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Fish bones (FBs) are aquatic by-products that are sources of antioxidant-active peptides, calcium dietary supplements, and biomedical materials. Usually, fermentation of these by-products via microorganisms brings desirable changes, enhancing their value. This study investigates the value addition of FB when fermented with Monascus purpureus (MP) for different time intervals, such as 3 days (F3) and 6 days (F6). The results indicate that the soluble protein, peptide, amino acid and total phenol content, as well as the antioxidant capacity (DPPH, ABTS+ radical scavenging activity, and relative reducing power), of F3 and F6 were significantly increased after fermentation. Furthermore, the ROS contents of F3 and F6 were reduced to a greater extent than that of hydrogen peroxide (H2O2) in Clone-9 cells. The MMP integrity, as well as the SOD, CAT, and GPx activity, of F3 and F6 were also increased significantly compared to the H2O2 in Clone-9 cells. Notably, F3 and F6 displayed significant reductions in ROS content, as well as elevate, SOD activity and MMP integrity in Clone-9 cells, when compared with the native FB. These results indicate that the FBs fermented with MP for 3 days (F3), and 6 days (F6) have antioxidant capacity, with possible applications as natural food supplements.

To obtain the angiotension-I converting enzyme inhibitor (ACEI), a fusion ACEI polypeptide encoded with 8 DNA sequences of GPL, GPM, IKW, IVY, IRPVQ, IWHHT, IYPRY and IAPG, which were selected and designed and cloned into pGAPZαC and then transformed into Pichia pastoris SMD1168H. After 3 days induction, the fraction with highest ACEI activity was expressed and purified using a Ni Sepharose™ 6 Fast Flow. The IC50 of recombinant ACEI polypeptide was 88.2 μM. A 128-fold increase of ACEI activity (0.69 μM) was obtained after pepsin digestion, which was equivalent to 0.022 μM of captopril. Reverse phase HPLC indicated all the 8 peptides contained in ACEI-hydrolysate after pepsin digestion. Keywords: Angiotesin I-converting enzyme inhibitory peptides, Cloning, Expression

To obtain a Pichia pastoris mutant with an Escherichia coli phytase gene, which was synthesized according to P. pastoris codon preference, a mature phytase cDNA of E. coli being altered according to the codons usage preference of P. pastoris was artificially synthesized and cloned into an expression vector of pGAPZαC. The final extracellular phytase activity was 112.5 U/mL after 72 h of cultivation. The phytase, with a molecular mass of 46 kDa, was purified to electrophoretical homogeneity after Ni Sepharose 6 Fast Flow chromatography. The yield, purification fold, and specific activity were 63.97%, 26.17, and 1.57 kU/mg, respectively. It had an optimal pH and temperature of 4.0-6.0 and 50 °C, respectively, and was stable at pH 3.0-8.0 and 25-40 °C. The purified recombinant phytase was resistant to trypsin, highly inhibited by Cu(2+), Zn(2+), Hg(2+), Fe(2+), Fe(3+), phenylmethylsulfonyl fluoride, and N-tosyl-l-lysine chloromethyl ketone, but activated by Mg(2+), Ca(2+), Sr(2+), Ba(2+), glutathione, ethylenediaminetetraacetic acid, and N-ethylmaleimide. It revealed higher affinity to calcium phytate than to other phosphate conjugates.

A cellulase-producing bacterium was isolated from soil and identified as Cellulomonas sp. YJ5. Maximal cellulase activity was obtained after 48 h of incubation at 30 degrees C in a medium containing 1.0% carboxymethyl cellulose (CMC), 1.0% algae powder, 1.0% peptone, 0.24% (NH4)2SO4, 0.20% K2HPO4, and 0.03% MgSO(4).7H2O. The cellulase was purified after Sephacryl S-100 chromatography twice with a recovery of 27.9% and purification fold of 17.5. It was, with N-terminal amino acids of AGTKTPVAK, stable at pH 7.5-10.5 and 20-50 degrees C with optimal pH and temperature of 7.0 and 60 degrees C, respectively. Cu2+, Fe2+, Hg2+, Cr3+, and SDS highly inhibited, but cysteine and beta-mercaptoethanol activated, its activity. Substrate specificity indicated it to be an endo-beta-1,4-glucanase.

The DNA encoding Pseudomonas aeruginosa keratinase was ligated into pRPA expression vector and transformed into Bacillus subtilis DB104. Recombinant keratinase (rK), secreted by B. subtilis after 72 h of incubation, was purified to electrophoretical homogeneity by nickel affinity chromatography and found to have a molecular mass of 33 kDa. The rK had an optimal pH and temperature at 8.0 and 60 degrees C, respectively, and was stable at pH 6.0-9.0 and 10-50 degrees C. It was strongly inhibited by Cu(2+), Fe(2+), Hg(2+), Fe(3+), ethylene glycol tetraacetic acid, and ethylene diamine tetraacetic acid but activated by Ca(2+), Mg(2+), Zn(2+), dithiothreitol, glutathione, and beta-mercaptoethanol. According to substrate specificity, the rK was considered to be a metalloprotease.

The DNA encoding keratinase from Pseudomonas aeruginosa was ligated into pET-43b(+) expression vector and transformed into Escherichia coli AD494(DE3)pLysS. After isopropyl beta-d-thiogalactopyranoside induction, the soluble recombinant keratinase was expressed in E. coli. The keratinase with a molecular mass of 33 kDa was purified to electrophoretical homogeneity after nickel affinity chromatography. It had an optimal pH and temperature of 8.0 and 50 degrees C, respectively, and was stable at pH 6.0-9.0 and 10-60 degrees C. It was highly inhibited by Cd(2+), Cu(2+), Hg(2+), Ni(2+), Fe(3+), ethylene glycol tetraacetic acid, ethylenediaminetetraacetic acid, and p-chloromercuribenzoate, but activated by Ba(2+), Ca(2+), Mg(2+), Mn(2+), Zn(2+), dithiothreitol, glutathione, and beta-mercaptoethanol. According to substrate specificity results, the purified keratinase was considered to be a metalloprotease.

Amylase, with MW of 59 kDa, was purified from small abalone Haliotis sieboldii by ammonium sulfate fractionation, CM Sepharose Fast Flow and Sephacryl S-100 HR chromatographies. The optimal pH and temperature of purified amylase were 6.0 and 37°C, respectively. The purified enzyme was stable at pH 6.0–8.0 and low temperatures. It was activated by Ba2+, Mg2+, Ca2+, Co2+, Ni2+, Mn2+, K+, Ag+, Na+ and Li+, but completely or partially inhibited by Al3+, Cu2+, Cd2+, Hg2+ and Zn2+. EDTA could completely inhibit, while iodoacetamide, N-ethylmaleimide and urea partially inhibit the purified amylase. According to the digestion mode of various polysaccharides, the purified enzyme was considered to be an α-amylase.

To investigate the biopreservative effectiveness of pediocin ACCEL on refrigerated seafoods, fresh fish fillets were immersed in various concentrations of pediocin ACCEL and then stored at either 4° or 0°C. Samples treated with nisin were used as a positive control. The aerobic plate counts (APC) of samples with bacteriocins were 2.0 log cfu/g after 2 days storage, while those with pediocin ACCEL occurred after 1 day storage at 4°C. In refrigerated seafoods, pediocin ACCEL and nisin suppressed the growth of inoculated Listeria monocytogenes during 2- and 1-week storage at 4°C, respectively. Compared with nisin, the pediocin ACCEL was considered to be more effective on the suppression of L. monocytogenes growth in refrigerated seafoods during 2-week storage at 4°C.

This study was conducted to optimize the cultivation conditions for Bacillus subtilis to produce proteases, amylases and cellulase, and to further investigate the hydrolysis ability against mackerel and asparagus. The extracellular enzymes from B. subtilis after 2 and 4 days incubation in a modified medium, containing 1% skim milk, 1% soya meal, 0.25% starch, 0.25% K2HPO4, 0.5% NaCl and 0.05% MgCl2 were collected for the hydrolysis of asparagus and minced mackerel, respectively. Except for the α,α-diphenyl-β-picrylhydrazyl (DPPH) scavenging ability of the hydrolyzed asparagus, the trolox equivalent antioxidation capacity and DPPH scavenging ability of both samples increased significantly (P

The recombinant goat lactoferrin (rGLF) was expressed in the methylotropic yeast Pichia pastoris using pGAPZalphaC vector, GAP as promoter, and Zeocin as the selective marker. After transformation of the GLF-pGAPZalphaC into Pichia pastoris X-33 expression host, the GLF-pGAPZalphaC vector was integrated into the GAP promoter locus of Pichia pastoris X-33 chromosome. The rGLF was expressed and secreted into the broth using alpha-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.

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.

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 mM) 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.

Pseudomonas fluorescens MACK-4, isolated from mackerel surface, has an ice-nucle- ating activity (INA). In addition to bacterial cells, this strain could also produce an extracellular ice- nucleating substance (INS) with a maximal INA at pH 6.0. The extracellular INS (EINS) was stable at pH 6-9 during 1-h incubation with 3-5% of saccharides including maltose, trehalose and sucrose at 15 ∞ C. However, glycerol dramatically lowered the INA in both bacterial cells and the EINS. The addi- tion of either the EINS or bacterial cells significantly elevated the ice-nucleating temperatures of pure water, full-cream milk, and 10% starch solution, but not orange juice and mackerel mince. The EINS produced from this strain could serve as crystal nuclei and accelerate ice crystallization during freezing.

sity in stained cells and increase in efflux were observed with the increase in bacteriocin concentrations up to sity in stained cells and increase in efflux were observed with the increase in bacteriocin concentrations up to sity in stained cells and increase in efflux were observed with the increase in bacteriocin concentrations up to sity in stained cells and increase in efflux were observed with the increase in bacteriocin concentrations up to 12.5 12.5 12.5 12.5 � g/mL. No further decreases in fluorescence intensity and cell numbers were obtained when the concentrag/mL. No further decreases in fluorescence intensity and cell numbers were obtained when the concentrag/mL. No further decreases in fluorescence intensity and cell numbers were obtained when the concentrag/mL. No further decreases in fluorescence intensity and cell numbers were obtained when the concentrag/mL. No further decreases in fluorescence intensity and cell numbers were obtained when the concentration of bacteriocins was over 12.5 tion of bacteriocins was over 12.5 tion of bacteriocins was over 12.5 tion of bacteriocins was over 12.5 tion of bacteriocins was over 12.5�

The NADPH-sulfite reductase from Escherichia coli was purified to electrophoretic homogeneity by ammonium sulfate fractionation, and DEAE Sephacel and Sephacryl S-300 HR chromatography. The recovery and molecular weight were 31.7% and 119000, while the optimal pH and temperature for the activity were 7.7 and 25°C, respectively. It was strongly inhibited by PCMB, KCN, Hg2+, Fe2+, Fe3+, Ca2+, Co2+, and Cu2+, moderately by NEM, PMSF, IAA, Cd2+, Zn2+, Mn2+, and Ba2+. Addition of purified reductase significantly increased the reactive SH and gel strength of surimi prepared from frozen mackerel. The data suggest the high potential of microbial NADPH-sulfite reductase in surimi processing using frozen fish or denatured muscle proteins as raw materials. Keywords: Escherichia coli, sulfite reductase, mackerel, purification, characterization

To develop a new processing technique, mackerel mince was homogenized to obtain different media with protein concentration of 90, 45, 22.5 mg/mL, respectively. Lactic acid bacteria (LAB) were inoculated to the media. During 48 h fermentation at 37 °C, rapid growth of LAB, decline in pH, suppression of main microflora, and increases in whiteness, Hunter L and sensory quality were observed and the VBN of fermented samples was still below 25 mg/100g. After 24 and 48 h fermentation, the sensory evaluation and photographic records indicated the high acceptability of the fermented products. From this study, various LAB fermented fish products can be produced and this technique has very high commercial potential.

To improve the functionality and quality of seafood, mackerel minces were fermented with lactic acid bacteria (LAB) - Lactobacillus plantarum CCRC10069, Lactococcus lactis subsp. lactis CCRC 12315, Lactobacilius heiveticus CCRC 14092, or their combination at 37 °C. Rapid growth of LAB, decline in pH, suppress of main microflors, increases in whiteness, Hunter L, nonproteinous nitrogen, sensory quality, and free amino acids related to taste were observed. However, VBN of samples fermented with LAB were still d ≤ 25 mg/100 g after 36 h fermentation. SDS-PAGE indicated the obvious degradation of water- and salt-soluble muscle proteins after 12 h fermentation. Animal test demonstrated the LAB-fermented mince has the functionality on reduction of blood pressure, glucose, and total cholesterol of SHR. Les bacteries lactiques peuvent inhiber la microflore naturelle et les bacteries indesirables des produits carnes. Pour ameliorer les proprietes fonctionnelles et la qualite des produits de la mer, cette etude evalue l'effet d'une fermentation lactique sur les caracteristiques de chair de poisson hachee dont le degre d'hydrolyse, la multiplication de la microflore naturelle, la texture, la flaveur, la couleur et les proprietes antihypertensives.

During 72 h fermentation at 37°C, rapid increase in lactic acid bacteria count (LAB), dramatic decline in pH and suppression in the growth of contaminating Pseudomonas, Staphylococcus, and Enterobacteriaceae of mackerel minces with Pediococcus pentosaceus strains L and S were observed (p 0.05) during 3 wk storage at 4°C, however, the growth of Enterobacteriaceae and Staphylococcus were effectively suppressed. The residual nitrite in sample decreased during processing and fermentation, and was minimally detected after 72 h fermentation. Suppression of VBN and psychrophilic microflora, and lowering residual nitrite suggest the potential of using these 2 strains in the fermented fish products. Lors de la fermentation du maquereau hache en presence de Pediococcus pentosaceus souches L et S, le nombre de bacteries lactiques augmente pendant les 72 premieres heures, provoquant la suppression de la croissance des contaminants pathogenes, Pseudomonas, Staphylococcus, et les Enterobacteriaceae. La pâte de poisson est ensuite transformee en saucisse et entreposee pendant 3 semaines a 4°. La croissance des pathogenes est toujours inhibee. Les nitrites diminue au cours de la fermentation. Les 2 souches de bacteries Pediococcus pentosaceus sont donc utilisables pour la fermentation des produits a base de poissons.

Goat lactoferricin (GLfcin), an antibacterial peptide, is released from the N terminus of goat lactoferrin by pepsin digestion. Two GLfcin-related cDNAs, GLfcin L and GLfcin S, encoding Ala20-Ser60 and Ser36-Ser60 of goat lactoferrin, respectively, were cloned into the pET-23a(+) expression vector upstream from (His)6-Tag gene and transformed into Escherichia coli AD494(DE3)pLysS expression host. After being induced by isopropyl-beta-D-thiogalactopyranoside (IPTG), two (His)6-Tag fused recombinant lactoferricins, GLfcin L-His*Tag and GLfcin S-His*Tag, were expressed in soluble form within the E. coli cytoplasm. The GLfcin L-His*Tag and GLfcin S-His*Tag were purified using HisTrap affinity chromatography. According to an antibacterial activity assay using the agar diffusion method, GLfcin L-His*Tag had antibacterial activity against E. coli BCRC 11549, Staphylococcus aureus BCRC 25923, and Propionibacterium acnes BCRC 10723, while GLfcin S-His*Tag was able to inhibit the growth of E. coli BCRC 11549 and P. acnes BCRC 10723. These two recombinant lactoferricins behaved as thermostable peptides, which could retain their activity for up to 30 min of exposure at 100 degrees C.

An acid protease from the broth of a 24 h cultivated Aspergillus niger BCRC 32720 was purified to electrophoretical homogeneity by CM Sepharose FF and Sephacryl S-100 HR chromatographs. The specific activity, purification fold, and yield were 23.29 kU/mg, 2.5, and 24.2%, respectively. Molecular mass (M) and N-terminal amino acid sequence were 47.5 kDa and SKGSAVTT, whereas the pH and temperature optima were at 2.5 and 50 °C, respectively. It was stable at pH 2.0-4.0 or ≤40 °C and activated by Fe(2+) and cysteine, but partially inhibited by phenylmethanesulfonyl fluoride and tosyllysine chloromethyl ketone and highly inhibited by Ag(+), Sn(2+), Fe(3+), Sb(3+), and pepstatin A. It was considered to be an aspartic protease.

Locust bean gum (LBG) was employed to screen mannanase-producing bacteria. The bacterium with highest mannanase ability was identified as Paenibacillus cookii. It revealed highest activity (6.67 U/mL) when cultivated in 0.1% LBG with 1.5% soytone and 0.5% tryptone after 4 days incubation at 27 °C. Its mannanase was purified to electrophoretical homogeneity after DEAE-Sepharose and Sephacryl S-100 separation. The purified mannanase, with an N-terminus of GLFGINAY, had pH and temperature optimum at 5.0 and 50 °C, respectively, and was stable at pH 5.0-7.0, ≤ 50 °C. It was strongly activated by β-mercaptoethanol, dithiothreitol, cysteine, and glutathione, but inhibited by Hg(2+), Cu(2+), Zn(2+), Fe(3+), PMSF, iodoacetic acid, and EDTA. According to substrate specificity study, the purified mannanase had high specificity to LBG and konjac.

A feather-degrading bacterium with high keratinase activity was isolated and identified as Bacillus licheniformis YJ4. After 72 h incubation in a medium (0.5% feather meal, 0.05% NH4Cl and NaCl, 0.04% K2HPO4, 0.03% KH2PO4, 0.01% MgCl2 and yeast extract, 0.1% rice husk) at 37°C, 2 keratinases (keratinase I and II) were purified to electrophoretical homogeneity by CM sepharose and Sephadex G-75 chromatographs. They were with molecular masses (M) of 35.5 and 32.8 kDa, isoelectric point (pI) of 6.63 and 6.50, respectively, and stable at pH 6.0-10.0 and 10-50°C. The optimal pH and temperature were similar, at 9.0 and 60°C, respectively. According to the effect of metal, inhibitor and reducing agent, and previous studies, the purified keratinases I and II were considered to be cysteine and serine proteases, respectively.

Broth incubated with Bacillus subtilis YJ1 at 37°C for 36 h was collected and removed bacterial cells by passing through a 0.45 µm membrane. A cellulase was purified to electrophoretical homogeneity by ammonium sulfate precipitation, Macro-Prep ion exchange and Bio-Gel P-100 chromatography. The recovery and purification fold were 9.7% and 289, respectively, after Bio-Gel P-100 chromatograph. The purified cellulase, with a molecular mass (M) of 32.5 kDa, had an optimal pH and temperature at 6.0 and 50-60°C, respectively. It was stable at pH 6.0-7.5 and

Fibrinolytic enzyme activity was observed during cultivation of Bacillus subtilis YJ1 in a medium containing 1% skim milk, 1% rice husk, 0.5% NaCl, and 0.25% glucose. It was purified to electrophoretical homogeneity after CM-sepharose FF chromatography. The specific activity and yield were 1791.9 FU/mg and 9.5%, respectively. This purified fibrinolytic enzyme had M of 27.5 kDa, optimal temperature and pH at 50 degrees C and 8.5, respectively. It was stable at pH 6.0-10.0 and 10-40 degrees C and inhibited by Fe(3+), Hg(2+), Cu(2+), Zn(2+), and PMSF. Compared the N terminal of amino acids and full DNA sequence with those in NCBI, it was considered to be a nattokinase.

To characterize the xylanase from Bacillus sp. YJ6, broth after 4 days incubation at 25 degrees C was collected and purified to electrophoretical homogeneity after Sephacryl S-100 HR chromatograph. About 3.5% recovery and 678.1 purification fold were achieved. The purified xylanase, with a Mw of 19 kDa, had an optimal pH and temperature at 5.0 and 50 degrees C, respectively, and was stable at pH 5.0-9.0 or

Recombinant keratinase (rK) from Pseudomonas aeruginosa was secreted by Pichia pastoris SMD1168H with a final yield of 580 mg/L (1.03 kU/mL) after 72 h of induction. The rK was purified after nickel affinity chromatography and was stable at pH 6.0-9.0 and 10-60 degrees C. It was nonglycosylated protein with a molecular mass of 33 kDa and had an optimal pH and temperature at 8.0 and 60 degrees C, respectively. Ba(2+), Ca(2+), Mg(2+), Mn(2+), Zn(2+), dithiothreitol, glutathione, and beta-mercaptoethanol activated, while Cu(2+), Fe(2+), Hg(2+), Fe(3+), ethylene glycol tetraacetic acid, ethylene diamine tetraacetic acid, and p-chloromercuribenzoate inhibited its activity. rK could hydrolyze broad substrates and cleave hydrophobic and aromatic amino acids at P(1) position, behaving as those from the wild type strain and E. coli transformant.

The Pediococcus pentosaceus ACCEL bacteriocin was purified to electrophoretical homogeneity by cell adsorption-desorption and Superose 12 fast performance liquid chromatography (FPLC). The purified bacteriocin, with a molecular mass of 17.5 kDa and an N-terminal sequence of -KYYGNGVTXGKHSXXVDXG-, belongs to class IIa and is designated pediocin ACCEL. It was inactivated by various proteases and stable at pH 2.0-6.0 and100 degrees C. More than 80% activity was left even after 15 min of heating at 121 degrees C and pH 2.0-4.0. Gram-positive food-borne pathogens were inhibited by this bacteriocin, but Gram-negative ones were not. According to the storage stability study, the purified pediocin was stable at pH6.0 and low temperature. No significant change in bactericidal activity was observed after freeze-drying and subsequent 1-month storage at room temperature.