Isolation of protoplasts from <Emphasis Type="Italic">undaria pinnatifida</Emphasis> by alginate lyase digestion

The aim of this study is to isolate protoplasts from Undaria pinnatifida. Protoplasts of the alga were isolated enzymatically by using alginate lyase, which was prepared by fermenting culture of a strain Vibrio sp. 510. Monofacterial method was applied for optimizing digestion condition. The optimum condition for protoplast preparation is enzymatic digestion at 28°C for 2h using alginate lyase at the concentration of 213.36 U (8 mL) every 0.5g fresh thalline with NaCl 50 and at the shaking speed of 150 r min⁻¹ during digestion. The protoplast yield can reach 2.62±0.09 million per 0.5 g fresh leave under the optimum condition. The enzyme activity is inhibited by Ca²⁺ and slightly enhanced by Fe²⁺ and Mn²⁺ at concentrations of 0.05, 0.08 and 0.10 mol L⁻¹.     

Optimization of Culturing Condition and Medium Composition for the Production of Alginate Lyase by a Marine Vibrio sp. YKW-34

Carbohydrases secreted by marine Vibrio sp. YKW-34 with strong Laminaria cell wall degrading ability were screened, and among them alginate lyase was found to be dominant. The effects of medium composition and culturing condition on the production of alginate lyase by marine Vibrio sp. YKW-34 in flask were investigated in this study. In the culture medium of marine broth, no alginate lyase was produced. The activity of the alginate lyase, after being induced, reached 5 UmL^-1. The best inoculum volume and inoculum age were 10% and 12h, respectively. The optimal temperature for alginate lyase production was 25℃. The fermentation medium was composed of 0.5% of Laminaria powder and 0.2% of KNO3 with an initial acidity of pH 8.0. Alginate could induce alginate lyase production but not as efficiently as Laminaria powder did. The addition of fucoidan, cellulose and glucose had negative effect on the alginate lyase production. Other kinds of nitrogen sources, such as yeast extract, beef extract and peptone, had positive effect on the growth of the microorganism and negative effect on alginate lyase production. In addition, the time course of alginate lyase production under the optimized condition was described. The optimal harvest time was 48 h.     

The extraction of pigments from fresh Laminaria japonica

The pigments in Laminariajaponica was extracted with six organic solvents and analyzed in spectroscopy analysis. The extractions conditions were screened by an orthogonal test and the quantity of extracted pigments was determined spectroscopically. The results show that： （1） among the six organic solvents, acetone was the most effective one for the extraction; （2） the optimum extraction conditions were as follows： the ratio of S/M （solvent volume/ material weight） was 30 ml/g; fresh seaweed was extracted 2 times in 2 h; （3） the average total content of pigments was 1.85 mg/g （calculated with dry L. japonica）.     

Effects of composition and structure of alginates on adsorption of divalent metals

Results of a series of experiments (on the adsorption of divalent metal ions by dried alginic acid, Na and Ca alginates of different composition and block structure) conducted in this systematic study of the effects of the composition and structure of alginates on the static adsorption equilibrium of divalent metal ions indicate that the properties of alginate adsorption to divalent metal ions are highly different, depending not only on the cations used, but also on the form and structure of the alginates. There is close correlation between the adsorption properties and the structure of the alginates. The selectivity coefficient of Na alginate for Cd-Sr ion exchange tends to increase with the increase of theM/G ratio in alginate, whereas the adsorption capacity of Ca alginate for Cu²⁺ ion decrease with the increase of the G-block or the average length of the G-block and the total adsorption capacity of alginic acid is found to vary in the same order as theF _MM(diad frequency) in alginate in the mixed solution of Sr²⁺, Ba²⁺ and Cd²⁺. Contribution No. 2166 from the Institute of Oceanology, Chinese Academy of Sciences.     

Production of the Blood Pressure Lowing Peptides from Brown Alga ( Undaria pinnatifida )

1 IntroductionIncreased blood pressure appears to be one of theprimary risk factors of circulatory organ diseases suchas encepharo-apoplexy, encepharo-infarction and cardi-ac infraction. Angiotensin-I-converting enzyme (ACE)plays an important role in the rennin-angiotensin sys-tem by regulating blood pressure. Antihypertensivedrugs such as captopril and enalapril are potent ACEinhibitors (Ondetti et al., 1977). Recently, severalinhibitory peptides derived from food proteins havebeen isolat…     

EFFECTS OF COMPOSITION AND STRUCTURE OF ALGINATES ON ADSORPTION OF DIVALENT METALS

Results of a series of experiments(on the adsorption of divalent metal ions by dried alginic acid, Na and Ca alginates of different composition and block structure) conducted in this systematic study of the effects of the composition and structure of alginates on the static adsorption equilibrium of divalent metal ions indicate that the properties of alginate adsorption to divalent metal ions are highly different, depending not only on the cations used, but also on the form and structure of the alginates. There is close correlation between the adsorption properties and the structure of the alginates. The selectivity coefficient of Na alginate for Cd-Sr ion exchange tends to increase with the increase of the M/G ratio in alginate, whereas the adsorption capacity of Ca alginate for Cu~(2+) ion decrease with the increase of the G-block or the average length of the G-block ((?)_G) and the total adsorption capacity of alginic acid is found to vary in the same order as the F_(MM)(diad frequency) in alginate in     

Optimized water vapor permeability of sodium alginate films using response surface methodology

The water vapor permeability （WVP） of films is important when developing pharmaceutical applications. Films are frequently used as coatings, and as such directly influence the quality of the medicine. The optimization of processing conditions for sodium alginate films was investigated using response surface methodology. Single-factor tests and Box-Behnken experimental design were employed. WVP was selected as the response variable, and the operating parameters for the single-factor tests were sodium alginate concentration, carboxymethyl cellulose （CMC） concentration and CaClz solution immersion time. The coefficient of determination （R2） was 0.97, indicating statistical significance. A minimal WVP of 0.389 8 g-mm/（m^2.h.kPa） was achieved under the optimum conditions. These were found to be a sodium alginate concentration, CMC concentration and CaCl2 solution immersion time at 8.04%, 0.13%, and 12 min, respectively. This provides a reference for potential applications in manufacturing film-coated hard capsule shells.     

STUDIES ON COLOR TYPE VARIANTSFROM MUTAGENIZED PROTOPLASTS OFPORPHYRA HAITANENSIS CHANG ET ZHENG& P. YEZOENSIS UEDA (RHODOPHYCEASE )

Isolated protoplasts from thalli of Porphyra haitanensis and Porphyra yezoensis were treated with colchicine or irradiated by ultraviolet (UV ). Several types of color variants were observed among the protoplast offspring. After treatment with colchicine: (1) 0.04-0.09% of red type variants in P. haitanensis were obtained; (2) The rate of red type variants and the variegated chimeral thalli composed of red type and wild type of sectors were 6.31- 1.11% in P. yezoensis. After irradiation with UV: (1) 3.5- 10.5% of red type variants in P. yezoensis were obtained: (2) 0.5-2-0% of red type variants and the variegated chimeral thalli composed of red type and wild type of sectors were obtained in P. haitanensis. Colchicine and UV's mutangenic effects on P. yezoensis protoplasts were stronger than those on P. haitanensis protoplasts. The most efficient concentration of colchicine was 0.05%. The optimal length of UV-radiation was 1/2 min (radiation distance 5 cm). The red type variants induced, by colchicine tr     

Study on the Tripolyphosphatase （TPPase） Property of Bighead Carp （Aristichthys nobilis） Myosin Subfragment-1

Myosin subfragment-1 was prepared from the myofibrils of bighead carp (Aristichthys nobilis). The myosin subfrag- ment-1 was proved to have the activity of tripolyphosphatase (TPPase) responding to the hydrolysis of sodium tripolyphosphate (STPP). The optimum temperature and pH for the TPPase of myosin subfragment-1 were 30℃ and pH 5.0, and at pH 8.0 the TPPase also showed a high activity. Mg2 was necessary to TPPase. The TPPase activity of myosin subfragment-1 was activated by Mg2 under low concentrations, but was inhibited when the concentration was over 17 mmolL-1. The TPPase activity was also affected by KCl. The optimum concentration of KCl for TPPase was 0.3 molL-1 under the condition of 17 mmolL-1 Mg2 . The TPPase activity was significantly inhibited by EDTA-Na2. Reagents such as KBr, KI and KIO3 could inhibit the TPPase effectively. K2Cr2O7 as well as KMnO7 and KNO3 exhibited weak inhibiting effects. The TPPase converted STPP to pyrophosphate (PP) and orthophosphate (Pi) stoichiometrically with a KM of 3.2 mmolL-1.     

Bioaccumulation of Pb^2＋ and Its Effects on Growth, Morphology and Pigment Contents of Spirulina （Arthrospira）platensis

A laboratory experiment was conducted to assess the bioaccumulation of Pb2 and its effects on growth,morphology andpigment contents of Spirulina (Arthrospira) platensis.The specimen cultured in Zarrouk liquid medium was treated with variousinitial metal concentrations (0,5,10,30,50 and 100 μgmL-1).The growth of S.platensis was adversely affected by Pb2 at high con-centrations (30,50 and 100 μg mL-1).However,at low concentrations (5 μg mL-1),Pb2 could stimulate its growth slightly.The pig-ment contents (chlorophyll α and β carotene) were decreased in a dose-dependent manner.The highest reductions (67% and 53%respectively in chlorophyll α and β carotene) were observed in 100 μg mL-1 treatment group.The LC50 (96 h) of Pb2 was measured as75.34μg mL-1.Apart from a few cases of filament breakages at elevated concentrations (50 and 100μg mL-1),morphological abnor-malities are not specific.Metal bioaccumulation increased with Pb2 concentrations,but decreased with exposure time.The maxi-mum accumulated amount was 188 mgg-1 dry weight.The bioconcentration factor (BCF) reached to a peak at day 2,followed by agradual reduction for all the exposure concentrations.S.platensis is able to tolerate considerably high Pb2 concentrations.Conse-quently it can be used as a potential species to remove heavy metal from contaminated waters.     

Purification and Characterization of a Novel Lipase from Antarctic Krill

Lipase from Antarctic krill,with a molecular weight of 71.27kDa,was purified with ammonium sulfate precipitation and a series of chromatographic separations over ion exchange(DEAE)and gel filtration columns(Sephacryl S-100),resulting in 5.2%recovery with a 22.4-fold purification ratio.The optimal pH and temperature for enzyme activity were 8.0 and 45℃,respectively.Purified lipase had Km and Vmax values of 3.27mmolL−1 and 2.4Umg−1,respectively,using p-nitrophenyl laurate as the substrate.Lipase activity was enhanced by adding Ca2+and Mg2+ions in the concentration ranges of 0–0.5mmolL−1 and 0–0.3mmolL−1,respectively,while the activity was inhibited by a further increase in these ion concentrations.Fe3+and Cu2+ions showed obvious inhibitory effects on enzyme activity,and the inhibition rates were 71.8%and 53.3%when the ion concentrations were 0.5mmolL−1.     

Mechanisms and factors affecting the adsorption of sodium alginate onto modified clays

Algal organic materials (AOMs) are one critical factor affecting the efficiency of modified clays used for the mitigation of harmful algal blooms (HABs). This study was conducted to develop a deeper understanding of the mechanisms and factors affecting the adsorption of AOMs onto modified clays. Sodium alginate (polysaccharide) and kaolinite modified with polyaluminium chloride (PACl) were used as AOMs and modified clay model substances, respectively, and the effects of modifier dosage, contact time, solution pH and ionic strength were investigated through batch adsorption experiments. Kinetics revealed that the alginate adsorption rate was described well by a pseudo-second order model. PACl effectively enhanced the adsorption capacity of kaolinite and increased the adsorption rate, and the optimum additive amount of PACl was 5%. The experimental data fitted both the Freundlich and Langmuir adsorption equations well. The adsorption thermodynamics for alginate onto modified clays suggests that alginate adsorption is a spontaneous process. The adsorption of alginate onto modified clays was highly dependent on pH, with a decrease in adsorption observed with increased pH to 9.48, but the opposite was true above pH 9.48. Finally, adsorption increased with increasing ionic strength.     

Production of the Blood Pressure Lowing Peptides from Brown Alga （ Undaria pinnatifida ）

Brown alga （ Undaria pinnatifida） was treated with alginate lyase and hydrolyzed using 17 kinds of proteases and the inhibitory activity of the hydrolysates for the angiotensin-I-converting enzyme （ACE） was measured. Four hydrolysates with potent ACE-inhibitory activity were administered singly and orally to spontaneously hypertensive rats （SHRs）. The systolic blood pressure of SHRs decreases significantly after single oral administration of the brown alga hydrolysates by pro- tease S ＇ Amano＇ （from Bacillus stearothermophilus） at the concentration of 10 （mg protein） （kg body weight）^ - 1. In the 17 weeks of feeding experiment, 7-week-old SHRs were fed standard diet supplemented with the brown alga hydrolysates for 10 weeks. In SHRs fed 1.0 and 0.1% brown alga hydrolysates, elevating of systolic bloodpressure was significantly suppressed for 7 weeks. To elucidate the active components, the brown alga hydrolysates were fractionated by 1-butanol extraction and HPLC on a reverse-phase column. Seven kinds of ACE-inhibitory peptides were isolated and identified by amino acid composition analysis, sequence analysis, and LC-MS with the results Val-Tyr, Ile-Tyr, Ala-Trp, Phe-Tyr, Val-Trp, Ile-Trp, and Leu-Trp. Each peptide was determined to have an antihypertensive effect after a single oral administration in SHRs. The brown alga hydrolysates were also confirmed to decrease the blood pressure in humans.     

Effects of heavy metal ions (Cu2+, Pb2+ and Cd2+) on DNA damage of the gills, hemocytes and hepatopancreas of marine crab, Charybdis japonica

There are rising concerns about the hazardous effects of heavy metals on the environment. In this study, comet assay and DNA alkaline unwinding assay were conducted on the tissues (gills, hepatopancreas, and hemocytes) of Charybdis japonica in order to illustrate genotoxicity of three heavy metal ions (Cu2+, Pb2+, and Cd2+) on the marine crabs C. japonica. The crabs were exposed to Cu2+ (10, 50, and 100 ?g.L?1), Pb2+ (50, 250, and 500 ?g L?1) and Cd2+ (5, 25, and 50 ?g L?1), and the tissues were sampled at days 0.5, 1, 3, 6, 9, and 15. DNA alkaline unwinding assay was used for testing the DNA single strand break in gills and hepatopancreas and comet assay was employed for testing the DNA damage in hemocytes. The results showed that the DNA damage (F-value) of gills in the crabs exposed to the three heavy metals was decreased gradually during the exposure periods and there was a dose-time response relationship in certain time, suggesting that the levels of DNA single strand break in all the experimental groups increased significantly compared to the controls. Changes of F-value in hepatopancreas of the crabs exposed to the three heavy metals were similar to those in gills except that the peak values were found in the 500 ?g L?1 Pb2+ treatment group at day 3 and the 50 ?g L?1 Cd2+ treatment group at day 9. The ranks of DNA damage in gills and hepatopancreas induced by the three heavy metal ions (50 ?g L?1, day 15) were Cd2+ >Pb2+ >Cu2+ and Pb2+ >Cu2+ >Cd2+. The levels of DNA damage in gills were higher than those in hepatopancreas in the same experimental group. It can be concluded that indices of DNA damage can be used as the potential biomarkers of heavy metal pollution in marine environment.     

生物采油解堵剂中产蛋白酶菌株的筛选及培养条件优化

为研究生物酶采油解堵剂中产蛋白酶菌株的初、复筛选及培养条件优化,从大庆原油样品中筛选菌种,通过水解酪素的透明圈实验及福林酚测蛋白酶酶活的方法进行菌株的初、复筛选;以蛋白酶酶活为优化指标,采用单因素实验对筛选的产蛋白酶菌株的培养基及培养条件进行优化,优化最适培养基:可溶性淀粉为15g/L,蛋白胨为20g/L,酵母膏为20g/L,NaCl为1.0g/L,CaCl2为0.02g/L,Na2HPO4为0.2g/L,NaH2PO4为0.1g/L;在初始pH为6.0、接种量为5%(体积分数)、温度为31℃、摇床转速为160r/min的条件下,培养72h后,菌株的蛋白酶酶活为551.0U/mL,为复筛选菌株的蛋白酶酶活的22.92倍,即为菌株生长繁殖及代谢的最佳条件,能够获得更高的蛋白酶酶活,有利于后续实验的进行.结果表明:菌株产蛋白酶对原油作用效果为发酵液表面张力从作用前的56.2mN/m降低到作用后的30.5mN/m,表面张力显著降低,还有降解降黏原油等效果,具有一定的研究价值.     

Simultaneous identification and quantification of tetrodotoxin in fresh pufferfish and pufferfish-based products using immunoaffinity columns and liquid chromatography/quadrupole-linear ion trap mass spectrometry

In this study,we established a comprehensive method for simultaneous identification and quantification of tetrodotoxin(TTX) in fresh pufferfish tissues and pufferfish-based products using liquid chromatography/quadrupole-linear ion trap mass spectrometry(LC-QqLIT-MS).TTX was extracted by 1% acetic acid-methanol,and most of the lipids were then removed by freezing lipid precipitation,followed by purification and concentration using irnmunoaffinity columns(IACs).Matrix effects were substantially reduced due to the high specificity of the IACs,and thus,background interference was avoided.Quantitation analysis was therefore performed using an external calibration curve with standards prepared in mobile phase.The method was evaluated by fortifying samples at 1,10,and 100 ng/g,respectively,and the recoveries ranged from 75.8%-107%,with a relative standard deviation of less than 15%.The TTX calibration curves were linear over the range of 1-1 000 ng/L,with a detection limit of 0.3 ng/g and a quantification limit of 1 ng/g.Using this method,samples can be further analyzed using an informationdependent acquisition(IDA) experiment,in the positive mode,from a single liquid chromatography-tandem mass spectrometry injection,which can provide an extra level of confirmation by matching the full product ion spectra acquired for a standard sample with those from an enhanced product ion(EPI) library.The scheduled multiple reaction monitoring method enabled TTX to be screened for,and TTX was positively identified using the IDA and EPI spectra.This method was successfully applied to analyze a total of 206 samples of fresh pufferfish tissues and pufferfish-based products.The results from this study show that the proposed method can be used to quantify and identify TTX in a single run with excellent sensitivity and reproducibility,and is suitable for the analysis of complex matrix pufferfish samples.     

Simultaneous determination of calcium and magnesium in water using artificial neural network spectro-photometric method

A new analytical method using Back-Propagation(BP) artificial neural networks and spectrophotometry for simultaneous determination of calcium and magnesium in tap water,the Yellow River water and seawater is established.By condition experiment,the optimum analytical conditions for calcium,magnesium and Arsenazo(Ⅲ) color reactions are obtained.Levenberg-Marquart(L-M) algorithm is used for calculation in BP neural network.The topological structure of three-layer BP ANN network architecture is chosen as 11-10-2(nodes).The initial value of gradient coefficient μ is fixed at 0.001 and the increase factor and reduction factor of μ take the default values of the system.The data are processed by computers with our own programs written in MATLAB 7.0.The relative standard deviations of the calculated results for calcium and magnesium are 2.31% and 2.14%,respectively.The results of standard addition method show that the recoveries of calcium and magnesium are 103.6% and 100.8% in the tap water,103.2% and 96.6% in the Yellow River water(Lijin district of Shandong Province),and 98.8%-103.3% and 98.43%-103.4% in seawater from Jiaozhou Bay of Qingdao.It is found that 14 common cations and anions do not interfere with the determination of calcium and magnesium under the optimum experimental conditions.The comparative experiments do not show any obvious difference between the results obtained by this new method and those obtained by the classical complexometric titration method in seawater medium.This method exhibits good reproducibility and high accuracy in the determination of calcium and magnesium and can be used for the simultaneous determination of Ca2+ and Mg2+ in tap water and natural water.     

Enhanced Biodegradation of High-Salinity and Low- Temperature Crude-Oil Wastewater by Immobilized Crude-Oil Biodegrading Microbiota

High salt and low temperature are the bottlenecks for the remove of oil contaminants by enriched crude-oil degrading microbiota in Liaohe Estuarine Wetland(LEW),China.To improve the performance of crude-oil removal,microbiota was further immobilized by two methods,i.e.,sodium alginate(SA),and polyvinyl alcohol and sodium alginate(PVA+SA).Results showed that the crude oil was effectively removed by the enrichment with an average degrading ratio of 19.42-31.45 mg(L d)^?1.The optimal inoculum size for the n-alkanes removal was 10%and 99.89%.Some members of genera Acinetobacter,Actinophytocola,Aquabac-terium,Dysgonomonas,Frigidibacter,Sphingobium,Serpens,and Pseudomonas dominated in crude-oil degrading microflora.Though the removal efficiency was lower than free bacteria when the temperature was 15℃,SA and PVA+SA immobilization im-proved the resistance to salinity.The composite crude-oil degrading microbiota in this study demonstrated a perspective potential for crude oil removal from surface water under high salinity and low temperature conditions.     

Bioflocculant Produced by Klebsiella sp. MYC and Its Appli-cation in the Treatment of Oil-field Produced Water

1IntroductionWater co-produced with oil and gas recovery istermed oil-field produced water.This water is consid-ered to be of the largest volume in the waste streamfromoil and gas production processes.The volume ofproduced water can be more than10ti mes t…     

Bioaccumulation of Pb<Superscript>2+</Superscript> and its effects on growth,morphology and pigment contents of <Emphasis Type="Italic">Spirulina</Emphasis> (<Emphasis Type="Italic">Arthrospira</Emphasis>) <Emphasis Type="Italic">platensis</Emphasis>

A laboratory experiment was conducted to assess the bioaccumulation of Pb²⁺ and its effects on growth, morphology and pigment contents of Spirulina (Arthrospira) platensis. The specimen cultured in Zarrouk liquid medium was treated with various initial metal concentrations (0, 5, 10, 30, 50 and 100 μg mL⁻¹). The growth of S. platensis was adversely affected by Pb²⁺ at high concentrations (30, 50 and 100 μg mL⁻¹). However, at low concentrations (5 μg mL⁻¹), Pb²⁺ could stimulate its growth slightly. The pigment contents (chlorophyll α and β carotene) were decreased in a dose-dependent manner. The highest reductions (67% and 53% respectively in chlorophyll α and β carotene) were observed in 100 μg mL⁻¹ treatment group. The LC₅₀ (96 h) of Pb²⁺ was measured as 75.34 μg mL⁻¹. Apart from a few cases of filament breakages at elevated concentrations (50 and 100 μg mL⁻¹), morphological abnormalities are not specific. Metal bioaccumulation increased with Pb²⁺ concentrations, but decreased with exposure time. The maximum accumulated amount was 188 mg g⁻¹ dry weight. The bioconcentration factor (BCF) reached to a peak at day 2, followed by a gradual reduction for all the exposure concentrations. S. platensis is able to tolerate considerably high Pb²⁺ concentrations. Consequently it can be used as a potential species to remove heavy metal from contaminated waters.