Bonnie Sun Pan

In-vitro digestibility simulating the proteolysis of feed protein in the midgut gland of grass shrimp (Penaeus monodon)

Abstract Due to feed ingestion, maximal organ weight was reached in 1 h after feeding for the foregut, and 5 h after feeding for the midgut where the highest protease activity was observed in the whole digestivetract of shrimp. The proteases in the midgut gland showed two pH optima at 7.5 and 4.0, that at pH 7.5 being higher. Feed proteins were digested in-vitro with crude enzyme extract from the midgut gland of grass shrimp at 30°C, pH 7.5 for up to 4 h and in two steps at pH 7.5 for 2 h followed by 2 h at pH 4.0. The latter increased feed protein hydrolysis to 1.12–1.56 times that at pH 7.5 for 4 h. The two-step hydrolysis by the shrimp midgut gland extract can serve as an in-vitro method to approximate feed protein digestibility. The tests showed the following order of digestibility: Artemia >predissolved casein>gluten, pelletized shrimp feed>unpelletized shrimp feed>brown fish meal>white fish meal>soybean meal>yeast, undissolved casein. They were significantly different ( P The protein digestibility correlated with the amount of lysine and arginine in the total amino acid composition of the protein sources ( r =0.99) indicating that trypsin plays an important role in digestion in shrimp. Aromatic amino acid content had a positive correlation with the digestibility of water-soluble or native protein sources, but a negative correlation with that of the heat-denatured protein sources, indicating that hydrophobic interaction in heat-processed proteins decreased their digestibility.

Digestive protease activities of juvenile and adult eel (Anguilla japonica) fed with floating feed

Two pepsins, designated P I and P II were isolated from the stomach of juvenile and adult eels. The optimal pH of P I was 1.5 for juvenile (50±5 g) and 2.0 for adult eel (150±10 g), while that of P II was 3.5 for both eels. P I in juvenile eel was 1.5 to 2.0 times more active than that in adult. NaCl at 0.14 to 0.21 mM activated P I activity to two times of that without the presence of salt. P II activity was not affected by salt. Trypsin at optimum pH 8.0 and chymotrypsin at optimum pH 7.0 seemed to correlate with the quantity of digesta in the organ while pepsin activity bore no correlation with digesta. The profile of post-feeding digestive proteases was different between feed in pelleted form and in paste form as a result of its susceptibility for proteases to diffuse into the feed digesta. These post-feeding observations were obtained during a 14-month feeding trial. The only difference between eels fed the float diet or the paste diet was the 24-h profile of the post-feeding digestive protease activity in the tissue and digesta of the gastrointestinal tract. It seems that the susceptibility of the ingested feed to be broken down into particles of smaller sizes, i.e., 0.1 mm diameter, for proteases to readily diffuse into the digesta was a determining factor for in vivo digestion in eel.

Identification of Sulfoglycolipid Bioactivities and Characteristic Fatty Acids of Marine Macroalgae

The fatty acid compositions of 21 species of marine macroalgae, including 5 species of Chlorophyta (green algae), 13 of Rhodophyta (red algae), and 3 of Heterokontophyta (brown algae), were collected from northeastern Taiwan to survey their functional lipids. The lipid contents of green algae ranged from 15.36 to 20.15 mg/g, dry basis (db), and were characterized by a high content of C18:2 and C18:3, red algae (18.57-28.34 mg/g db) were high in C20:4 and C20:5, and brown algae (13.11-19.56 mg/g db) were high in C18:4, C20:4, and C:20:5. All algal lipids contained fatty acids of odd-number carbons, C17:0, and C17:1. Red algae had relatively higher levels of polyunsaturated fatty acids (PUFAs) and were richer in eicosapentaenoic acid (EPA) than green and brown algae. A red alga, Porphyra crispata , was extracted with ethanol and separated on a hydrophobic column (Diaion HP-20 column) to obtain sulfoglycolipids (sulfoquinovosyldiacylglycerols, SQDGs). The main fatty acids in SQDGs were palmitic acid (C16:0), 33.3%; EPA (C20:5), 30.0%; arachidonic acid (C20:4), 12.7%; oleic acid (C18:1), 7.52%; and stearic acid (C18:0), 6.83%. The n-3/n-6 ratio was 1.9, whereas the authentic standard, spinach SQDG, did not contain n-3 fatty acids. Sulfoglycolipids inhibited the growth of human hepatocellular carcinoma cell line (HepG2). The IC50 was 126 μg/mL, which is lower than that of the spinach SQDG (255 μg/mL).

Changes in composition and proteolytic enzyme activities of artemia during early development

Abstract 1. 1. The proximate composition, total and free amino acids, and proteases of Artemia nauplii were determined during early development. 2. 2. Moisture increased from 71.0% to 80.8%, crude protein decreased from 13.2% to 8.8%, crude fat and ash varied slightly. 3. 3. The total amino acids decreased. Free amino acids changed in three patterns. 4. 4. Trypsin, chymotrypsin, carboxypeptidase A, B and cathepsin B and C increased in activity. The activity of trypsin was lower, while cathepsin B and C were the highest. 5. 5. The protease activities were maximal at pH 7.5 and 8.0, and at 45°C on casein. 6. 6. The optimal pH for carboxypeptidase A was 4.0, for carboxypeptidase B was 4.5, for trypsin and chymotrypsin were 7.0–7.5. The protease(s) active at pH 9.0–9.5 were to be determined.

Inhibition of 12- and 15-lipoxygenase activities and protection of human and tilapia low density lipoprotein oxidation by I-Tiao-Gung (Glycine tomentella).

I-Tiao-Gung, Glycine tomentella, has been used extensively as a traditional herbal medicine to relieve physical pain, but its bioactivity has not been studied systematically. Ninety-five percent ethanol extracts of G. tomentella (GT-E) showed antioxidant activity in human plasma by prolonging the lag phase (+Tlag) of Cu2+-induced, LDL oxidation and were dose dependent. The+Tlag of LDL combined with 3.2 μg/mL GT-E was similar to that with 2.0 μM (ca. 0.5 μg/mL) Trolox. A similar inhibitory effect was found toward tilapia plasma LDL. In addition, GT-E inhibited tilapia thrombocyte (nucleated platelet), 5-, 12-, and 15-lipoxygenase (LOX). The IC50 values were 0.43, 0.72, and 0.42 μg/mL, respectively, whereas the IC50 values for nordihydroguaiaretic acid (NDGA) on 5-, 12-, and 15-LOX were 2.3, 1.6, and 1.7 μg/mL, respectively. The IC50 value for cyclooxygenase-2 (COX-2) inhibition by GT-E was 42.0 μg/mL, whereas the IC50 value by indomethacin as a positive control was 0.61 μg/mL. The prevention of LDL oxidation and the dual inhibition of LOX and COX-2 are indicative of the possible roles of I-Tiao-Gung in antiatherosclerosis and anti-inflammation.

Isoflavone-rich extracts from wooly glycine Glycine tomentella inhibits LPS-induced TNF-α expression in a macrophage cell line of Atlantic salmon (Salmo salar L.)

The immunomodulatory effects of an isoflavone-rich extract from the root of wooly glycine Glycine tomentella (GTE) were studied in a macrophage-like cell line from Atlantic salmon (TO cells). The TO cell line was stimulated with defined concentrations of lipopolysaccharide (LPS) from Escherichia coli (serotype O127:B8) for defined time periods to induce expression of pro-inflammatory enzymes and cytokines. Cyclooxygenase-2 (COX-2), 5-lipoxygenase (5-LOX), tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) were measured by real-time PCR methods and combined with analyses of eicosanoid production in cell extracts and evaluation of molecules of the TNF-alpha cell signaling pathway. The results showed that TNF-alpha was strongly induced by LPS, while GTE (25miicrog/ml) inhibited 67% of the TNF-alpha response when added to the cells together with LPS. Incubation of LPS in combination of GTE in TO cells caused increased intracellular prostaglandin E2 (PGE2), and reduced activation of p38 MAP kinase compared to LPS alone. GTE seemed to arrest NADPH oxidation, the coenzyme for carbonyl reductase and the prostaglandin-E2 9-reductase converting PGE2 to PGF2. We suggest that the mechanism of increased intracellular PGE2 levels following GTE treatment is caused by reduced breakdown of PGE2. GTE did not inhibit the other pro-inflammatory responses in LPS stimulated cells studied herein. IL-1beta and COX-2 showed moderately increased levels of expression likely caused by the increased PGE2.

The Involvement of Proteins and Nonprotein Nitrogen in Postmortem Changes in Seafoods

Many nitrogenous compounds are involved in postmortem reactions in the muscles of animals, primarily as enzymes catalyzing the catabolysis of different tissue components. Thus, they contribute to changes in color, flavor, and rheological properties of the products. Furthermore, proteins and NPN participate also as substrates in reactions leading to the formation of products affecting the pH in the fish tissues. On the other hand, the rate and extent of changes in proteins are influenced by the products of degradation of saccharides and organic phosphates, as well as by the concentration of free ions, whereas these depend on the antemortem condition of the fish and on the temperature in the tissues after catch. A positive relationship of the enthalpy of activation of fish myofibrillar ATPase and the habitat temperature was demonstrated, from large negative values for cold-adapted species to high positive values for tropical species (Johnstone and Goldspink, 1975).

Phenolic acids identified in sorghum distillery residue demonstrated antioxidative and anti-cold-stress properties in cultured tilapia, Oreochromis mossambicus.

This study aimed to identify the bioactive compounds and evaluate the anti-cold-stress function of the sorghum distillery residue (SDR) using tilapia as an alternative animal model. The highest contents of water-soluble bioactive compounds in SDR were polyphenols, followed by tannins, anthocyanins, and flavonoids. SDR was extracted with double-distilled water, 95% ethanol, and ethyl acetate, separately. The ethanol extract (SDR-E) yielded the highest polyphenol content [15.03 mg/g of SDR dry weight (dw)], of which the EC50 value of R,R-diphenyl-β-picrylhydrazyl (DPPH) radical scavenging efficiency was 0.56 ± 0.04 mg/mL. The SDR-E suppressed the oxidation of low-density lipoproteins (LDLs) more efficiently than that of other extracts. Tilapia fed a diet containing 3.6% SDR-E decreased accumulative mortality during cold stress, of 46.2%. The accumulative morality of the control was 92.9%. The phenolic acids identified in SDR included gallic acid (0.36 ± 0.08 mg/g of SDR dw), 3,4-dihydroxybenzoic acid (0.16 ± 0.12 mg/g of SDR dw), and 4-hydroxybenzoic acid (0.49 ± 0.23 mg/g of SDR dw). Diets supplemented with 0.5% 4-hydroxybenzoic acid fed to tilapia showed a lower mortality rate than that fed 1.0% 4-hydroxybenzoic acid, comparable to that of the tilapia fed 20% SDR. The latter showed lower mortality than that of the control. These results suggested that 4-hydroxybenzoic acid is one of the major anti-cold-stress compounds in SDR.

Purification and characterization of a fish scale-degrading enzyme from a newly identified Vogesella sp.

The objective of the present study is to purify and characterize the fish scale-degrading enzyme from Vogesella sp.7307-1, which was newly identified and isolated from fish scales. The enzyme from Vogesella sp.7307-1 was assayed with casein and confirmed as a protease. Crude protease was extracted, isolated, and purified 35.7-fold with 19.6% recovery using 20-80% saturation of ammonium sulfate fractionation, Q FF ion exchange chromatography, and Superdex 200 gelfiltration. The molecular weight of the purified enzyme was 119 kDa. The Km and Vmax were 0.067 mM and 425.5 U/mg-min, respectively using azo-casein as substrate. The optimum pH of the purified enzyme was 7.5, and the optimum temperature was 50 °C. The enzyme was stable at temperatures below 55 °C and pH range 7.5 to 9.0. The enzyme activity of the purified protease was completely inhibited by EDTA (ethylene diamine teraacetates), indicating the enzyme was a metalloprotease. Hydrolysates from fish scales treated with protease 7307-1 were found having low molecular weight peptides (<1 kDa). The protease 7307-1 is a promising enzyme for preparing smaller peptides from fish scales.

Anti-stress effects of Glycine tomentella Hayata in tilapia: inhibiting COX-2 expression and enhancing EPA synthesis in erythrocyte membrane and fish growth.

The objective of this study was to elucidate the in vivo effects of the ethanol extract of wooly Glycine tomentella Hayata (GTE) root on tilapia to elucidate whether GTE has antistress activity. Tilapia as an animal model were fed with or without GTE, then injected with lipopolysaccharide (LPS) or ammonium chloride (NH(4)Cl). The tilapia were exposed to 100 mg/L of aqueous NH(4)Cl, and/or acute cold stress. Growth parameters of the tilapia were measured during the feeding trials. Tilapia injected with GTE (20 μg/g of fish), NH(4)Cl (100 μg/g of fish) and/or LPS (1 μg/g of fish) were then sampled 2 h poststimulation. GTE significantly inhibited cyclooxygenase-2 expression and hemoglobin (Hb) dimer formation (36 kDa). GTE also improved growth and blood viscosity and upregulated eicosapentaenoic acid content of erythrocytes. The in vivo results indicated that GTE (20 μg/g of fish) can be applied as a stress-tolerance enhancing agent for the aquaculture industry.