Expression and purification of goat lactoferrin from Pichia pastoris expression system

ABSTRACT:  The recombinant goat lactoferrin (rGLF) was expressed in the methylotropic yeast Pichia pastoris using pGAPZαC vector, GAP as promoter, and Zeocin as the selective marker. After transformation of the GLF-pGAPZαC into Pichia pastoris X-33 expression host, the GLF-pGAPZαC vector was integrated into the GAP promoter locus of Pichia pastoris X-33 chromosome. The rGLF was expressed and secreted into the broth using α-factor preprosequence. SDS-PAGE and PAS staining analysis indicated that the rGLF could be purified to electrophoretic homogeneity by heparin-Sepharose 6 Fast Flow affinity chromatography and glycosylated by the expression host. The yield of purified rGLF was approximately 2.0 mg/L of culture broth. The N-terminal sequence was identical to the native goat lactoferrin (nGLF). The iron-binding behavior, papain-inhibiting property, and thermal stability of the purified rGLF were comparable to nGLF. This is the 1st report of intact goat lactoferrin expression using the P. pastoris system.     

Effect of Combining Proteolysis and Lactic Acid Bacterial Fermentation on the Characteristics of Minced Mackerel

ABSTRACT: To improve the quality of fish muscle, mackerel muscle protein was hydrolyzed by proteases from Aspergillus oryzae , and then fermented by lactic acid bacteria (LAB). The highest protease activities were obtained from A. oryzae after 72 h incubation at 25°C. Acidic protease activity was much higher than neutral and alkaline proteases. SDS-PAGE indicated the degradation of muscle proteins after 1 or 2 h hydrolysis by A. oryzae proteases at 50°C. During 48 h fermentation by Pediococcus pentosaceus L and S at 37°C, rapid growth of LAB, decline in pH, and suppression in the growth of microflora, Enterobacteriaceae, Staphylococcus , and Pseudomonas , occurred while increases in whiteness, nonprotein nitrogen, sensory quality, and free amino acids were observed. These data suggested that the acceptability of LAB -fermented mackerel hydrolysates could be substantially improved.     

Effect of Monascus Fermentation on the Characteristics of Mackerel Mince

ABSTRACT: To improve the characteristics of fish muscle, Monascus purpureus or Monascus species was inoculated in 2.1% rice powder broth containing 30% minced mackerel tissue to produce a fermented fish product. Enterbacteriaceae, Staphylococcus , and Pseudomonas were not present during the 7 d of fermentation at 25 °C. The activities of both amylase and acidic proteases were detected after 1 and 2 d of fermentation, respectively, and further increased during fermentation. The volatile base nitrogen of all Monascus fermented samples were lower than the limit of food regulation in Taiwan (< 25 mg/100 g) even after 7 d of fermentation. Hydrolysis of the muscle protein and accumulation of free amino acid were observed. The fermented fish products had good flavor and color that were highly likeable to the sensory panel.     

Improvement of the Functionality of Minced Mackerel by Hydrolysis and Subsequent Lactic Acid Bacterial Fermentation

ABSTRACT: To improve functionality and add nutritional value to mackerel, its muscle protein was hydrolyzed by the proteases from Aspergillus oryzae , and further fermented by lactic acid bacteria (LAB). Compared with minced mackerel, the antioxidative ability against linoleic acid (from 10% to 42%), α,α-diphenyl-β-picrylhydrazyl scavenging capability (from 38% to 85%), reducing power (from 0.58 to 0.83), Fe²⁺ chelating capability (from 42% to 72%), and trolox equivalent antioxidant capacity (from 2.5 to 3.5 m M ) of the hydrolysate significantly increased ( P < 0.05) after 1 h of hydrolysis and 12 h of LAB fermentation. Both hydrolysate and LAB fermented samples could stimulate the proliferation of both human hybridoma HB4C5 and mouse macrophage J774.1 cells. These data suggest that the enhancement of cell viability and antioxidative ability could be substantially improved after hydrolysis and 12 h of LAB fermentation.     

Purified NADPH-Sulfite Reductase from Saccharomyces cerevisiae Effects on Quality of Ozonated Mackerel Surimi

The NADPH-sulfite reductase from Saccharomyces cerevisiae was purified to electrophoretic homogeneity by ammonium sulfate fractionation, DEAE Sephacel, Sephacryl S-300 and DEAE Sephadex A-50 chromatography. Optimal pH was 7.3 and temperature 25°C. It was inhibited by IAA, PCMPS, PMSF, NEM, PCMB, cyanide and most divalent metal ions. For the ozonated mackerel surimi ground with purified reductase, the reactive SH increased from 2.29∞10⁵ to 4.46∞10⁵mole/g and gel strength from 256.7 to 360.5g·cm. According to SDS-PAGE, the recovery of myosin heavy chain was observed on the ozonated mackerel surimi with addition of NADPH-sulfite reductase.     

Comparative Study on Antibacterial Effect of Pediocin ACCEL and Nisin Against Fluorescencestained Listeria monocytogenes BCRC 14845

ABSTRACT: Effects of pediocin ACCEL (Class IIa, pediocin-like bacteriocins) and nisin on carboxyfluorescein diacetate (cFDA) -stained Listeria monocytogenes BCRC 14845 was carried out. Decrease of fluorescence intensity in stained cells and increase in efflux were observed with the increase in bacteriocin concentrations up to 12.5 μg/mL. No further decreases in fluorescence intensity and cell numbers were obtained when the concentration of bacteriocins was over 12.5μg/mL. The leakage of efflux was further confirmed by the release of intracel-lular ultraviolet-absorbing substances. Comparing the leaking time of fluorescence, the intensity of cFDA in nisin-treated cells within 1 min incubation was almost comparable to that in pediocin-treated samples after 40 min incubation. Considering the leakage of cFDA, UV-absorbing substances, changes in cell numbers during incubation of cFDA-stained cells, and restoration ability, nisin seemed to reveal mainly bacteriostatic effect and partially bactericidal effect on L. monocytogenes and to cause easier-to-repair membrane damage. However, pediocin ACCEL appeared to have both bacteriostatic and bactericidal effects on L. monocytogenes and to cause more difficult-to-repair membrane damage.     

Hydrolysis of Chlorella by Cellulomonas sp. YJ5 cellulases and its biofunctional properties

Both 10% and 20% (w/w) Chlorella suspensions were hydrolyzed by 150 to 350 U/mL of cellulases from a 3-d cultivation of Cellulomonas sp. YJ5. Higher chlorophyll, reducing sugars and soluble proteins, and lower residual insoluble solid were observed on both samples after 30-min hydrolysis by various concentrations of cellulases at 50 °C. Decrease in insoluble solid, increases in soluble proteins, peptides and chlorophyll contents, and microscopic observation indicated obvious lysis of cell walls occurred during 60- to 180-min hydrolysis. Significant increases in soluble proteins, peptides, Fe(2+) chelating ability, trolox equivalent antioxidation capacity (TEAC), and reducing power was obtained after 3-h hydrolysis by 150 U/mL of cellulase. These data suggested that cellulolysis technology has high application potential in Chlorella industry.