Enzymatic Hydrolysis Of Casein Research Articles

Screening of Enzyme Producers with Keratinase Activity among Marine Actinobacteria

About 2 million tons of feathers are produced annually around the world as a by-product of poultry farming. Due to the lack of funds and the complexity of processing, they have become one of the main environmental pollutants. The biodegradation of feathers by keratinolytic microorganisms has proven to be an effective, environmentally friendly and cost-effective method of bioconverting feather waste into a nutritious, balanced and easily digestible product that contains free amino acids, peptides and ammonium ions. Aim. To investigate the ability of marine actinobacteria to synthesize enzymes with keratinolytic activity and to study some of the physicochemical properties of the most active enzyme preparation. The object of the study was 10 strains of actinobacteria isolated from bottom sediments in the area of the Pradneprovsky trench of the Black Sea shelf. Methods. Caseinolytic (general proteolytic) activity was determined by the Anson method modified by Petrova, based on the quantitative determination of tyrosine, which is formed during the enzymatic hydrolysis of casein. Keratinase activity was determined by UV absorption at 280 nm of the hydrolysis products of keratin-containing raw materials. The cultivation of actinobacteria was carried out in a liquid nutrient medium with the addition of defatted chicken feathers as the main source of carbon and nitrogen. Results. The ability to hydrolyze keratin was found in five cultures. Moreover, all the strains studied were practically unable to break down casein. The Acty 9 strain (12 U/ml) showed the highest keratinase activity. Additional introduction of NaCl to the nutrient medium did not have a positive effect on the enzymes synthesis. The study of the physicochemical properties of the enzyme preparation Acty 9 showed that the pH and thermooptimum were 9.0 and 60°C, respectively. It retained 100% of the initial activity in the range of pH 7.0–10.0 after 3 h and 95% activity at pH 8.0 after 24 h of incubation. The studied enzyme preparation was thermostable, since it remained active for 3 h at 50°C and 1 h at 60°C. Conclusions. The extracellular keratinase synthesized by actinobacterium Acty 9 is promising for further research, since the enzyme is pH and thermostable and is not inferior in its physicochemical properties to those previously described in the literature.

Encapsulation of chlorine-containing carbamates in polypeptide nanoparticles prepared by enzymatic hydrolysis of casein

Encapsulation of biologically active β-oxalylamino substituted O-ethylcarbamate in micelles of non-hydrolyzed and hydrolyzed casein is performed. The AFM studies show that the controlled hydrolysis of casein by trypsine allows preparation of monodisperse nanoparticles with a mean size of 22 nm and with a dispersion of 5 nm. The high-precision coulometric method for the chlorine detection shows that, as compared with initial casein micelles, the formed nanoparticles involve appreciable amounts of chlorine-containing alkylcarbamate. Micellar solubilization allows the preparation of the colloidal form of alkylcarbamate form, which is suitable for its practical application.

A tryptic hydrolysate from bovine milk αs1-casein enhances pentobarbital-induced sleep in mice via the GABAA receptor

Studies have shown that enzymatic hydrolysis of casein, the primary protein component of cow’s milk, produces peptides with various biological activities, and some of these peptides may have sleep-promoting effects. In the present study, we evaluated the sedative and sleep-promoting effects of bovine αS1-casein tryptic hydrolysate (CH), containing a decapeptide αS1-casein known as alpha-casozepine. CH was orally administered to ICR mice at various concentrations (75, 150, 300, or 500mg/kg). An hour after administration, assessment of its sedative (open-field and rota-rod tests) and sleep-potentiating effects (pentobarbital-induced sleeping test and EEG monitoring) were conducted. Although a trend can be observed, CH treatment did not significantly alter the spontaneous locomotor activity and motor function of mice in the open-field and rota-rod tests. On the other hand, CH (150mg/kg, respectively) enhanced the sleep induced by pentobarbital sodium in mice. It also promoted slow-wave (delta) EEG activity in rats; a pattern indicative of sleep or relaxation. These behavioral results indicate that CH has sleep-promoting effects, but no or has minimal sedative effects. To elucidate the probable mechanism behind the effects of CH, we examined its action on intracellular chloride ion influx in cultured human neuroblastoma cells. CH dose-dependently increased chloride ion influx, which was blocked by co-administration of bicuculline, a competitive GABAA receptor antagonist. Taken together, the results of the present study suggest that CH has sleep-promoting properties which are probably mediated through the GABAA receptor–chloride ion channel complex.

A hydrophobic peptide fraction that enhances the water dispersibility of curcumin

The present study describes the complexation between curcumin (Cur) and a peptide mixture (Pep). Pep was prepared by enzymatic hydrolysis of casein and used as an excipient for poorly water-soluble Cur. An aqueous solution of Pep and an acetone solution of Cur were mixed and lyophilized to obtain a white-yellow powder of the peptide and Cur complex (Cur-Pep). The water dispersibility of Cur was enhanced by the complexation with Pep. Pep was fractionated using ammonium sulfate precipitation and ultrafiltration to identify which peptides preferentially interact with Cur. Relatively hydrophobic peptides with high molecular weights (>5 kDa) were more effective in enhancing the water dispersibility of Cur than other fractions. Cur-Pep dispersed under acidic and neutral conditions, at which amphoteric Pep is positively or negatively charged. Cur-Pep exists as a hydrocolloid with particle size 160–330 nm in aqueous media.

Enhancing the water dispersibility of paclitaxel by complexation with hydrophobic peptides

The complex between paclitaxel (Ptx) and a peptide mixture (Pep) was prepared to enhance of the water-dispersibility of Ptx. Pep was prepared by enzymatic hydrolysis of casein, followed by fractionation using ammonium sulfate precipitation and ultrafiltration. The Ptx and Pep complex (Ptx–Pep) was prepared by mixing an ethanol solution of Ptx and an aqueous solution of Pep followed by lyophilization. The water dispersibility test of Ptx–Pep prepared using different fractions of Pep demonstrated that a fraction (Pep-A), containing relatively hydrophobic peptides with high molecular weights, was effective in enhancing the water dispersibility of Ptx. The sequences of the major peptides in Pep-A were identified by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry with “LIFT” technique. The water dispersibility of the complex between Ptx and Pep-A (Ptx–Pep-A) was independent of pH, even though it is positively or negatively charged under strongly acidic and neutral conditions. As the particle size of Ptx–Pep-A in aqueous media was 147–215nm, Ptx–Pep-A was present as a hydrocolloidal material in aqueous media.

Facile Synthesis of Zirconia‐Modified Magnetic Nanoparticles for Purification of Phosphopeptides

AbstractBecause of the low stoichiometry from enzymatic hydrolysis of casein, purification of casein phosphopeptides (CPPs) is necessary prior to application in infant formula or nutritional foods. In this study, functional zirconia‐modified magnetic nanoparticles (Fe3O4@ZrO2) were prepared and applied for purification of CPPs. The physical shape, crystalline structures and chemical groups were characterized by transmission electron microscopy, X‐ray diffraction and Fourier transform infrared. Fe3O4@ZrO2 nanoparticles had nano‐dimension and good dispersion. The purification studies showed that the high selectivity of Fe3O4@ZrO2 was obtained at pH 4.5. Then the structures of reused Fe3O4@ZrO2 were characterized to study the stability of material. The purity of CPPs isolated by Fe3O4@ZrO2 was analyzed to study the affinity force of material. All results confirmed that Fe3O4@ZrO2 nanoparticles had good stability and high affinity force for CPPs after 15 repeated cycles, and the maximum loading amount of material was 105.53 mg/g with lower N/P (5.94) of CPPs.Practical ApplicationsCasein phosphopeptides can enhance the absorption and utilization of calcium, but it needs to be purified before used as calcium nutritional supplements. Most of existing purification methods are two‐step methods, which have limitations in food application. Compared with traditional methods, the separation method using Fe3O4@ZrO2 nanoparticles can one‐step purify phosphopeptides from real samples, and shows good performance. The purification process using Fe3O4@ZrO2 nanoparticles has the simpler procedures, higher enrichment efficiency and shorter production cycle. It means that the purification process is easy to expand the scale of production for improving the yield and quality of phosphopeptides, which can satisfy the demand of market. The results demonstrate that Fe3O4@ZrO2, which has good stability and high affinity force for metal oxide affinity peptides, is one new type of extraction materials and possesses wide application prospects in food industry.

Casein peptides with inhibitory activity on lipid oxidation in beef homogenates and mechanically deboned poultry meat

Bioactive peptides obtained by enzymatic hydrolysis of casein may have antioxidant activity. In this work, casein peptides were obtained using the proteolytic enzymes Alcalase and Flavourzyme. Casein was hydrolyzed for 4 h at 50 °C and pH 8, and the resulting peptides were analyzed. The enzymatic hydrolysis with Flavourzyme resulted in higher concentration of soluble protein and free amino acids, and produced peptides with lower molecular mass than those obtained with Alcalase, as observed by gel permeation chromatography and polyacrylamide gel electrophoresis. Casein peptides obtained with Flavourzyme also exhibited greater antioxidant capacity using the ABTS radical method. Casein peptides (20 mg ml−1) effectively inhibited lipid peroxidation in ground beef homogenates and mechanically deboned poultry meat. Casein peptides may be useful in meat processing as another naturally occurring antioxidant, helping to prevent off-flavor formation in meat products and increasing shelf life.

In vitro evaluation of feed-grade enzyme activity at pH levels simulating various parts of the avian digestive tract

The activities of β-glucanase, xylanase, amylase, α-galactosidase and protease were measured at their published optimum pH levels and at pH levels of 3.0, 6.0, 6.5, 7.0 and 7.5 to simulate pH levels of the gizzard, the diet, the crop, and the proximal and distal parts of small intestine, respectively. The activity of β-glucanase was determined by measuring reducing sugars after incubation of β-glucan. Xylanase activity was assayed by measuring xylose after hydrolysis of xylan. The activity of amylase was measured through hydrolysis of soluble starch. The assay of α-galactosidase was based on a hydrolysis of p-nitrophenyl-α- d-galactoside followed by measurement of liberated p-nitrophenol. The activity of protease was assayed by measuring tyrosine after enzymatic hydrolysis of casein. β-Glucanase had high activity at pH levels of 3.0–7.0. Xylanase had no enzyme activity at pH 3.0, but had high activity at pH levels of 6.0–7.0. Amylase had high activity at pH levels of 6.0 and 6.5 but had no or very low activity at pH 3.0, 7.0 and 7.5. α-Galactosidase had high activity at pH 6, but not at other pH levels tested. Protease had either no or very low activity at all pH levels except at pH 3.0. These results suggest that the pH levels commonly found in the avian digestive tract may be a limiting factor for maximum activity of the exogenous enzymes, such as amylase, α-galactosidase and protease.

Synergistic action of an X-prolyl dipeptidyl aminopeptidase and a non-specific aminopeptidase in protein hydrolysis.

Non-specific monoaminopeptidase (AP; E.C. 3.4.11) and X-prolyl dipeptidyl aminopeptidase (X-PDAP; E.C. 3.4.14.5), both from Aspergillus oryzae, demonstrate strong synergism in hydrolyzing proline-containing peptides. Incubation of AP alone with the peptide Ala-Pro-Gly-Asp-Arg-Ile-Tyr-Val-His-Pro-Phe does not generate free amino acids. However, when AP and X-PDAP are added in combination, complete and immediate hydrolysis of all peptide bonds, other than X-Pro bonds, is observed. In the enzymatic hydrolysis of casein, soy, and gluten, degree of hydrolysis (DH) values of 54, 54, and 47% were achieved, respectively, when subtilisin (E.C. 3.4.21.62) was supplemented with AP. Addition of a third enzyme, X-PDAP, resulted in significantly higher DH values of 69, 72, and 64%, respectively, establishing the utility of this synergism in protein hydrolysis.

Casein Hydrolysate as a Rapid and/or Enteric Dissolving Additive for Oral Drugs

ABSTRACTTwo types of casein hydrolysates, casein A (mean peptide length 3.3) and casein B (mean peptide length 17.4) were prepared by the enzymatic hydrolysis of casein, and their effects on in vitro dissolution rates and oral bioavailability of drugs were evaluated. The in vitro dissolution behavior of the kneaded mixture of three drugs (diclofenac acid, diazepam, and prednisolone) with caseins A and B were significantly improved compared to the drugs alone, even at 1:1 weight ratio of drug and casein hydrolysate, even though casein A and casein B did not interact with drug molecules in the kneaded mixture. Only diclofenac, an acidic drug, showed an increased dissolution rate with added casein hydrolysates, and a more rapid dissolution with casein A than with casein B was observed. When the dissolution of prednisolone from kneaded mixture was compared at pH 1.2 and 6.8, the dissolution rate of prednisolone from the casein A kneaded mixture was considerably higher than that of prednisolone powder at both pHs, and the rate from the casein B kneaded mixture was higher only at pH 6.8. The plasma concentration-time profile showed that prednisolone was completely and rapidly absorbed from the casein A kneaded mixture as well as the prednisolone solution. In addition, prednisolone in the kneaded mixture with casein B was more difficult to absorb up to 1 hr after administration in comparison to prednisolone powder. The slow and lowered absorption of prednisolone by casein B might be explained by conversion of casein B to a shorter soluble peptide in the gastrointestinal tract and by the slow dissolution of prednisolone at acidic conditions. The toxicological tests revealed that casein hydrolysate is a safe drug carrier. Consequently, casein hydrolysates might be safely used to control the dissolution rate and bioavailability of a variety of drugs, depending on the peptide length of the casein fragments.

Enzymatic Hydrolysis of Casein: Effect of Degree of Hydrolysis on Antigenicity and Physical Properties

ABSTRACTCasein was enzymatically hydrolyzed for potential use in a hypoallergenic infant formula. Samples from the digest at different hydrolysis time (HT) intervals were tested for antigenicity, hydrophobicity, net charge (zeta potential), and emulsifying activity. Casein antigenicity loss could be approximated by three‐phased first‐order kinetics. Extensive antigenicity loss occurred during the first 10% of HT and relatively small changes occurred during the remaining 90% of HT. Hydrophobicity and emulsifying activity decreased, while the magnitude of the net charge increased (P <0.01) with increasing degree of hydrolysis. Strong correlations (r2≥ 0.89) were found among hydrophobicity, emulsifying activity and zeta potential.