Shao-Chi Wu

Antivirus and Prebiotic Properties of Seaweed-oligosaccharide- lysates Derived from Agarase AS-II

The seaweed-oligosaccharide-lysates (SwOSLys) Gra-C108, Mon-C108, Gra-AS-II, or Mon-AS-II are seaweed polysaccharide extracts (SwPSExts) Gra (Gracilaria sp.) or Mon (Monostroma nitidum), as digested by either crude MAEF108-agarases or Agarase AS-II produced from marine bacteria Aeromonas salmonicida MAEF108. Different concentrations of Gra, Mon, Gra-C108, Mon-C108, Gra-AS-II, or Mon-AS-II were cultured with different cell densities of BHK-21 kidney cells, in which the SwPSExts or SwOSLys samples showed no cytotoxicity, and the cell viability was higher than 78%. The six seaweed samples displayed an ability to decrease the effects caused by Japanese encephalitis virus (JEV, Beijing-1 strain) infection. The six seaweed samples were tested as a carbon source for bifidobacteria growth to evaluate the prebiotic potentials. Viable bacteria counts of Bifidobacterium ( B.) pseudolongum subsp. pseudolongum BCRC 14673 could increase more than 3 log CFU/ml in 24 h incubation, when Gra-C108 is utilized. Mon and its agarase lysates can be used by bifidobacteria as a carbon source. B. adolescentis BCRC 14608 and B. pseudolongum subsp. pseudolongum BCRC 14673 could utilize the six seaweed samples to lower pH. B. longum BCRC 11847 displayed an increased pH-lowering ability when utilizing the Gracilaria sp. (red algae) than Mon. nitidum (green algae) as based samples.

Growth and Survival of Streptococcus faecalis and Lactobacillus rhamnosus during Agar Oligosaccharides Fermentation and Storage

To evaluate the performance of two strains of lactic acid bacteria (LAB) that could grow in agar oligosaccharides solutions (AOsS). And during storage at 4℃, the changes in pH values, titratable acidity (TA), and LAB count in fermented AOsS were monitored. The results of six agar polysaccharide extract solutions (APsS) or six AOsS inoculated with Streptococcus (Strept.) faecalis BCRC13076 and/or Lactobacillus (Lact.) rhamnosus BCRC14068 indicated that their titratable acidity (TA) increased, pH decreased, optical density (OD) at 600 nm increased, and their LAB counts increased during the fermentation period (0-24 h). During storage at 4℃, six APsS or six AOsS fermented with Strept. faecalis BCRC13076 and/or Lact. rhamnosus BCRC14068 exhibited decreasing pH, increasing TA, decreasing viability, and an increasing reduction in sugar contents from 7 to 14 days. Six AOsS showed better growth with Strep. faecalis BCRC13076 and/or Lact rhamnosus BCRC14068 than did APsS.

Study on Ethanol Fermentation from Monostroma nitidum Hydrolysate Solution

One percent (w/v) Monostroma (M.) nitidum solution was hydrolyzed using 1 unit/ml five enzymes MA103-agarases, MAEF108-agarases, α-amylase, β-galactosidase, or cellulase, respectively, at 40℃ for 24 h. The reducing sugar content of the 1% (w/v) M. nitidum solutions all reached the stationary phase after enzymatic digestion for 6 h. MAEF108-agarases and cellulase showed to have a better reducing sugar content than the rest. When 0.5 to 3.0% M. nitidum solution were digested using 10 units/ml MAEF108-agarases and 10 units/ml cellulase, the reducing sugar contents (3.97-6.56 mg/ml) greater than 2.0% had no significant difference (p ＜ 0.05). Ten Saccharomyces (S.) cerevisiae were tested for fermented ethanol from a M. nitidum hydrolysate solution, and four S. cerevisiae: BCRC20581, BCRC21686, BCRC21824, and BCRC21962 had the highest ethanol content (5.91, 6.30, 6.57, and 6.18%, respectively) among them. Next, the above four strains were arranged in pairs for ethanol producing starter groups; S5: S. cerevisiae BCRC21686 and BCRC21962, S6: S. cerevisiae BCRC21824 and BCRC21962 had the highest ethanol content (6.94 and 6.87%) of the six groups. As a result, the ethanol producing starter groups S5 and S6 as described in this study should be used in the future for making ethanol from a M. nitidum hydrolysate solution.

Antioxidative Properties of Oversulfation Seaweed Oligosaccharides

The aim of this study is to separate 8 individual seaweed oligosaccharides (SOSs) with different degree of polymerization (DP) from hydrolysate of seaweed polysaccharide (SPSs) of Gracilaria sp., Sargassum sp., and Ulva lactuca. Next, using chlorsulfonic acid-N,N-dimethylformamide (DMF) method to increase sulfate contents from 14.3% to 28.9% of these 8 individual SOSs. In the evaluation of the antioxidative effects of 8 individual SOSs and their oversulfated SOSs, the scavenging effect on DPPH free radical were not correspond to sulfate contents. On the other hand, the chelating ability on ferrous ions of oversulfated SOSs had differences on statistical with their individual SOSs. Especially, Gra-II-DMF, Sar-IIDMF, and Ulva-I-DMF possess 89.5%, 88.9%, and 86.9% chelating ability on ferrous ions, respectively.

The Antimutagenic Properties of Seaweed Oligosaccharides, Evaluated Using Salmonella Typhimurium TA98

Porphyra (Por.) dentate of ten seaweed polysaccharide extracts (SPEs) have the greatest antimutagenic effect on Salmonella (Sal.) typhimurium TA98 under the direct action of the mutagen, 4-NQO; and the indirect action of mutagens, B[a]P. The antimutagenic effects of ten SPEs were related to their polyphenol contents. Under the influence of the directly acting mutagen, 4-NQO, the antimutagenic effects on 60 SOLs were shown to range from 12 to 94%. Under the influence of indirectly acting mutagens B[a]P, the antimutagenic effects on 60 SOLs were from 18 to 89%. The antimutagenic effects of different fractionations from SPE of Por. dentate digestive, by MAEF-108 agarases, demonstrated not only a major contribution from polyphenols, but also a minor contribution from neoagarobiose.

Purification and Characterization of Aeromonas salmonicida MAEF108 Agarase AS-II

Agarase AS-II was purified from the four fractions having agarase activities of marine bacteria Aeromonas (A.) salmonicida MAEF108 crude agarases. The culture suspension of A. salmonicida MAEF108 was centrifuged, ultrafiltrated, then applied to DE-52 ion-exchange chromatography to obtain Agarase AS-II, which was 44.03 fold after purification and estimated as a 95.4 kDa single protein. The optimum temperature and pH for the 0.2% agarose degrading activity of Agarase AS-II was 45°C and 7.0. Agarase AS-II maintained 60% residual activity when stored below 40oC for 2 h; the stability of Agarase AS-II was optimal in pH 6.0-8.0 phosphate buffer. The addition of 5 mM CaCl2 in assay buffer contributed 30% higher relative activity than when no metal ions were added. The activation energy and Km of Agarase AS-II was 46.24 J mol^(-1)K^(-1) and 4.39% of agarose, respectively. The Agarase AS-II had substrate specificity on Rhodophyta and its refined products. It could degrade the substrate to final products, such as neoagarohexaose (N6) and neoagarotetraose (N4), indicating Agarase AS-II could be regarded as a β-agarase, which would directly degrade agarose to produce N6 and N4. In addition, Agarase AS-II has the abilities to biosynthesize N4 and galactose oligomers from neoagarobiose (N2) and galactose, respectively.

Biological Activities of Monostroma nitidum Wines

The subject of this research is to investigate the changes on antioxidative properties and antimutagenicity of the Monostroma (M.) nitidum wine, which made from M. nitidum (green alga) via fermentation with yeasts to produce green algal wine. As to the ability of chelating on ferrous ions of M. nitidum wines, the tested results indicated that M. nitidum wines were inferior to M. nitidum extract and its hydrolysate solution. The α-diphenyl-β-picrylhydrazyl (DPPH) free radical scavenging effect of M. nitidum wines ranged from 65.3% to 69.1%, which were better than M. nitidum extract and hydrolysate solution. The Trolox equivalent antioxidant capacity (TEAC) of M. nitidum wines increased with an increase in their ethanol content, and the M. nitidum wines with 20% sucrose added and fermented by the S6 yeast group obtained the highest TEAC (1.9 mM). The inhibitions of M. nitidum wines against the mutagenicity induced by 4-NQO were evaluated by Salmonella typhimurium TA98 and TA100, reaching 37-48% and 52-63% inhibition of mutagenesis, respectively. The inhibition effect of M. nitidum wines against the mutagenicity induced by indirect-acting mutagen Benzo[a]pyrene (B[a]P) with S9 mix were evaluated by Sal. typhimurium TA98 or TA100, reaching 28-45% and 37-55% inhibition of mutagenesis, respectively. The antioxidative abilities and antimutagenicity activities values were correlated with the soluble total polyphenols content in M. nitidum wines.

Antioxidative Properties of Two Lactic Acid Bacteria Fermented Agar Oligosaccharides

The aim of this study is to assess the potential of the lactic acid bacteria fermentation products (LABFPs) of agar polysaccharide (AgPS) and enzymatically prepared agarooligosaccharide-lysates (AgOSLys) for future utility as antioxidant additives in health foods. The antioxidative properties of thirty six LABFPs from six commercial agars or their AgOSLys produced by two lactic acid bacteria (LAB) strains, Enterococcus (Ent.; formerly Streptococcus) faecalis BCRC13076 and/or Lactobacillus (Lb.) rhamnosus BCRC14068 were evaluated by a series of antioxidative assays. The fermentation products of AgOSLys (AgOS-LABFPs) showed substantial reducing power, an increased chelating effect on ferrous ions, an increased inhibition effect on the hemoglobin-catalyzed peroxidation of linoleic acid, and an increased scavenging capacity towards hydrogen peroxide, compared with the fermentation products of AgPS (AgPS-LABFPs). However, AgOS-LABFPs showed a reduced activity toward α,α-diphenyl-β- picrylhydrazyl (DPPH) radicals. Among the thirty six LABFPs tested, only two exhibited a scavenging effect upon hydroxyl radicals. These findings indicate that AgPS-LABFPs and AgOS-LABFSs may be potentially useful as antioxidative food components.

Purification and Characterization of Agarase PV-1 from Pseudomonas vesicularis MA103

The agarases producing strain Pseudomonas (P.) vesicularis MA103 is an agar-liquefying strain. The crude MA103-agarases is concentrated by ultrafiltration, chromatography by ion-exchange, and Bio-Gel P-100 to obtained Agarase PV-1. The optimum pH and temperature for Agarase PV-1 were 6.0 and 40℃; however 80.0％ of the enzyme activity was inactivated in 15 min under 50℃. The molecular mass of Agarase PV-1 was 62.9 kDa and under sodium ion could enhance 20％ relative agarase activity. The best substrate specific activities of Agarase PV-1 toward brand Himedia(superscript TM) agar were 1.4-time digestion activity compare to agarose, respectively. Hydrolytic products of agarose digested by Agarase PV-1 were analyzed by HPLC, and the results showed that they were oligosaccharides, withe molecular size between neoagarohexaose and neoagarobiose. Products of neoagarohexaose reacted with Agarase PV-1 analyzed by HPLC were oligosaccharides, with molecular size larger than neoagarohexaose.

Studies on Lactic Acid Bacteria Fermented Seaweed Polysaccharide Hydrolysates Solution of Grateloupia filicina

The purpose of this study is to develop the enzyme-digested hot water polysaccharide extract of Grateloupia (G.) filicina fermented by lactic acid bacteria (LAB). Polysaccharide extracts of G. filicina were digested by 500 units of cellulase and 500 units of agarases produced from Pseudomonas vesicularis MA103. Six LAB fermentation solutions were obtained from media: (1) the hydrolysate of 0.5% aqueous solution of the polysaccharide extracts of G. filicina digested by 500 unit agarase and cellulase or (2) this G. filicina hydrolysate added 0.5% sucrose, 1% beef extract, and 0.5% yeast extract. Each of these two media was fermented in three LAB groups: group A: 5% Lactobacillus (Lb.) rhamnosus BCRC14068, group B: 5% Enterococcus (Ent.) faecalis BCRC13076, group C:2.5% Lb. rhamnosus BCRC14068 and 2.5% Ent. faecalis BCRC13076. The results of the pH values of the six LAB fermentation solutions showed a decrease as the fermentation time increased. The pH value of the LAB fermentation solution that the LAB group C cultured in the G. filicina hydrolysate with added carbon and nitrogen source (L-Gra- CN-F(subscript C5)) was below 4.6 with a fermentation time of 9 hr. The titratable acidity was 0.74% and lactic acid bacteria count was 9.39 log CFU/ml. The results could have potential for use in the future development of lactic fermentative G. filicina oligosaccharide hydrolysate health foods.