Vis spectrometry, dynamic light scattering (DLS), and scanning electron microscopy (SEM) were used to characterize the ß-CN-Pt(II) complex . What was apparent in this study was that the solubility of Pt (II) complex increased in the presence of ß-CN. Furthermore, fluorescence spectroscopy results revealed the binding of the ß -CN micelle to the platinum complex at pH 7.0. The tryptophan fluorescence intensity further revealed that the optimal loading molar ratio of ß-CN: Pt (II) complex was 1:3 (with ß-CN at 1 mg/mL). Under these conditions, the optimal nano-vehicle was formed based on the DLS results. Results from the DLS and SEM analyses are proof for the formation of the ß-CN-Pt(II) complex nanoparticles with a very good colloidal stability and an average particle size of 250 nm. Finally, the cytotoxicity of free- and encapsulated-Pt (II) complex was evaluated using colorectal carcinoma HCT116 cells, as a cancer model cell line, because platinum drugs have been used mostly for treatment of Gastrointestinal cancers. Results indicated that the cytotoxicity and cellular uptake of the drug was enhanced when entrapped in ß -CN nanoparticles. Polymeric micelles are internalized into the cells via fluid-state endocytosis. These findings suggest that ß-CN is an excellent nano-vehicle for targeted delivery of platinum drugs, which are generally recognized as safe (GRAS) and potentially useful in pharmaceutical industries.

Synthesis and biological response of casein-based silica nano-composite film for drug delivery system.

Casein possesses many interesting properties that make it a good candidate for conventional and novel drug delivery systems. In this study, casein-based silica nano-composite was prepared via double in situ method, and the as-prepared latex particles were evaluated in terms of their morphology and size through transmission electron microscopy (TEM). The film morphology was investigated by scanning electron microscopy (SEM) and energy dispersive X-ray (EDX), and the mechanical property and response behavior of the films as a function of silica content were discussed. Ibuprofen was used as the model drug. The drug load and release properties were studied by solid-state nuclear magnetic resonance (solid-state NMR), Fourier transform infrared (FT-IR), SEM and in vitro test. The composite latex particle showed a stable core-shell structure, and the film exhibited a regular surface with even SiO2 distribution. The drug load efficiency of the composite films increased with adding silica because of the adsorption of the drugs on the silica. In an acidic release medium, the ibuprofen-loaded composite showed a slower drug release dependent on the silica content. These behaviors were most likely due to the reduced diffusion rate of the drug through the composite microsphere, which resulted from the interaction between the silica and the drug.

Microwave initiated synthesis of polyacrylamide grafted casein (CAS-g-PAM)--its application as a flocculant.

Modification of the milk protein 'casein' has been carried out through microwave initiated graft copolymerization of acrylamide. The synthesis was optimized in terms of microwave irradiation time and monomer (acrylamide) concentration. The synthesized graft copolymers have been characterized by intrinsic viscosity measurement, elemental analysis, FTIR spectroscopy, scanning electron microscopy (SEM) and number average molecular weight determination through osmometry; taking the starting material (casein) as reference. Further, flocculation efficacy of various grades of the grafted product were studied and compared to that of the starting material 'casein' by Jar test and settling test procedure, in 1% coal-fine suspension.

Extensive manipulation of caseicins A and B highlights the tolerance of these antimicrobial peptides to change.

Caseicins A and B are low-molecular-weight antimicrobial peptides which are released by proteolytic digestion of sodium caseinate. Caseicin A (IKHQGLPQE) is a nine-amino-acid cationic peptide, and caseicin B (VLNENLLR) is a neutral eight-amino-acid peptide; both have previously been shown to exhibit antibacterial activity against a number of pathogens, including Cronobacter sakazakii. Previously, four variants of each caseicin which differed subtly from their natural counterparts were generated by peptide synthesis. Antimicrobial activity assays revealed that the importance of a number of the residues within the peptides was dependent on the strain being targeted. In this study, this engineering-based approach was expanded through the creation of a larger collection of 26 peptides which are altered in a variety of ways. The investigation highlights the generally greater tolerance of caseicin B to change, the fact that changes have a more detrimental impact on anti-Gram-negative activity, and the surprising number of variants which exhibit enhanced activity against Staphylococcus aureus.

Formation of fibrous materials from dense calcium caseinate dispersions.

Application of shear and cross-linking enzyme transglutaminase (Tgase) induced fibrous hierarchical structures in dense (30% w/w) calcium caseinate (Ca-caseinate) dispersions. Using Tgase was essential for the anisotropic structure formation. The fibrous materials showed anisotropy on both micro- and macroscale as determined with scanning electron microscopy (SEM) and mechanical analyses, respectively. SEM revealed protein fibers with a diameter of approximately 100-200 nm; visually, we observed fibers of about 1 mm. Both shear and Tgase affected the reinforcement of the fibers to a large extent, whereas the mechanical properties in the direction perpendicular to the shear flow remained constant. Shearing Ca-caseinate without Tgase yielded a slightly anisotropic layered structure. Both cross-linking in the absence of shear and cross-linking during mixing resulted in gels without alignment. The formation of shear- and enzyme-induced anisotropic structures was explained by aligning of protein aggregates due to shear and concurrent solidification of the aligned protein aggregates.

Chemical synthesis and structure elucidation of bovine kappa-casein (1-44).

The caseins (alphas1, alphas2, beta, and kappa) are phosphoproteins present in bovine milk that have been studied for over a century and whose structures remain obscure. Here we describe the chemical synthesis and structure elucidation of the N-terminal segment (1-44) of bovine kappa-casein, the protein which maintains the micellar structure of the caseins. kappa-Casein (1-44) was synthesised by highly optimised Boc solid-phase peptide chemistry and characterised by mass spectrometry. Structure elucidation was carried out by circular dichroism and nuclear magnetic resonance spectroscopy. CD analysis demonstrated that the segment was ill defined in aqueous medium but in 30% trifluoroethanol it exhibited considerable helical structure. Further, NMR analysis showed the presence of a helical segment containing 26 residues which extends from Pro8 to Arg34. This is the first report which demonstrates extensive secondary structure within the casein class of proteins.

Efectos de la sal sobre la solubilidad y las propiedades emulsionantes de la caseína y sus hidrolizados trípticos / Effect of salt on the solubility and emulsifying properties of casein and its tryptic hydrolysates

En este trabajo se presentan los resultados de una investigación sobre los efectos de la adición de NaCl (0,02 mol/L) sobre algunas propiedades funcionales de la caseína (CA) y de sus hidrolizados trípticos (TH). Se determinaron la solubilidad, la capacidad emulsionante (CE), el índice de actividad emulsionante (IAE) y la estabilidad de la emulsión (EE) con dos valores de pH (4,0 y 5,0). Los resultados demostraron que el procedimiento era benefi cioso para la solubilidad de CA y TH, siendo más intensa con pH 5,0 y 4,0, respectivamente. También se observó un efecto positivo de la adición de NaCl en CA y TH, con ambos valores de pH, consiguiéndose los mejores resultados con pH 5,0. La EE se vio ligeramente afectada por la presencia de sal, y sólo aumentó en algunas muestras de CA y TH. Por el contrario, los valores del IAE de la caseína se redujeron al añadir NaCl con pH 4,0 y 5,0, mientras que las de TH se vieron afectadas positivamente por este tratamiento con ambos valores de pH

This work reports an investigation about the effect of NaCl addition (0.02 mol/L) on some functional properties of casein (CA) and its tryptic hydrolysates (TH). The solubility, the emulsifying capacity (EC), the emulsifying activity index (EAI) and the emulsion stability (ES) were determined at two pH values (4.0 and 5.0). The results showed that this procedure was benefi cial for the solubility of CA and TH, being more intense at pH 5.0 and 4.0, respectively. Also, a positive effect of NaCl addition was observed for CA and TH, at both pH values, and the best results for both samples were achieved at pH 5.0. The ES was slightly affected by the presence of salt and only for some samples of CA and TH it was increased. Contrarily, the EAI values of casein were reduced with the addition of NaCl at pH 4.0 and 5.0, while those of TH were positively affected by this treatment at both pH values

Modelos estructurales de la micela de caseína / Structural models for the casein micelle

La micela de caseína constituye un sistema coloidal muy estable en la leche. Este hecho tiene importantesimplicaciones prácticas relacionadas con la formación de los geles de caseína, así como con la estabilidad delos productos lácteos durante su tratamiento térmico, concentración y almacenamiento. Por este motivo, lamicroestructura de la micela de caseína ha sido intensamente estudiada durante las últimas cinco décadas. Apesar de ello, existen todavía muchas lagunas sobre su estructura y estabilidad, como se refleja en los resultadosderivados de recientes investigaciones basadas en el empleo de novedosas técnicas analíticas

Casein micelle constitutes a very stable milk colloidal system. This has significant practical implicationsespecially regarding to casein gel formation and stability of dairy products during heating, concentration andstorage. For that reason, the microstructure of the casein micelle has been intensively investigated during thelast five decades. However, specific questions about micelle structure and stability are still arising as new instrumentalmethodologies become available

Prevention of intestinal infection by glycomacropeptide.

The preventive effects of glycomacropeptide (GMP) against intestinal infection were investigated, and conjugates of GMP with xylooligosaccharide (XOS) and carboxymethyldextran (CMD) were prepared by the Maillard reaction to enhance the effect of GMP. The binding ability of GMP to intestinal pathogenic bacteria was evaluated by a binding assay with biotinylated bacteria. GMP showed the ability to bind to Salmonella enteritidis and enterohemorrhagic Escherichia coli O157:H7 (EHEC O157). This binding ability was decreased by a sialidase treatment and completely eliminated by periodate oxidation. These results indicate that such carbohydrate moieties as sialic acid in GMP are involved in binding to S. enteritidis and EHEC O157. The preventive effect of GMP on the adhesion of pathogenic bacteria to Caco-2 cells was also investigated. GMP showed an inhibitory effect on the adhesion of EHEC O157 in a dose-dependent manner, although it was not a potent inhibitor of the adhesion of Salmonella infection. However, in the case of Salmonella infection, GMP-XOS and GMP-CMD significantly suppressed IL-8 production which was the index of infection. Our results indicate GMP to be a promising agent for preventing intestinal infection.

Application of triazine "superactive esters" in the repetitive synthesis of oligopeptides. Part 1. Synthesis of [54-59] fragment of human beta-casein.

A new synthetic protocol which considerably improves the classic REMA procedure is proposed herein. The offered modification is based on the application of triazine "superactive esters" as the superior substitutes for mixed anhydrides, which have been used as acylating reagent in the classic procedure. The improved repetitive procedure in solution was applied to the preparation of [54-59] fragment of human beta-casein. The structure and high purity of the intermediates, as well as of the final products, was confirmed by FAB-MS, 1H-NMR and HPLC.

Efficacy of human beta-casein fragment (54-59) and its synthetic analogue compound 89/215 against Leishmania donovani in hamsters.

The characteristic feature of visceral leishmaniasis (VL) is the profound impairment of immune system of the infected host, which contributes significantly to the partial success of antileishmanial chemotherapy. Since in VL, cure is the combinatorial effect of drug and immune status of the host, the rationale approach towards antileishmanial chemotherapy would be to potentiate the immune functioning of the host to extract desired results. Towards this direction several rationally designed analogues of human beta-casein fragment (54-59) were evaluated for their ability to stimulate the non-specific resistance in hamsters against Leishmania donovani infection. By virtue of being derived from the food protein casein derivatives may be devoid of unwanted side effects associated with the substances of microbial origin, e.g. muramyl dipeptide (MDP). Out of this one peptide Val-Glu-Gly-Ile-Pro-Tyr (compound 89/215) had been reported to have such activity. In this communication, the prophylactic and therapeutic efficacy of the peptide along with its natural sequence has been evaluated in detail against experimental VL in hamsters. Their use as an adjunct to chemotherapy was also explored. Human beta-casein fragment, compound 89/215 and MDP were tested in vivo at various dose levels wherein compound 89/215 showed superiority over MDP at 3 mg/kg x 2 given intraperitoneally (i.p.). Compound 89/215 sensitized peritoneal macrophages acquired considerable resistance and only 24% of the cells were found infected in comparison to control peritoneal macrophages where 76.4% of the cells were found infected. Similarly, the efficacy of sodium antimony gluconate (SAG) in hamsters pretreated with compound 89/215 enhanced significantly (P < 0.001). This peptide also exhibited considerably good therapeutic efficacy when evaluated either alone or in combination with SAG in established infection of L. donovani.

Effects of bovine beta-casein (1-28) and its chemically synthesized partial fragments on proliferative responses and immunoglobulin production in mouse spleen cell cultures.

Effects of bovine beta-casein (1-28) having a phosphoserine-rich region (Glu14-SerP-Leu-SerP-SerP-SerP-Glu-Glu21) and its chemically synthesized partial fragments on proliferation of lymphocytes and immunoglobulin production were investigated in mouse spleen cell cultures. The parent fragment 1-28 and all fragments containing SerP-Leu-SerP and/or SerP-SerP-SerP had a significant mitogenic effect, stimulated proliferation of lymphocytes induced by lipopolysaccharide, phytohemagglutinin, or concanavalin A, and increased immunoglobulin (IgG + IgM + IgA) or IgA levels in the cell cultures. In contrast, dephosphorylated beta-casein (14-21) and SerP-SerP amide had hardly any immunoregulatory activity. On the other hand, SerP-Leu-SerP amide reacted little with antibodies specific to bovine beta-casein (1-28), but beta-casein (14-21), and SerP-SerP-SerP amide obviously reacted with the antibody. These results confirm that the immunoregulatory activity of casein phosphopeptides is attributable to SerP-X-SerP, which may well be available as a non-allergic food ingredient having an adjuvant activity for mucosal IgA responses.

Synthesis and immunomodulatory activity of novel analogues of human beta-casein fragment [54-59].

Three novel analogues of human beta-casein fragment [54-59] have been synthesized and tested for their immunomodulatory activity. Interestingly, human beta-casein fragment [54-59] has been found to be increased nitric oxide release from neutrophils. The obtained analogues have shown less immunomodulatory activity than native hexapeptide.

Conformational analysis of the hydrophobic peptide alphas1-casein(136-196).

Hydrophobic interactions are important in the self-association of milk proteins, including alphas1-casein. The extent to which casein interaction sites are influenced by local secondary structure is not widely known. Both primary amino acid sequence and local secondary structure are shown to affect the self-association of the hydrophobic peptide alphas1-casein(136-196). The peptide is aggregated at low concentrations (7 microM and above), as determined by 1H nuclear magnetic resonance (NMR) measurements at pH 6.0 in phosphate buffer. Increase in temperature is shown to induce side chain mobility (melting) as indicated by both 1H NMR and near-UV circular dichroism (CD) measurements. As determined by far-UV CD, there is also a loss in the global amount of extended structure with increasing temperature, while beta-turn structures and some aromatic dichroism are conserved at temperatures as high as 70 degrees C. Similar retention of structure occurs at pH 2 and in 6 M guanidine HCl. The observed stability of beta-turns and some side chains in alphas1-casein(136-196) supports previous assumptions that hydrophobic, proline-based turns are important interaction sites in the self-association of alphas1-casein, and possibly in the formation of the calcium transport complexes, the casein micelles. It may be speculated that these areas of the peptide represent a 'molten globule-like', heat stable, core structure for alphas1-casein.