Isolation,Identification and in vitro Antimicrobial Susceptibility of Pathogenic Aeromonas veronii from Soft-shelled Turtles

In order to effectively control diseases caused by Aeromonas veronii,pathogenic bacteria were isolated and incubated from infected soft-shelled turtles with traditional bacterial isolation method. Four strains of pathogenic bacteria were isolated and identified with traditional biochemical identification method and modern molecular biological identification techniques. According to the results, four strains of pathogenic bacteria were identified as A. veronii biovar sobria. Drug sensitivity test and in vitro antimicrobial test against Bacillus amyloliquefaciens were performed with agar diffusion method. The results showed that B. amyloliquefaciens and several antibiotics such as ofloxacin and gentamicin exerted strong antimicrobial effects on four isolates. B. amyloliquefaciens could be used for the prevention and control of diseases caused by A. veronii in aquaculture.

A Simple Method for the Isolation and Purification of 2,4-Dihydroxy-7-Methoxy-2H-1,4-Benzoxazin-3(4H)-One (DIMBOA) from Maize (Zea mays L.) Seedlings

2,4-Dihydroxy-7-methoxy-2H-1,4-benzoxazin-3（4H）-one （DIMBOA）, the dominant benzoxazinoid hydroxamic acid in maize （Zea Mays L.）, serves as important factors of resistance against insects and microbial diseases, allelochemicals used in competition with other plants. In this paper, a novel and simple method for the isolation and purification of DIMBOA from maize seedlings was developed. Frozen shoots from 7-d-old maize seedlings （1 000×g） were firstly defrosted and then were directly homogenized and extracted with ethyl acetate. The macerate was allowed to stand at room temperature （25±2）°C for 1 h to allow enzymatic release of DIMBOA from DIMBOA-glucoside. Then the ethyl acetate phase was filtered, dried and evaporated to dryness. The resulting light-tan, semicrystalline residue was stored at -20°C for 24 h. Upon recrystallization from acetone-hexane, a relative higher yield （0.58 g） of pure DIMBOA crystals was obtained compared with the yield afforded by Woodward methodology （0.26 g）.

Isolation, Identification and Antimicrobial Test in vitro of Aeromonas from Hemibarbus maculatus Bleeker

[Objective] The aim was to quickly find high free drugs to prevent Hemibarbus maculates Bleeker from dying. [Method] The conventional biochemical methods and molecular biological identification method was used to do the isolation and identification of bacteria from H. maculates, and agar diffusion method was used for the susceptibility test and in vitro becteriostasis experiment. The toxins were detected using the plate method, and the intraperitoneal injection method was used to do animal experiments. [Result] Twelve strains of isolated bacteria were obtained, which could uniformly decompose glucose, maltose, but were unable to break down lactose, and they could reduce the nitrate, and were negative in hydrogen sulfide and indole. A clear band was found in 685 bp of the isolated bacteria. And the isolated bacterial all generated hemolysins. Some strains produced protease. The isolated bacterial were resistant to penicillin G, amoxicillin, but sensitive to gentamicin, ciprofloxacin, and could be inhibited by the Bacillus amyloliquefaciens. All experimental animals died within 12 hours. [Conclusion] The 12 isolates were Aeromonas veronii, Aeromonas caviae and Aeromonas hydrophila, respectively, and the deaths of H. maculates were caused by the mixed infection of Aeromonas veronii, Aeromonas caviae and Aeromonas hydrophila. The first choice drug for the treatment was ciprefloxacin, and Bacillus amyloliquefeciens could be used as the drug for ecological prevention and control.

Isolation, Purification and Cryopreservation of Cells from Neonatal Bovine Testis

The development and application of spermatogonial stem cell technology have an important significance in animal cloning, preservation of endangered species and spermatogenesis research. In this study, the seminiferous epithlium cells were isolated and purified, the ceils were cryopreserved after identification, and the effects of different purification and cyopreservation methods on bovine testicular cells were studied. The results showed that there were spermatogonial stem cells and sertoli cells in the neonatal bovine seminiferous tubules, differential adherent selection methods could effectively separate these two cell types. Spermatogonial stem cells were positive after AKP, C-kit, and OCT-4 identification; sertoli ceils were positive after oil red O and vimentin identification. Frozen stock solution supplemented with 10% DMSO had the best effect in spermatogonial stem cell cryopreservation, while fxozen stock solution supplemented with 10% of ethylene glycol and 0.1 mmol. L^-1 trehalose had the best effect in sertoli cells crvooreservation.

Isolation and Purification of Unstable Iridoid Glucosides from Traditional Chinese Medicine by Preparative High Performance Liquid Chromatography Coupled with Solid-phase Extraction

An efficient preparative method was successfully developed for isolation and purification of unstable components from medicinal plant extracts, using a combined method of preparative high performance liquid chro-matography(HPLC) and solid-phase extraction(SPE). The aim of this study was to obtain an effective method with high preparative efficiency and importantly to avoid the transformation of unstable compounds. The preparative HPLC system was based on an LC/MS controlled four-channel autopurification system. The SPE method was performed with a C18 packing material to trap the target compounds and to remove the acidic additive derived from the mobile phase. Using this method, the unstable iridoid glucosides(IGs) as model compounds were successfully isolated and purified from the extract of Hedyotis diffusa Willd. Six IGs(including one new minor IG) and one nucleotide compound were simultaneously obtained, each with a purity of 91% as determined by HPLC. The structures of the isolated compounds were identified by UPLC/Q-TOF MS, UV, 1D and/or 2D NMR. It was demonstrated that the combination of preparative HPLC with SPE is a versatile tool for preparative purification of unstable compounds from complex natural products.

Isolation and Purification of Myxobacteria in Chengdu

[ Objective] To lay the basis for further studying bioactive substances in myxobacteria. [ Method] A total of 35 samples including fresh water, mud, rabbit dung, sheep dung, weathered rock and rotten wood were collected from districts and counties of Chengdu. After pretreatment, the myxobacteria were isolated and purified using different methods. Vegetative cells, myxospores, fruiting body and colony morphology of these myxobacteria strains were observed. E ResultJ Forty-two myxobacteda strains were isolated and purified. They were classified into seven genera including Myxococcus, Angiococcus, Chondromyces, Sorangium, Melittangium, Stigmatella and Archangium. The plating efficiency of myxobacteria was highest in the fresh water samples. [ Conclusion] Abundant myxobacteria resource is found in fresh water.

Isolation and Preparative Purification for Ginkgolides A and B

In this paper a simple preparative method for isolation and purification of ginkgolides A and B was developed,As starting material,a commercially available standardized ginkgo extract (EGb761,containing 24% flavonoid and 6% terpene trilactones) was used,After a pretreatment step,optimized by the uniform design method ,the concentrated intermediate extract with high content of GA and gb(+90%) was separated into the individual terpenes by preparative liquid chromatography eluted with petroleum ether-ethylacetate,Analysis of products was carried out by means of HPLC-ELSD(evaporative light -scattering detector),The results show that ginkgolides A and B are obtained in higher yield and better purity.

STUDIES ON THE ISOLATION PURIFICATION AND CHARACTERIZATION OF THE FUCOIDAN-GALACTOSAN SULFATE (FGS) FROM LAMINARIA JAPONICA

FGS, isolated from the water solution of enzymolyzed laminaria japonica, is a mixture of acid heteroglycans. Four fractions F1, F2, F3, and F4 were obtained from FGS by ion exchange chromatography. After further purified by gel filtration chromatography on a sepharose 2B and 6B column, we obtained F9, F10, F11, and F12. They showed single band when identified by electrophoresis. The molecular weight of F9, F10, F11, and F12 was estimated to be 216, 120, 138 and 140KD respectively. They containedа-glucosidic bond by IR and 1H-NMR analysis. The typical absorption peaks of these polysaccharides were showed in UV and IR spectra. These polysaccharides contained rha, fuc, man, gal, and uronate when identified by paper chromatography (p.c) and gas chromatography (GC). The molar ratio of these sugars was also assayed.

The Isolation, Purification and Identification of Fumitremorgin B Produced by Aspergillus fumigatus

Twenty-six strains of Aspergillus fumigaius were screened for toxigenicity for fumitremorgins A and B. Twenty-three of 26 strains can produce fumitremorgin B in rice medium determined by TLC and HPLC, and no fumitremorgin A was detected. The strains of no.C4104 and no. 3656 were inoculated onto 5 kg of rice media and incubated in a modified procedure. Finally, 4.0 g of fumitremorgin B was obtained after extraction and purification by modified methods, and was confirmed by TLC, HPLC, spectral analysis together with other physicochemical analysis. This is the first report of the preparation of fumitremorgin B in China.

Isolation,Purification and Immunomodifying Activity of Polysaccharides from Phellinus igniarius

Phellinus igniarius polysaccharides were obtained by hot water extraction of mushroom fruit bodies followed by ethanol precipitation,and further purified by anion exchange and gel filtration chromatography.Immuno-enhancing activity was evaluated by determining the effect of different polysaccharide fractions on mouse spleen lymphocyte proliferation in vitro.Three crude polysaccharide preparations,designated PIP30,PIP60 and PIP80 according to the percentage concentration of ethanol required for precipitation from hot aqueous extracts,promoted lymphocyte proliferation to varying degrees.Purification of polysaccharide present in PIP30 and PIP60 was carried out using anion exchange chromatography.Proliferation rates in samples treated with 200 μg/mL of fractions P30U,P30W and P30S1(obtained following anion exchange chromatography of PIP30) were approximately 5-,4-,and 3.5-fold higher,respectively compared with negative controls.Similarly,marked increases(3.4-and 2.8-fold) in cell proliferation rates compared with negative controls were observed with 200 μg/mL concentrations of P60W and P60S2(obtained following anion exchange chromatography of PIP60),respectively.Two purified polysaccharide preparations,P60W1-1 and P60S1-1,were obtained following gel filtation chromatography of fractions P60W1 and P0S1,respectively.Fraction P60W1-1(10-100 g/mL) enhanced splenocyte proliferation 0.5-1.4-fold compared with negative controls,whereas no effects were observed with P60S1-1.

Isolation and Purification of Bovine Colostrum sIgA and IgG

To further utilize bioactive substance such as bovine colostrum sIgA and IgG, sIgA and IgG were isolated and purified simultaneously by salting out, ultrafiltration and gel chromatography, etc. The analysis of results were showed quantitatively by nonhydrogenized SDS-PAGE, and quanlities of sIgA and IgG were respectively detected by Western Blot. The results showed that the purity and yield of bovine colostrum sIgA were 85.3% and 42.8%, respectively, while the purity and yield of bovine colostrum IgG were respectively 97.2% and 64.4%. This preparative method provides theoretical and experimental foundation for sIgA industrial production.

Isolation and Purification of Antibacterial Peptides from Mussel

[ Objective ] This study aimed to extract antibacterial peptides from mussel. [ Method ] Blue mussels were used as raw materials for direct extraction of antibacterial peptides by using O. 5 % acetic acid, and the antibacterial peptides were isolated and purified by Sephacryl S-100 polyacrylamide gel chromatography. The fractions were collected for measurement of antibacterial activity and minimum inhibitory concentration for a variety of bacterial species by filter paper diffusion assay. Molecular weight of the antibacterial peptides was determined by SDS-PAGE. Variation of antibacterial activity of antibacterial peptides was measured at 100 ~C under conditions of different processing time and different pH. [ Result] The O. 5% acetic acid was used for crude extraction of antibacterial peptides as extrac- tion solution and led to relatively high extraction efficiency. By using Sephacryl S-100, the antibacterial peptides could be purified as a single substance. The isola- ted and purified antibacterial peptides of mussel had relatively strong antibacterial properties with molecular weight of 5 908, showing heat-resistance acid-alkaline resistance. [ Conclusion] This study laid the theoretical foundation for large-scale production of antibacterial peotides.

Isolation and Purification of Enzymatic Hydrolysates of Yam(Dioscorea opposita Thunb.)Proteins

Using yam （Dioscorea opposita Thunb. ） as the experimental material, enzymatic hydrolysates of yam proteins were prepared with alkaline protease, which were then isolated and purified by cellulose DE-52 anion exchange chromatography Sephadex G-50 gel chromatography. According to the results, four absorp- tion peaks were obtained by cellulose DE-52 anion exchange chromatography, and fractions at each absorption peak were collected. Specifically, the reducing ability of four peaks demonstrated a descending order of peak 2 （P2） 〉 peak I （ P1 ） 〉 peak 3 （P3） 〉 peak 4 （P4）. By Sephadex G-50 gel chmmatography, P1 and P2 were isolated and purified with distilled water and Tris-HC1 buffer, and two absorption peaks were obtained, respectively.

AN EFFICIENT METHOD FOR ISOLATION AND PURIFICATION OF THE PANCREATIC ISLETS

On the base of the one step, operator independent method which was set up by Christophe A.E., the pancreas was infused with cold University of Wisconsin(UW) solution for the preservation, digested by the collagenase P, circuited with HBSS+5%fetal calf serum(FCS)+10mmol/L Hepes solution, and separated with the stainless steel mesh. The number of the collected islets were 400000～1800000 per pancreas, i.e. about 12150/g pancreas. After purification, the recovery was 350000～1700000 per pancreas, i.e. about 10250/g pancreas, the recovery rate was above 80%, and the purity of the final preparation was above 95%. The insulin secretion in the response to the high concentration glucose (22 mmol/L) stimulation was apparently different on the 1,3,5 day of the cultural islets, which the high level of insulin was three times the low level (5.5 mmol/L) on the 5th day, and the insulin level of the double stimulation under perfusion conditions is apparently higher than low glucose. The result demonstrated that the purified islets were functionally alive. Histological studies also show that the shape of islets are complete, and the β cell was specially stained by the dithizone (DTZ). The Trypan Blue staining had shown the living cell was above 90%. In conclusion, the new method was highly practical and yielded higher concentration of active pancreatic islets.

ISOLATION AND PURIFICATION OF METHANE IVf ONOOXYGENASE FROM METHYLOMONAS SPECIES GYJ3

A three—component enzyme system that catalyzes in vivo the oxidation of CH_4 to CH_3OH has been purified with high specific activity from an unusual type I methanotroph through the use of stabilizing reagents.

A Review on the Isolation and Purification of D-allulose

D-allulose has very little content in nature,and it needs to be synthesized artificially and meet the purity requirements of industrial grade.The basic physical and chemical properties of D-allulose,its preparation methods and many different ways of isolation and purification were described.In order to achieve the goal of industrial production of D-allulose as soon as possible,the research progress of D-allulose isolation and purification technologies at home and abroad in recent years was classified and discussed,so as to provide useful reference for the practical improvement of D-allulose isolation and purification process technologies.

Apoptosis and oxidative injury of donor islets during isolation and purification

Objective To observe the changes of islet cell apoptosis and oxidation - antioxidation before transplantation,and to explore pathways of islet protection. Methods Fifteen human pancreases were perfused with Hanks solution containing collagenase,then digested and isolated.

Study on Purification and Properties of Antibacterial Substance from Antagonistic Bacteria P72 against Wheat Scab

[Objective] The study aimed to explore the antibacterial activity and stability of antagonistic bacteria P72 against wheat scab.[Method] The Bacillus subtilis P72 was inoculated into fermentation media with the inoculation amount 5%,then cultured on shaking table at 28 ℃ for 48 h and centrifuged at 8 000 r/min for 10 min.The supernatant of fermentation broth was purified and then the genetic stability,thermal stability and pH stability were detected.[Result] By DEAE-52 ion exchange chromatography,the protein eluted by 0.5 mol/L NaCl solution possessed the strongest antagonistic ability against wheat scab pathogen.The SDS-PAGE electrophoresis showed that the molecular weight of the purified protein with antibacterial effect was 40 kD.By stability test,the antibacterial substance produced by P72 strain showed heritable antagonistic activity,high stability below 60 ℃,stability to acid and unstability to alkali.[Conclusion] The antagonistic bacteria P72 had strong antagonistic ability to wheat scab pathogen,stable antibacterial activity and thermal stability,so it would possess a wide development prospect.

Isolation of Plasma and Thylakoid Membranes from the Heterocystous Cyanobacterium Anabaena sp. PCC 7120

利用水溶性多聚体双相法分离蓝细菌Anabaenasp .PCC 712 0质膜和类囊体膜两种膜系统。吸收光谱分析表明 ,质膜相和类囊体膜相的主要色素分别为类胡萝卜素和叶绿素。SDS_凝胶电泳显示这两种膜系统蛋白组成有很大差别。这种分离方法容易操作 ,对研究蓝细菌的膜蛋白和膜脂非常有用。

Extraction and purification of TGFβ and its effect on the induction of apoptosis of hepatocytes

AIM To extract and purify the transforming growth factor β (TGF β), and to demonstrate its biological activity in vivo and induction of apoptosis of hepatocytes in vitro.METHODS TGF β was isolated from fresh bovine platelets by acid/ethanol extraction method and purified with ion exchange and gel chromatography. The extracted TGF β was injected subcutaneously to mice, and its biological activity in vivo was observed 72 hfs post-injection by HE staining. The morphological changes were observed by HE staining and the occurrence of apoptosis was detected by TUNEL method after the human normal hepatic cell line QZG was treated with 8 μg@L 1 TGFβ for 12 hrs in vitro.RESULTS The molecular mass 25 ku TGF β protein was successfully extracted. It was able to induce localized granulation tissue formation in vivo. TGF β-treated hepatocytes showed obvious apoptotic morphological changes, including the pyknosis and dense-stained nuclei and cytoplasm, the fragmentary, annular or crescent nuclei, and the "bubbling" cytoplasm. Moreover, its apoptotic rate was significantly higher than that of the control group (P＜0.05).CONCLUSION Biological active TGF β protein is extracted and purified successfully from bovine platelets, and it is able to induce the apoptosis of hepatocytes.