Chorng-Liang Pan

Algal-oligosaccharide-lysates prepared by two bacterial agarases stepwise hydrolyzed and their anti-oxidative properties

Twenty algal-oligosaccharide-lysates (AOLs), derived from six agars and four algal polysaccharide extracts (APEs), were treated first with agarases with 250 or 500 agarase activity units (AU), which were produced from the agar-liquefying bacterial strain Pseudomonas vesicularis MA103, named MA103-agarases. The AOLs were then treated with agarases (250 or 500 AU) derived from the agar-softening bacterial strain Aeromonas salmonicida MAEF 108, named MAEF 108-agarases. Anti-oxidative properties of the AOLs were evaluated by five in vitro methods. The AOL obtained from the APE of Porphyra dentate, digested by 250 AU of MA 103-agarases, and by 250 AU of MAEF 108-agarases, designated as A250-Por, showed better results than the 19 other AOLs. This result is in accordance with the level of soluble total polyphenols (STP) of A250-Por, which was also higher than the remainder of the AOLs tested. The AOL derived from the APE of P. dentate, digested by 500 AU of MA103-agarases and then 500 AU of MAEF108-agarases, and designated as B500-Por, displayed the second highest data in four potential evaluation methods, except in H2O2 scavenging capacity. In this study, certain agars or APEs digested by specific agarases can present an increasing antioxidative capacity. These agars include Bitek agar, Agar powder, Bacteriological, Agar Bacteriological, and Guanghui agar, plus APEs of Gracilaria sp. and Monostroma nitidum decomposed stepwise by two agarases. The fraction of polyphenols (<1 kDa) that were derived from A250-Por showed anti-oxidative activities on α, α-diphenyl-β-picrylhydrazyl (DPPH) assay and reducing power determination, while the remaining four agar-lytic fractions obtained from A250-Por did not exhibit anti-oxidative activity. This phenomenon may suggest that anti-oxidative properties of AOLs originate in polyphenols. Algal-oligosaccharide-lysates may have potential use as a health food.

Separation and quantification of neoagaro- and agaro-oligosaccharide products generated from agarose digestion by β-agarase and HCl in liquid chromatography systems

A series of neoagaro-oligosaccharides (NAOS) were separated and isolated by beta-agarase digestion and agaro-oligosaccharides (AOS) by HCl hydrolysis from agarose with defined quantity and degree of polymerization (DP). Profiles of the oligomer length in the crude product mixtures were monitored by two high-performance liquid chromatography (HPLC) systems: size-exclusion chromatography (SEC) and NH2-column chromatography (NH2-HPLC), coupled with an evaporative light-scattering detector (ELSD). Calibration curves were established separately to identify the DP and quantify the amount of the oligomer products analyzed in the two systems. Each system was optimized to generate a spectrum of saccharide oligomers with various DP, where the reaction yield for NAOS was 52.7% by 4U/mg beta-agarase and for AOS was 45.6% by 0.4M HCl. SEC resolved the product in size ranges consisting of DP 1-22 for NAOS and DP 1-14 for AOS. NH2-HPLC clearly resolved both distinct saccharide product sizes within DP 12. The optimized system was connected with a fraction collector to isolate and quantify these individually separated products. The total product yields of the recovered NAOS of DP 1-22 and AOS of DP 1-14 by the SEC system were 84.7% and 82.9%, respectively. NH2-HPLC recovered NAOS and AOS, both with a DP of 1-10 with total product yields of 48.9% and 90.0%, respectively. Isolated NAOS and AOS product fractions were inspected by (1)H NMR spectroscopy and ESIMS spectrometry to confirm structure, molecular mass, and purity. This study established feasible systems for the preparation and qualitative and quantitative measurements, as well as for the isolation of various sizes of oligomers generated from agarose.

Identification of cell wall antigens associated with a large conjugative plasmid encoding phage resistance and lactose fermentation ability in lactic streptococci

In order to begin to analyze the gene products encoded by phage resistance plasmids in lactic streptococci, we identified phage-resistance plasmids by screening resistant strains from commercial starter cultures for the ability to carry out unselected cotransfer, by conjugation, of phage resistance with lactose fermentation ability (lac+). In this fashion, we identified a large (90 kilobases) plasmid, pCLP51R, that encodes the lac+ marker, resistance to a lytic phage called LP10G (1pr+), high-frequency conjugal donor ability (hft+), and clumpy growth of host bacteria in broth culture (clu+). The mechanism of resistance conferred by this plasmid appears to involve interference with a step in the phage replication cycle that occurs after the initial attachment of the phage. Polyacrylamide gel electrophoresis of cell surface extracts of isogenic strains, carrying or lacking pCLP51R, combined with immunoblotting analysis, showed that there were several plasmid-related differences in the banding pattern of low molecular weight proteins and that the plasmid resulted in production of several unique antigenic polypeptides in the size range of 15-30 kd, as well as modification of chromosomally encoded antigens to different molecular weight forms.

Monitoring and preparation of neoagaro- and agaro-oligosaccharide products by high performance anion exchange chromatography systems

A series of neoagaro-oligosaccharides (NAOS) were prepared by β-agarase digestion and agaro-oligosaccharides (AOS) by HCl hydrolysis from agarose with defined quantity and degree of polymerization (DP). Chain-length distribution in the crude product mixtures were monitored by two high performance anion exchange chromatography systems coupled with a pulsed amperometric detector. Method 1 utilized two separation columns: a CarboPac(™) PA1 and a CarboPac(™) PA100 connected in series and method 2 used the PA100 alone. Method 1 resolved the product in size ranges consisting of DP 1-46 for NAOS and DP 1-32 for AOS. Method 2 clearly resolved saccharide product sizes within DP 26. The optimized system utilizing a semi-preparative CarboPac(™) PA100 column was connected with a fraction collector to isolate and quantify individually separated products. This study established systems for the preparation and qualitative and quantitative measurements as well as for the isolation of various sizes of oligomers generated from agarose.

Heat shock and cold shock treatments affect the survival of Listeria monocytogenes and Salmonella Typhimurium exposed to disinfectants.

The foodborne pathogens Listeria monocytogenes and Salmonella Typhimurium were subjected to heat shock at 48°C for 10 and 30 min, respectively, and then cold shocked at 15°C for 3 h. The effect of these shocks on the viability of test organisms exposed to chlorine dioxide and quaternary ammonium compounds was then determined. After exposure to the disinfectants, the viable population of each test organism, regardless of heat shock or cold shock treatment, decreased as the exposure period was extended. Both heat shock and cold shock treatments reduced the susceptibility of L. monocytogenes to both disinfectants at 25°C. However, for Salmonella Typhimurium, exposure to the chlorine dioxide disinfectant or quaternary ammonium compounds at 25°C significantly reduced (P < 0.05) survival of heat-shocked cells but significantly increased (P < 0.05) survival of cold-shocked cells compared with control cells. Survival of both L. monocytogenes and Salmonella Typhimurium generally was reduced after exposure to disinfectants at 40°C compared with 25°C.

Evaluation of HPSEC-ELSD method for precise measurement of β-agarase activity.

β-agarase activity was monitored by traditional reducing sugar content methods: Somogyi-Nelsons arsenomolybdate, Millers dinitrosalicylic acid and Kidby and Davidsons ferricyanide methods, as well as by high-performance size exclusion chromatography coupled with a refractive index detector and an evaporative light scattering detector (ELSD). Calibration curves were established separately for each method to measure the amounts of the neoagaro-oligosaccharides (NAOS) in the reaction mixtures, which are the products from 1-10 units (U) of β-agarase cleavage activity on agarose. Product quantities from each monitoring method were compared with the isolated NAOS products. The graphs plotted by agarase activity unit and product concentration clearly displayed that the ELSD method closely followed the results of the isolated products. The percentage deviation of results measured by the five methods away from those of the isolated NAOS product mixture amounted to -13.1-35.1, -21.1-25.5, -27.1-23.81, 6.1-24.3 and 16.2-22.8%, respectively. When the loss during product isolation, about 15-17%, was taken into account, the high precision of the ELSD method was confirmed. HPSEC-ELSD methods also accurately measured the enzyme kinetics as well as enabling partial identification of oligosaccharides assembled in the NAOS product mixture. This study established the HPSEC-ELSD system as an alternative method for monitoring agarase activity.

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.