Solubilization and identification of hen eggshell membrane proteins during different times of chicken embryo development using the proteomic approach.

A fertilized chicken egg is a unit of life. During hatching, transport of nutrients, including calcium, have been reported from the egg components to the developing embryo. Calcium is mobilized from the eggshell with the involvement of Ca(2+)-binding proteins. In addition, other unknown proteins may also play some important roles during embryo developing process. Therefore identification and prediction of biological functions of eggshell membrane (ESM) proteins during chick embryo development was conducted by proteome analysis. Comparison of different lysis solutions indicated that the highest ability to extract ESM proteins could be obtained with 1 % sodium dodecyl sulfate in 5 mM Tris-HCl buffer pH 8.8 containing 0.1 % 2-mercaptoethanol. In this study fertilized Cornish chicken eggs were incubated at 37 °C in humidified incubators for up to 21 days. At selected times (days 1, 9, 15 and 21), samples were taken and the ESMs were carefully separated by hand, washed with distilled water, and air-dried at room temperature. The ESM proteins were then solubilized and analyzed by proteome analysis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis combined with high performance liquid chromatography and mass spectrometry revealed 62 proteins in the ESM; only keratin is known ESM protein, 8 of which are egg white proteins and related while 53 others have not previously been reported. Some differences in the types of proteins and their molecular functions were noted in ESM at different incubation times. One protein which was present only at days 15 and 21 of egg incubation was identified as a calcium binding protein i.e. EGF like repeats and discoidin I like domain 3 (EDIL3 homologous protein).

Proteolytic activity from chicken intestine and pancreas: extraction, partial characterization and application for hyaluronic acid separation from chicken comb.

BACKGROUND: The potential utilization of viscera and combs, by-products from the chicken-processing industry, was investigated. Chicken combs have been reported to consist of hyaluronic acid (HA) bound to protein. Thus proteases extracted from broiler viscera were applied to digest combs to separate HA, a high-value ingredient widely used in pharmaceutical and medical products. RESULTS: The highest activity of crude protease extracted from chicken intestine and pancreas was 0.35 U mg(-1) enzyme at 60 °C and pH 7.5. pH stability of the enzyme was attained from pH 6 to 8, while its thermal stability declined from 30 to 50 °C, with complete activity loss occurring after 30 min at temperatures above 60 °C. Therefore the optimal conditions for broiler comb digestion by crude protease in this study were chosen as pH 7.5 and 50 °C. The results showed that the yield of crude enzyme-extracted HA was lower (P < 0.05) than that obtained by commercial papain digestion. Similar identity of extracted HA and HA standard was verified by cellulose acetate electrophoresis and Fourier transform infrared spectroscopy. CONCLUSION: The crude enzyme extract from chicken intestine and pancreas had high proteolytic activity and could be used successfully to separate HA from broiler combs. The method provides an appropriate means to add value to poultry-processing waste.

Temperature effects on type I pepsin-solubilised collagen extraction from silver-line grunt skin and its in vitro fibril self-assembly.

BACKGROUND: Fish skin is a potential source of collagen. Increasing the extraction temperature increases the yield of collagen. However, it may also result in degradation of the peptide chains, thus damaging the 3D structure of collagen that is vital for its application as a biomaterial. This work investigated the effects of extraction temperature on the yield and characteristics, including fibril self-assembly, of type I pepsin-solubilised fish skin collagen. RESULTS: Pepsin-solubilised collagens were extracted from fresh skin of silver-line grunt at 4, 10, 20 and 28 °C for 6 h. Extraction at 10 °C gave the highest yield of collagens (439.32 ± 96.43 mg g(-1) fresh skin, dry basis), which were identified as type I and comprised ß, α1 and α2 subunits. Extraction at higher temperatures (20 and 28 °C) resulted in the formation of low-molecular-weight peptide fragments, thus reducing the yield of the resultant type I collagen. The denaturation temperatures of collagens extracted at 4 and 10 °C, as determined by thermal analysis using differential scanning calorimetry, were 39.5 and 37.5 °C respectively. In vitro fibril self-assembly of 1 mg mL(-1) collagen solution (pH 6) incubated at 25 °C was only observed with collagens extracted at 4 and 10 °C. The 10 °C collagen not only showed a higher rate of self-assembly, but its matrix also had a larger fibril diameter of 0.50 ± 0.07 µm (compared with 0.41 ± 0.07 µm for the 4 °C collagen) after 4 h of incubation. CONCLUSION: The results indicated strong effects of extraction temperature on the yield and characteristics of the collagen obtained. Extraction of pepsin-solubilised collagen from silver-line grunt skin at 4-10 °C gave a high yield of type I collagen with molecular integrity suitable for tissue-engineering applications.

Chicken eggshell matrix proteins enhance calcium transport in the human intestinal epithelial cells, Caco-2.

Chicken eggshell powder has been proposed as an attractive source of calcium for human health to increase bone mineral density in an elderly population with osteoporosis. However, factors affecting calcium transport of eggshell calcium have not yet been evaluated. Chicken eggshell contains about 1.0% (w/w) matrix proteins in addition to a major form of calcium carbonate (95%, w/w). In this study, we found that soluble eggshell matrix proteins remarkably enhance calcium transport using in vitro Caco-2 cell monolayers grown on a permeable support. The total calcium transport across Caco-2 monolayers showed an increase of 64% in the presence of 100 microg/well soluble eggshell matrix proteins. The active enhancer with a molecular mass of 21 kDa was isolated by reversed phase high-performance liquid chromatography and did not correspond to any previously identified protein. The N-terminal sequence was determined to be Met-Ala-Val-Pro-Gln-Thr-Met-Val-Gln. The possible mechanisms of eggshell matrix protein-mediated increase in calcium transport and the potential significance of eggshell calcium as a nutraceutical are discussed.

Fermented pork sausage fortified with commercial or hen eggshell calcium lactate.

The effects of adding commercial-grade and eggshell calcium lactate on the microbiological and physicochemical properties of Nhams (Thai-style fermented pork sausage) were studied. The Nham calcium levels were 150, 300 and 450 mg/100 g. Compared to controls (no added calcium), calcium fortification did not affect the number of lactic acid bacteria or the colour value. The shear force of Nhams fortified with eggshell calcium lactate decreased (P<0.05) from 32.2 N in the controls to 19.5-22.8 N in Nhams fortified with eggshell calcium lactate. However, Nhams fortified with commercial calcium lactate had the same shear force as the controls. Sensory scores of sour taste, flavour and overall acceptance were not different between the control and calcium-fortified Nhams at a calcium level of 150 mg/100 g.