副溶血性弧菌细胞猝灭及代谢物组提取方法的优化

以气相色谱-质谱联用(GC-MS)为分析方法,比较了液氮和75%甲醇(-80℃)两种溶液对副溶血性弧菌细胞的猝灭效果,以及氯仿、75%冰甲醇、水、甲醇-氯仿-水(10∶3∶1,V/V)、甲醇-氯仿-水(3∶1∶1,V/V)、甲醇-氯仿(1∶1,V/V)6种提取剂对副溶血性弧菌代谢物组的提取效果。结果表明,用75%甲醇(-80℃)猝灭副溶血性弧菌时,出现了代谢物泄漏现象,而液氮猝灭则不存在这个现象;检索发现,采用75%冰甲醇提取获得了47种代谢物,峰面积标准偏差为8.02%,其它5种提取剂获得代谢物种类少于40种,且重现性差。比较色谱峰数量、面积和重现性后发现,液氮猝灭、75%冰甲醇提取适于副溶血性弧菌代谢物组提取。

荜茇乙醇提取物在牙膏中的防龋应用

《本草纲目》记载荜茇具有治疗牙痛之功效。研究了荜茇乙醇提取物在牙膏中的应用,介绍了荜茇中胡椒碱的提取方法、牙膏中有效成分胡椒碱的检测,并用抑制变形链球菌的替代实验法进行了防龋测试,初步证明荜茇乙醇提取物在牙膏中作为防龋剂是可行的。

A Method Suitable for Extracting Genomic DNA from Animal and Plant——Modified CTAB Method

[Objective] The study aimed to introduce a rapid and effective method that is suitable for extracting genomic DNA from animal and plant. [ Method ] The genomic DNAs were extracted from tender leaves of 24 peanut cuhivars and from the liver, lung and kidney of white mouse through the specifically modified CTAB method. The DNAs were run on agarose gel, next detected by DNA/Protein analyzer. Finally PCR amplification was conducted to detect the quality of DNAs extracted using the modified CTAB method. [ Result] The clear and orderly bands were observed in gel detection, and the values of OD200/OD200 for DNAs extracted via modified CTAB method were between 1.77 - 1.83. The DNAs performed well in PCR amplification. [ Conclusion] The DNAs extracted by modified CTAB method could satisfy the requirement of PCR amplification.

A simple and rapid method for extracting bacterial DNA from intestinal microflora for ERIC-PCR detection

AIM: To develop a simple and convenient method for extracting genomic DNA from intestinal microflora for en- terobacterial repetitive intergenic consensus (ERIC)-PCR detection. METHODS: Five methods of extracting bacterial DNA, including Tris-EDTA buffer, chelex-100, ultrapure water, 2% sodium dodecyl sulfate and 10% Triton-100 with and without sonication, were compared with the commercial fecal DNA extraction kit method, which is considered as the gold standard for DNA extraction. The comparison was based on the yield and purity of DNA and the indexes of the structure and property of micro-organisms that were reflected by ERIC-PCR. RESULTS: The yield and purity of DNA obtained by the chelex method was similar to that obtained with the fecal DNA kit. The ERIC-PCR results obtained for the DNA extracted by the chelex method and those obtained for DNA extracted with the fecal DNA kit were basically the same.CONCLUSION: The chelex method is recommended for ERIC-PCR experiments in view of its simplicity and cost- effectiveness; and it is suitable for extracting total DNA from intestinal micro-organisms, particularly for handling a large number of samples.

Behavior of different phosphorus species in suspended particulate matter in the Changjiang estuary

Suspended particulate matter （SPM） collected in the Changjiang （Yangtze River） estuary in June 2006 was separated into five fractions via water elutriation： clay-very fme silt （〈8 μm）, fine silt （8-16 μm）, medium silt （16--32 μm）, coarse silt （32~53 μm） and sand （〉63 μm）. The SPM and fractionated particles were sequentially analyzed by a modified SEDEX sequential extraction method to obtain six species of phosphorus： exchangeable or loosely-sorbed P, organic P, Fe-bound P, authigenic P, detrital P and refractory P. The results indicated that all particulate phosphorus species except for detrital P were negatively correlated to particle size; a high detrital P content was found in coarse silt and very coarse silt. From the inside of the river mouth to the gate of the fiver mouth, organic P, Fe-bound P and refractory P in the suspended particles decreased and a higher amount of exchangeable P appeared around the gate of the fiver mouth. From the gate of the river mouth to the sea, exchangeable P and organic P in suspended panicles increased distinctly. The total particulate P flux into the estuary from the Changjiang River was about 45.45×10^8μmol/s during sampling. Of this, about 8.27×10^8μmol/s was associated with the ＂truly suspended＂ fraction. The bio-available particulate P flux was about 13.58×10^8μmol/s. Of this, about 4.24 ×10^8μmol/s w as transported by ＂truly suspended＂ particles.

Identification of Volatile Compounds in Codfish(Gadus) by a Combination of Two Extraction Methods Coupled with GC-MS Analysis

Codfish is a kind of abyssal fish species with a great value in food industry. However, the flavor of codfish, especially the unpleasant odor, has caused serious problems in its processing. To accurately identify the volatile compounds in codfish, a combination of solid phase micro-extraction(SPME) method and simultaneous distillation extraction(SDE) method was used to extract the volatiles. Gas chromatography-mass spectrometry(GC-MS) along with Kovats indices(KI) and authentic standard compounds were used to identify the volatiles. The results showed that a total of 86 volatile compounds were identified in codfish, of them 24 were extracted by SDE, 69 compounds by SPME, and 10 compounds by both SDE and SPME. Seventy volatile compounds were found to have specific odors, of them 7 typical compounds contributed significantly to the flavor of codfish. Alcohols(i.e.,(E)-2-penten-1-ol and 2-octanol), esters(i.e., ethyl butyrate and methyl geranate), aldehydes(i.e., 2-dodecenal and pentadecanal) contributed the most to fresh flavor while nitrogen compounds, sulphur compounds, furans, as well as some ketones(i.e., 2-hydroxy-3-pentanone) brought unpleasant odor, such as fishy and earthy odor. It was indicated that the combination of multiple extraction methods and GC-MS analysis can enhance the accuracy of identification, and provide a reference for the further study on flavor of aquatic products.

The extraction of pigments from fresh Laminaria japonica

The pigments in Laminaria japonica 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 2h; (3) the average total content of pigments was 1.85 mg/g (calculated with dry L. japonica).

Improvements on environmental DNA extraction and purification procedures for matagenomic analysis

Our previously described environmental DNA extraction method has been widely used in environmental microbial community analysis. However, residual humic substances may remain with obtained environmental DNA, which interferes downstream molecular analyses. To remedy this situation, two DNA extraction buffers （PIPES and Tris-HCl） and four purification strategies including our new modified low melting point gel purification method and three commercial kits from QIAEX, Omega and Promega were evaluated with diverse soil samples. The PIPES buffer （pH 6.5） is found to be more effective for removing the humic substances, but it leads to lower DNA yield and causes more severe DNA shearing than using the Tris-HC1 buffer （pH 8.0）. Gel purification and the Promega purification kit achieve much higher DNA recoveries than QIAEX or Omega kit, and higher purity of DNA is obtained by gel purification than by the Promega kit with both DNA extraction buffers mentioned above. Considering all results together, two alternative methods for DNA extraction and purification are proposed： one uses Tris-HCl buffer extraction and gel purification as the primary approach when the amount of soil or biomass is not a major concern, and the other uses PIPES buffer extraction and the Promega kit purification when severe DNA shearing and/or limited biomass occurs. Purified DNA samples by both methods are amenable for use as templates for whole community genome amplifications and PCR amplifications of bacterial 16S rRNA genes. It is demonstrated that these two alternative methods could be applied to a wide variety of environmental samples.

Comparison of Different Extraction Approaches for Heavy Metal Partitioning in Sediment Samples

Three extraction methods, ultrasonic assisted extraction (USE), microwave assisted extraction (MSE), and conventional single extraction (CSE), in conjunction with the modified three-stage BCR sequential extraction procedure (SEP) were applied to examine the contents of Cd, Cu, Cr, Ni, Pb and Zn from lake sediment samples, to know whether these techniques can reduce extraction time and improve reproducibility. The SEP and developed alternative single extrac- tion methods were validated by the analysis of certified reference material BCR 601. By the use of optimized sonication and microwave conditions, steps 1, 2 and 3 of the BCR sequential extraction methods (excluding the hydrogen peroxide digestion in step 3, which was not performed with sonication and microwave) could be completed in 15-30 min and 60- 150 s, respectively. The recoveries of total extractable metal contents in BCR 601, obtained by three single extractions ranged from 93.3%-102%, 88.9%-104% and 81.2%-96.2% for CSE, USE and MSE, respectively. The precision of the single extraction methods was found in the range of 3.7%-9.4% for all metals (n = 6).

A marked enhancement in production of amylase by Bacillus amyloliquefaciens in flask fermentation using statistical methods

A total of 126 bacterial strains were isolated from soil samples. Among them, 11 isolates were found positive for amylase production. Strain YL produced the largest zone of clearance on plate assay. The isolate YL was identified as Bacillus sp. based on morphological and physiochemical characterization. According to 16S rRNA gene sequencing data, the closest phylogenetic neighbor of strain YL was Bacillus amyloliquefaciens （99.54%）. After that, an optimization of culture conditions was carried out for the improvement of a-amylase production. Response surface methodology （RSM） was applied to evaluate the effect of medium components including wheat bran, cottonseed extract, yeast extract, starch, NaC1 and CaCl2. Three variables （wheat bran, cottonseed extract, and starch）, which were identified to significantly affect amylase production by Plackett-Burman design were further optimized using response surface methodology of Box-Behnken design （BBD）. The optimal concentrations estimated for each variable related to the maximum of amylase activity （86 kU/mL） were 10.80 g/L wheat bran, 9.90 g/L cottonseed extract, 0.5 g/L starch, 2.0 g/L yeast extract, 5.00 g/L NaCl and 2.00 g/L CaC12. The fermentation using optimized culture medium allowed a significant increase in amylase production （by 3-fold）. The improvement in the a-amylase production after optimization process can be considered adequate for large-scale applications.

Anti-promastigote Activity of the Amazon Plants

This study evaluated in vitro activity of ethanol extract, fractions, and isolated substance from Amazon species against promastigotes of L. amazonensis. The ethanol extracts were concentrated and fractionation. The anti-promastigote activity was evaluated through the cell viability assessment method （MTT）. The ethanol extract, fractions, and isolated substance from Himatanthus articulatus and Parahancorniafasciculata were inactive in promastigote ofL. amazonensis, as the ethanol extract ofPhysalis angulata. The hexane fractions from different parts ofMontrichardia linifera showed anti-promastigote activity probably due to the presence of steroids and terpenes. All species in studies were inactive, except ofM. linifera. The few polar constituents can be responsible for the activity. Therefore, the isolation and purification of the active on L. amazonensis promastigotes are urgently required.

Variations in Laboratory-Scale Actinomycete Communities Exposed to Cadmium as Assessed by Denaturing Gradient Gel Electrophoresis Profiles

The actinomycete populations and functions in cadmium （Cd） contaminated soil were investigated by the cultivation- independent molecular methods. The genomic DNA was extracted and purified from soil adulterated with various con- centrations of Cd in the laboratory. The partial 16S rDNA genes were amplified by polymerase chain reaction （PCR） using specific primers bound to evolutionarily conserved regions within these actinomycete genes. The diversity in PCR- amplified products, as measured by denaturing gradient gel electrophoresis （EGGE）, was used as a genetic fingerprint of the population. Principle component analysis and Shannon-Weaver diversity index （H） analyses were used to analyze the DGGE results. Results showed that the two principal components accounted for only a low level of the total variance. The value H in contaminated soil was lower than that in the control at later stages of cultivation, whereas at earlier stages it was higher. Among the six sampling time points, the first, fifth and sixth weeks had the highest values of H. Significantly negative correlations between bioavallable Cd concentration and H values existed in the samples from weeks 2 （R = 0.929, P 〈 0.05） and 4 （R = 0.909, P 〈 0.05）. These results may shed light on the effect of Cd on the soil environment and the chemical behavior and toxicity of Cd to actinomycetes.

Validation of Monomeric Anthocianin Determination Method for Bilberry Juice and Marc Extracts

The solid waste generated in industrial berry juice production was considered as a low cost raw material for the extraction of natural antioxidants. Berries contain phenolic compounds with high antioxidant potential, including anthocianin. Quantitative determination method for monomeric anthocianins in bilberry juice and marc was validated. An official method from Association of Analytical Communities was used to determine anthocianins in juice and marc extracts by measuring light absorption for solutions with pH values 1.0 and 4.5 at 520 nm and 700 rim. Results were expressed as cyanidin-3-glucoside equivalent, as it is the most common anthocianin pigment. Calibration curve was obtained, linearity was checked, working interval and accuracy was determined and samples were tested. Mentioned method was evaluated as appropriate for quantitative anthocianin analysis in bilberry juice and marc. Necessity of calibration curve was approved using extinction coefficient of cyanidin-3-glucoside instead. Method assures adequate precision and accuracy as well.

Investigation of Biochemical Properties and Fractional Composition of Amaranth Oil

The aim of the study is to conduct comprehensive experiments and developments of innovative technologies to produce the oil extract of amaranth for their application as food additives in the food industry. To achieve the aim, the following tasks were identified： （I） study the physicochemical, biochemical properties and the composition of amaranth seeds; （2） improving the methods for producing the oil extract of amaranth. This article is devoted to complex research of processes of manufacture of vegetable oils. Authors have developed scientific bases of improvement and an intensification of technological processes for production of oil from amaranth seeds. The authors have developed a new technology for extracting of oil from amaranth seeds using hexane as a solvent. The proposed method provides to obtain oil without great technical effort and keeps it all the nutrients that promote the highest taste of the product and with little labor and time costs.

A Comparative Study of Soluble Protein Extractions of Populus deltoides × （ Trichocarpa × Deltoides） for 2-DE

Background： The disclosure of the poplar genome strengthens its position as well-established model organism. Populus has been subject of several proteome studies, but up to date no comparative study was performed on the extraction method of soluble proteins for this species. The extraction is the most critical step in two-dimensional gel electrophoresis and each extraction method has its advantages, disadvantages and limitations. Therefore protein extraction methods should be optimized for each tissue before starting an experimental setup. In prospect of future DIGE （Differential Gel electrophoresis） experiments for the investigation of the effects of cadmium and inoculation with plant growth promoting bacteria at the proteome level, the aim of this study was to optimize an extraction method for soluble proteins of poplar leaves and roots. Results： The acetone-phenol extraction method was found to be the most suited, rendering a high spot number and low background interference. During further optimization, several critical steps in the extraction method were revealed. Conclusion： Aiming to optimize the extraction of soluble leaf and root proteins of Populus deltoides × （trichocarpa× deltoides） compatible with DIGE analysis, a protocol rendering high reproducibility, low background interference and a high spot number was established, however no novel insights were acquired.

Comparison of alternative extraction methods for secretome profiling in human hepatocellular carcinoma cells

Secreted proteins are important sources for early detection and diagnosis of disease, and as such have received considerable attention. The extraction of low concentration proteins from large volumes of culture media, which are rich in salts and other compounds that interfere with most proteomics techniques, presents a problem for secretome studies. Ultrafiltration, precipitation, and dialysis are three major extraction methods that can be used to overcome this problem. The present study for the first time, compared the merits and shortcomings of these three methods, without bias. Centrifugal ultrafiltration provided the best extraction efficiency, and precipitation provided the highest number of identifiable proteins. The three methods yielded closely related, but different, information on the secretome; thus, they should be considered complementary or, at least, supplementary methods. Three hundred and sixty unique proteins were identified, including 211 potential secreted proteins. Compared with previous studies, this study also identified 42 new secreted proteins. The present study not only offers a reference for the selection of secretome extraction methods, but also expands the secretome database for the investigation of hepatocellular carcinoma.