Polycyclic aromatic hydrocarbons (PAHs), a kind of persistent organic pollutants in soil, are of great concern due to their carcinogenic, mutagenic and teratogenic characteristics. PAHs are mainly derived from incomplete combustion processes and pyrolysis of organic materials. The occurrence, source, transport and fate of PAHs in various environments have been reported extensively, while the determination method of PAHs varied in the past reports. Thus the accurate analysis of PAHs is a vital step for further research. The PAHs are usually extracted and purified by organic solvent prior to determination. In order to improve determination accuracy and recovery rate, the pretreatment conditions for the determination of 16 PAHs in paddy soils are of great significance.
On the basis of former work, in order to get the highest extraction efficiency and recovery rate, we compared two different extract solvent mixtures, four different clean-up columns and three different elution solutions, and also compared the elution volume and temperature of nitrogen blowing to optimize the determination method for trace PAHs in paddy soils under the laboratory condition. Soil samples were taken from paddy field in Wenling City of Zhejiang Province, and the polluted soil samples were made by adding PAHs standard solution. The analysis of target compounds was performed by gas chromatography-mass spectrometry (GC-MS).
The results showed that the soil samples were extracted ultrasonically three times with V(n-hexane):V(acetone)=1:1 mixture, followed by clean-up with C18 solid phase extraction (SPE) column and 8 mL of V(n-hexane):V(dichloromethane)=7:3 mixture elution solution, and the nitrogen blowing temperature was 20 ℃, which was the optimal pretreatment procedure. The correlation coefficients (R²) for the tested 16 PAHs were 0.999 0-0.999 9 within the range of 10-1 000 μg/L. The method detection limits were in the range of 0.022-0.470 μg/kg. The average recovery rates of the spiked samples ranged from 70.2%-110.8% with relative standard deviation (n=5) of 1.8%-9.8%.
In sum, the above results suggest that the established method in this study is accurate, sensitive and reliable; meanwhile, it can also reduce the cost of organic reagent. This method is suitable for the analysis of trace PAHs in paddy soils.

With the development of intensive livestock farming, a large number of heavy metals such as Cu and Zn are widely used in feed. However, the accumulations of heavy metals in animal manures have become serious environmental problems, which may even pose long-term threats to ecosystems and humans.
This study focused on the effects of different passivators and vermicomposting on fractionations of heavy metals (Cu and Zn) and their bioavailability during pig manure vermicomposting. In the plot experiment treatments, calcium-magnesium phosphate, bentonite, biochar, enzymes, EM bacteria, fly ash and zeolite were added to the pig manure as heavy metal passivators, while vermicomposting treatment without passivator was set as a control.
The results indicated that the biochar, enzymes, EM bacteria, fly ash and zeolite treatments had a passivation effect on Cu fractionation. Meanwhile, the bioavailable Cu was converted into immobilized Cu, and there was a significant difference between the treatment group and the control (P＜0.05). In terms of the distribution rates of available Cu and Zn, biochar was considered as the best passivator for available Cu with up to 42.34% reduction of the distribution rate, and enzymes were considered as the best passivator for available Zn (P＜0.05) with a maximum reduction (3.77%) of the distribution rate. The concentrations of various Cu forms in each fraction for the pig manure vermicompost were in the order of oxidizable Cu＞residual Cu＞reducible Cu＞exchangeable Cu, while the concentrations of various Zn forms represented in the order of exchangeable Zn＞reducible Zn＞oxidizable Zn＞residual Zn. Oxidizable Cu was the dominant speciation for Cu with a fraction up to 50%, while exchangeable and reducible Zn were the dominant speciations for Zn (＞80%) in the pig manure vermicompost. Based on the Community Bureau of Reference (BCR) sequential speciation analysis, the bioavailability of Cu and Zn decreased after vermicomposting. The effect of passivators was different on the bioavailability of Cu and Zn, which showed that Zn was mostly available while Cu was mostly unavailable to plants after vermicomposting. Therefore, more attention should be paid to the risk of environmental pollution caused by Zn in compost application.
In conclusion, this research provides theoretical and technical information on vermicomposting of pig manure, which is of great significance to pollution prevention and risk management for heavy metals.

Contamination by trace elements resulted from abandoned mines presented a serious environmental concern and posed a significant threat to the environment and human health. Consequently, there has been an increasing effort for developing cost-effective technologies for minimizing the mobility of trace metals and their bioavailability in contaminated mine-tailing soils. Although the mechanisms involved in immobilization of heavy metals using phosphorus amendments have been intensively investigated, the implementation of this technology in the field for remediation of soils and vegetables contaminated by lead and zinc mining tailings is limited.
In this study, a field demonstration of this control technology was conducted at lead and zinc mining tailings heavily contaminated by lead (Pb). The main objective of this field experiment was to evaluate the effects of three different kinds of phosphorus fertilizers on pH and in-situ heavy metal immobilization of the soil, including single superphosphate (SSP), phosphate rock (PR), and calcium-magnesium phosphate (CMP), observe the changes of water-soluble fractionation in the contaminated soil in relation with Pb accumulation by cabbage, and evaluate the feasibility using phosphorus fertilizers for in-situ immobilization of heavy metals in the contaminated soil.
The three phosphorus fertilizers were added to the soil at a phosphorus equivalent application rate of 50, 300 and 500 g/m², respectively. The correlation between soil pH and water-soluble heavy metals (Pb, Zn, Cu, and Cd), and the correlation between water-soluble heavy metals and heavy metals uptake in cabbage were elaborated in this study. The efficiency of the three different phosphorus fertilizers in decreasing the bioavailability of heavy metals in soil was also evaluated.
It was showed that the addition of different phosphorus fertilizers and (SSP, CMP and PR) could decrease the watersoluble heavy metals (Pb, Zn, Cu and Cd) and heavy metals uptake by cabbage, and also change the pH values of soil. A negative correlation was observed between the pH values in soil and water-soluble heavy metals (Pb, Zn, Cu and Cd). The addition of PR at a phosphorus equivalent application rate of 500 g/m² was the most effective in reducing the water-soluble Pb and Cd (both of the water-soluble Pb and Cd had 66.7% reduction), compared with the other treatments. The addition of CMP at a phosphorus equivalent application rate of 500 g/m² was the most effective in reducing the water-soluble Zn and Cu (the water-soluble Zn and Cu in soil had 97.1% and 88.9% reduction, respectively). The Pb in the cabbage was reduced most significantly with the addition of PR at a phosphorus equivalent application rate of 500 g/m², which had 62% reduction. The addition of CMP at a phosphorus equivalent application rate of 500 g/m² was the most effective in reducing the Zn, Cu and Cd in the cabbage (the Zn, Cu and Cd in the cabbage had 57.4%, 49.7% and 46% reduction, respectively).
In conclusion, it is effective and feasible to use phosphorus fertilizers for controlling accumulation of heavy metals in cabbages of contaminated mine-tailings, and CMP will be a more effective amendment.

Currently， developing genetic linkage map mostly use the derivedpopulations from crossing of two homogenous parents， which only covers limited genetic diversity and is inappropriate for some species， such as tobacco with lower diversity in genome. It is very general that there are no sufficient polymorphic markers to construct linkage map and ineffective to conduct marker-assisted selection （MAS） and quantitative trait locus (QTL) mapping based on lower density linkage map. This study proposed a method for developing genetic linkage map based on a four-way cross population. Computer simulation was conducted to investigate the feasibility and effectiveness of the method and a supporting program was designed. The main procedures and features of the proposed method were summarized as follows: 1） estimating genetic distance of any paired markers based on maximum likelihood method; 2） splitting all markers into different groups （linkage group） by cluster analysis based on genetic distance of markers; 3） for each linkage group， two end markers were first determined， then the marker order could be determined by inserting other markers in appropriate position by distance analysis of any three neighboring markers. Monte Carlo simulation showed that the proposed method is feasible， effective， and applicable in other derived populations from crossing of two homogenous parents.

Complete chloroplast genome sequence is very useful for studying the evolution of species. To get chloroplast genome sequences， purification of the chloroplast or PCR amplification prior to sequencing is commonly involved in conventional approaches. Advances in DNA sequencing technology provide new opportunities to obtain chloroplast genome sequence from the wholegenome highthroughput sequencing data without purification of the chloroplast. In this study， we finished the complete chloroplast genome sequence of Dongxiang wild rice based on highthroughput sequencing data from their fresh green leaves. The chloroplast genome was 134 537 bp in size， and had a typical quadripartite structure with the large single copy （LSC， 80 585 bp） and small single copy （SSC， 12 346 bp） regions separated by two copies of an inverted repeat （IR， 20 803 bp each） region. One hundred and fiftytwo chloroplast genes were successfully annotated. A phylogenetic tree was constructed based on the chloroplast genomes of Dongxiang wild rice， Indica， Japonica and 10 other genera of grasses using the neighborjoining method. The result showed that Dongxiang wild rice had a closer relationship with Bambusa oldhamii and Panicoideae. Furthermore， the SNPs of 22 rice accessions were identified using the chloroplast genome of Dongxiang wild rice as a reference sequence and a phylogenetic tree was constructed based on these SNPs. The result illustrated that Indica had a closer relationship with wild rice-I， while Japonica was closer to wild rice-III， suggesting that Indica and Japonica were domesticated independently from different wild rice populations．

Camellia sinensis cv. Longjing 43 is a domestic variety of tea species and an important economic crop in China. In this study, we developed a rapid method to get the chloroplast (cp) genome and sequenced the entire cp genome sequence of C. sinensis cv. Longjing 43. The C. sinensis cv. Longjing 43 cp genome was 157 085 bp in length, which contained a large singlecopy (LSC, 86 642 bp) region, a small single-copy (SSC, 18 283 bp) region, and two inverted repeat (IR, each with a size of 26 080 bp) regions. With the cp genome of Korean C. sinensis cultivar as a reference, 134 chloroplast genes were successfully annotated. There were 15 genes with non-synonymous mutations in the coding region and more than 100 polymorphic sites in the non-coding region, which could be the DNA markers for the determination of different C. sinensis varieties. We also investigated the relationship of 12 C. sinensis varieties in China based on several cp genomic regions, which contain many variant sites. The result showed that these varieties were divided into two groups with Lingyunbaimaocha in one group and the other 11 in another group. Among the other 11 varieties, the Longjingchangye, Longjingyuanye, Longjingguazi, and Zhongcha 102 had a closer relationship and were formed into one cluster with 100% support rate, demonstrating the reliability of the method that used the cp genome sequences to investigate the genetic relationships.

The ratio of FEV1 (forced expiratory volume in one second) to FVC (forced vital capacity) is an index for pulmonary obstruction measurement and one of the most significant predictors for chronic obstructive pulmonary disease (COPD), which is a heritable multi-factorial disease. We present genome-wide association study (GWAS) to map the genetic architecture of this trait and investigate the networks between the external factors (smoking and gender) and genetic factors. By using a mixed linear model and a conditional model, we conducted GWAS in a cohort suffered COPD from the U.S. National Heart, Lung and Blood Institute. Among 561 467 single nucleotide polymorphisms, we found 12 significant quantitative trait SNPs (QTSs) fitted the full model. And for each of them, we demonstrated the mechanisms and relationship between pulmonary function and genes detected. STIM2 and MRE11A (PEW-value<1×10-5) showed unambiguous evidence of association with COPD. APOL3 (PEW-value<1×10-5) was influenced by different genders in different ways and previous studies also implicated its associations with smoking behavior. The variation of genes MRE11A and DNAJC15 was related to lung adenocarcinoma, which is a serious complication of COPD. The significant epistasis effects of these genes suggested the possibility of multiple functional polymorphisms. These associations offer mechanistic insight into pulmonary function regulation and networks between genetics factors and environmental factors, which indicate potential ways for interventions to COPD and many other respiratory diseases.

Obesity has been reported as an increasingly prevalent and highly heritable health problem， resulting in increased risk for several common diseases. Despite the consensus view， that epistasis and gene-environment interactions have a prominent role in its pathogenesis， they are largely ignored in the current genome-wide association study （GWAS）. A new approach based on a linear mixed model was conducted for GWAS to detect plausible genes and potential interactions among genes， impacts of smoking and gender on body mass index （BMI）. We conducted analysis based on database of genotype and phenotype （dbGaP） from chronic obstructive pulmonary disease （COPD） study， and identified 20 genes and two pairs of epistasis associated with BMI， some of which were smoking-influencing and gender-specific. Bioinformatics analysis revealed a complex gene network among the identified genes connected with BMI and other related diseases. These findings highlight that personalized measures including lifestyle modifications such as smoking is essential for prevention and treatment of obesity.

The dualism of genetic predisposition and gender influences has long been a hot topic in the development of chronic obstructive pulmonary disease (COPD), and smoking is considered a primary risk factor for this lung disease. This paper aimed to detect susceptibility genes for COPD with the data downloaded from dbGaP. A linear mixed model was employed to conduct association-mapping QTSs (quantitative trait single-nucleotide polymorphisms) because of its effectiveness in unbiased estimation of random effects with unbalanced data and in controlling population stratification. The primary focus of the study is to identify genetic risk factors that determine susceptibility for COPD and COPD-related phenotypes with the goal of providing insight into clinically relevant COPD subtypes. By comparing the conditional model excluding the cofactor smoking with the full model, we can detect related QTSs, which will reveal the gene expression on COPD caused or suppressed by smoking. As a result, there are significant genes with high heritability: TNS1 and DGKH were not caused by smoking, MACROD2 and CNIH3 were due to smoking, and LINC00426, METTL4 and GSDMC were suppressed by smoking.

Plant germplasm is an important gene resource for plant breeding and genetic improvement of traits. Core collection， as a representative subset of germplasm， provides an effective entry to investigate genetic diversity and screen potential breeding material. Most agronomic traits of tobacco often exhibit strong genetic variation which can be decomposed into genotypic and genotype × environment interaction variations. A tobacco collection of 805 accessions were evaluated in two locations， Fuquan and Jinsha City of Guizhou Province， China. The genetic variations of 12 agronomic traits were analyzed and the genotypic values of each accession were predicted by a mixed linear model approach. Significant genotypic variations were detected for 11 traits， as well as genotype × environment interaction for 10 traits. Based on the predicted genotypic values， the combination strategies of sampling and clustering methods in the procedure of stepwise clustering for constructing core collection were screened in terms of magnitude of genetic variation captured by sampled subsets. Finally， the subset （S1C3_10）， sampled by a centroid method combined with preferred sampling at 10% proportion， was determined to be a core collection of the tobacco germplasm. The representative and validation of the core collection was examined visually by the accession distribution pattern in the plot established by first two principal components， as well as by the correlation coefficients in terms of magnitude and significance between the core and the initial collections. The results showed that the initial collection was well represented by the core collection， and some potential breeding materials for high yield breeding program of tobacco， such as Y177 and Y178， were included in the core collection. In sum， the constructed core collection will facilitate tobacco breeder to access to whole resources and promote the utilization of genetic diversity in future breeding program of tobacco in China．

Programmed cell death （PCD） is crucial for plant growth and development. It maintains plant normal physiologic metabolism by eliminating damaged， diseased and old cells. Numerous studies have demonstrated that cytoplasmic male sterility （CMS） in plant was associated with programmed cell death （PCD）. The proper timing of PCD is very important for floral development. Based on RNA-Seq （RNA sequencing） result， we found that programmed cell death 5 gene pcd5， cytochrome oxidase gene cox1 and malate dehydrogenase gene mdh were all down-regulated in CMS-C line of maize. In this study， real-time fluorescence quantitative polymerase chain reaction （RT-qPCR） was done to further validate the expression of pcd5，cox1 and mdh genes. These analyses will contribute to better understand CMS-C mechanisms. The CMS-C line （C48-2） and its maintainer line （N48-2） were planted in the same research field of Sichuan Agricultural University. Total RNA of anther was extracted from N48-2 and C48-2 at uninucleate stages using Trizol kit. The RNA was reversely transcribed into a cDNA rst strand using PrimeScript RT reagent kit （TaKaRa）， and the genomic DNA was completely removed from total RNA before reverse transcription. The genomic DNA was extracted from leaf using cetyltrimethyl ammonium bromide （CTAB） method. To verify the expression levels of pcd5， cox1 and mdh genes， the RT-qPCR was performed using a commercial kit （AccuPower 2X Greenstar qPCR Master Mix， Bioneer）. Specific primers were designed in conserved region of coding sequence （CDS）， and the 18S and β-actin were assigned as internal control genes for RT-qPCR. PCR and RT-PCR amplification of pcd5 were performed with TAR HS DNA polymerase （TaKaRa） using specific primer. To verify the results of pyrosequencing， high resolution melting curve （HRM） was performed using a commercial kit （AccuPower 2X Greenstar qPCR Master Mix， Bioneer）. HRM primers were designed in the mutant region based on RT-qPCR. All reactions were performed in triplicate． Gene expression analysis showed that the pcd5， cox1 and mdh genes were down-regulated in C48-2 in comparison to N48-2， and the result was consistent with RNA-Seq result. The results of PCR revealed that the coding region of genomic DNA had no difference between C48-2 and N48-2. An insertion mutation was detected in C48-2 by RT-qPCR and pyrosequencing， which was also proved by HRM. As a consequence， the mutant pcd5 transcript may be associated with the down-regulated in C48-2. In conclusion， the differential expression of pcd5， cox1 and mdh genes may interfere with the programmed cell death， and the abortion of CMS-C microspores might be closely related to the cell death. The findings provide an improved sight for investigating the relationship of PCD and pollen abortion in maize

The mismatch repair (MMR) is a major pathway in DNA repair system，which is critical for maintaining genome stability and DNA replication fidelity，as it is responsible for the recognizing and repairing erroneous insertions, deletions and mismatch of bases newly arising during DNA replication and genetic recombination, as well as during the repair of some forms of DNA damage. The major components in the MMR system include MutS, MutH, and MutL in Escherichia coli. In eukaryotes, homologues of MutS and MutL have been found，but not of MutH. Based on homology， several MutS homologues have also been identified and cloned from Arabidopsis thaliana. Amongst these homlogues MSH2 complexes with MSH6, MSH3 or MSH7 form MutSα, MutSβ and MutSγ heterodimers to recognize different types of mutations, respectively. Mutation or disruption of MMR gene causes significantly increased frequencies of point mutations and microsatellite instability, thus to enhance genetic diversity for plant breeding. Rice is an important food crop and a prominent molecular model species for monocotyledonous plants. Some MMR genes have been annotated in Rice Annotation Project Database, one of which is Os09g24220, a homologue to Msh6 (At4g02070) in the MMR system of A. thaliana. However, no information is available for the Os09g24220 gene function and its mutator phenotype in rice. We reasoned that, if the disrupted Os09g24220 gene can enhance genetic diversity, offspring exhibiting mutations in agronomic traits could be generated using this approach. Herein, we mainly conducted molecular characterization of Tos17 insertion mutants of the Os09g24220 gene and analyzed their agronomic traits, to provide a foundation for the function studies of the Os09g24220 gene and exploitation of the mutant in rice mutation breeding. Three insertion mutants, NF9010, NF7784 and ND6011 with the Tos17 insertion at the 1st, 8th exons and 3′-UTR (untranslated region), respectively, were characterized by triple-primer PCR and RT-PCR. The Tos17 insertion in Os09g24220 gene was confirmed and all three insertion mutants were homozygous. In both NF9010 and NF7784 mutant seedlings, partial Os09g24220 mRNA transcripts were detected with the primer sets that amplified upstream region of the insertion site, but it was not detectable with the primer sets at the downstream region or across the insertion site, indicating that NF9010 and NF7784 lack full-length, functional Os09g24220 mRNA despite the existence of truncated transcripts. However, all Os09g24220 transcripts were detected with primer sets that amplified the upstream, internal and downstream fragments of whole functional region in ND6011 mutant, but no transcript was detected with the primer set across the insertion site, suggesting that ND6011 has the functional mRNA, but not full-length mRNA. Agronomic traits, such as plant height, number of productive panicles, panicle length, seed-setting rate and mass of 1 000 grains, of the insertion mutants were checked and compared with those of their wild type, Nipponbare. Results indicated that at least one trait for each mutant changed significantly. The mutant ND6011 just showed significantly (P<0.05) decreased seed-setting rate, but both NF7784 and NF9010 displayed significant (P<0.01) reduction in ear length and seed-setting rate, moreover plant height of NF9010 was also significantly lowered (P<0.05). In conclusion, some agronomic traits of Tos17 insertion mutants of the Os09g24220 gene changed significantly in rice plants, showing the mutator phenotype. Furthermore, different insertion mutants displayed different mutator phenotypes, which are related to effects on Os09g24220 gene function of different Tos17 insertion sites. These findings provide foundations for investigating the function of the Os09g24220 gene in DNA repair and exploiting such mutants in induced mutation breeding in rice.

Cymbidium ensifolium is one of Cymbidium genus, having elegant shape, beautiful appearance and fragrant aroma. Because of these features, this species gets with extremely high ornamental value. Owing to the lack of its genomic resource, the development and application of molecular marker is still limited. With the development of RNA-Seq technology, the transcriptomic data gradually accumulate and become a useful resource to explore marker with low cost and high efficiency.
Here, the transcriptome in C. ensifoliumwas subjected to RNA-seq. Illumina sequencing was performed at Shanghai Majorbio Bio-pharm Biotechnology Co., Ltd. (Shanghai, China) according to the manufacturer‘s instructions (Illumina, San Diego, CA). Highquality reads were assembled de novo using Trinity with optimized Kmer length of 25. The program Msatcommander was used to analyze microsatellite (as called simple sequence repeat, SSR) frequencies. The minimum numbers of repeats for SSR detection were as follows: six repeats for di-SSRs; and four for tri-, tetra-, penta- and hexa-SSRs. Single nucleotide polymorphisms (SNPs) were detected and filtered using SAMtools and VarScan. The open reading frame (ORF) and untranslated region (UTR) within the isogene were identified using Trinity software. Isogenes containing SSR and SNP were annotated on the basis of BLAST similarity searches.
All high-quality reads were assembled into 101 423isogenes, with total residues of 139 385 689.The isogenes averaged 1 374bp, ranging from 351 bp to 17 260 bp, and 70 583 isogenes, accounted for 69.60%, were about 600 bp. In total, 17 793SSRs and 16 676 SNPs were identified within transcriptomic database. The density of SSR and SNP was 1.28 SSRs/10 kb and 1.20 SSRs/10 kb, respectively. Among these SSRs, tri-SSR was the most types, followed by diSSR, except mono-SSR. Di-SSR and tri-SSR accounted for 20.46% and 21.98% in all SSRs, respectively. The location of SSR was also estimated. The estimated locations were obtained for 7 936 SSRs, but sequence information could not be determined for the remaining 6 586SSR regions as it extended over both estimated coding and non-coding regions. We found that most tri-SSRs and hexa-SSRs occurred more frequently in coding regions. In contrast, di-SSR, tetra-SSR, and penta-SSR, were more likely to appear in UTR rather than coding regions. Among these SNPs, C/T was the most common base substitution, followed by A/G. The two kinds of substitutions, C/T and A/G, accounted for 30.80% and 28.81% in all SNPs, respectively.
The number of isogenes containing SSR and SNP was 13 768 and 7 519, respectively. These isogenes were annotated by Clusters of Orthologous Groups (COG), Gene Ontology (GO) database and Kyoto Encyclopedia of Genes and Genomes (KEGG) database, respectively. A large number of them were annotated with crucial genes that were associated with important biological functions. There were 1 748SSR and 1 932 SNP isogenes assigned into 23 COG classifications, respectively. There were 4 994 SSR and 4 819SNP isogenes classified into 80 and 78 GO terms, respectively. There were 2 107SSR and 2 188 SNP isogenes involved in 300 and 308 KEGG pathways, respectively.
The numerous SSRs and SNPs identified in this study will contribute to marker development. The annotation of isogenes containing SSR and SNP will help in constructing genetic maps and exploring the associations between these markers and the interesting traits. The map will in turn accelerate research on genomics and functional genomics of C. ensifolium.

Crocodile is covered in treasure. Its leather has a high reputation in the world, and its armour contains a lot of bone collagen, protein, calcium, phosphorus and so on, and its gallbladder contains more than 20 kinds of bile acids and bilichols, which has a great medicine value. Its blood with antibacterial and antitumor activity is getting the attention of researchers both at home and abroad. There has been growing interest in commercial marketing of the crocodiles meat for human consumption in China, Thailand, America and Australia, which are all artificially breeding Siam alligator, Estuarine crocodile and Nile crocodile etc. Siam alligator is also called Siam freshwater crocodile, Singapore small crocodile, and is commonly known as Thai crocodile. It is getting more and more attention in China. With the increased amount of breeding, the deep processing for the meat of Siam alligator will be the focus of future research. Hence, our objectives were to identify the volatile components of Siam alligator muscle and evaluate its nutritional value. Odors in the muscle of Siam alligator were collected and determined by solid phase microextraction (SPME) and gas chromatographymass spectrometry (GCMS) before and after deodorization, and the nutritional components in the muscle of Siam alligator were analyzed by common methods. The result showed that there were 72 kinds of volatile compounds detected, in which hexaldehyde was the main component of the odors, along with others constituted the peculiar smell of Siam alligator meat. The contents of moisture, protein, fat and ash in Siam alligator meat were 76.8%, 19.8%, 2.0% and 1.0% respectively. Sixteen types of amino acids in muscle were contained with accounting for 70.44% of the muscle dry matter content and including seven essential ones for human being. The constitutional rate of the essential amino acids was in accordance with the FAO (Food and Agriculture Organization) standard. According to the nutrition evaluation in amino acid score (AAS) and chemical score (CS)，the essential amino acid index (EAAI) was 60.63%. The muscle also contained a variety amount of unsaturated fatty acids, in which the contents of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were 1.44% and 2.96%, respectively. The Siam alligator meat also contained rich minerals and trace elements, especially the most calcium content. Consequently, the meat of Siam alligator is a kind of highquality one with high protein and low fat, rich in a variety of unsaturated fatty acids and minerals.

Squid (Dosidicus gigas) has become one of important aquatic protein resources nowadays because of its appetizing, nourishing quality, low price as well as large fishing amount. However, because of its high insoluble myostromin (11.0%) and connective tissue with longitudinal, radial and circular orientations in squid muscle, its meat was sensitive to heat processing which made the traditional squid product taste tough and hard, as a result hindering greatly the edibility of old people and infant. Until now, the methods of activating endogenous protease or adding exogenous enzymes were usually used for improving the quality of squid meat, while the enzymic method would damage its appearance and was harmful for maintaining the quality. Ultrahigh pressure technology is one kind of physical technology with high efficiency. Previous studies had found that ultrahigh pressure technology could improve the quality of meat. This study aims to evaluate the quality of squid meat processed by ultrahigh pressure based on fuzzy mathematic method and to find out the optimal ultrahigh pressure processing conditions. Based on weight analysis of sensory evaluation about the color, texture, flavor and taste of treated squid slice by ultrahigh pressure processing, a sensory rating system was established. An overall rating by fuzzy mathematics comprehensive evaluation method was also made on the pressure, the processing time, and the temperature of ultrahigh pressure. At the same time, the texture and color of squid slice treated by ultrahigh pressure were also investigated to verify the feasibility and accuracy of fuzzy mathematics comprehensive evaluation method. The results showed that the quality weight collection of squid slice was K=(color 0.25, texture 0.15, flavor 0.35, taste 0.25), and the sensory quality appeared to be the best when the squid slice was treated by 300 MPa at 25 ℃ for 10 min by fuzzy mathematics comprehensive evaluation method. Meanwhile, the texture and whiteness of treated sample by instrument measurement were influenced in descending order as follows: pressure > temperature > time, and the optimal condition was 300 MPa at 25 ℃ for 10 min, which was correspondence with the result of fuzzy mathematics sensory evaluation. Under this optimal condition, the squid meat was with the best elasticity, the lowest shear force, and the optimal sensory quality. It is concluded that the ultrahigh pressure treatment can improve the quality of squid meat, and it is further confirmed the feasibility and accuracy of the fuzzy mathematics sensory evaluation and instrumental analysis.

Obesity and disorder of lipid metabolism, also called dyslipidemia, have been one of the most important diseases threating people health worldwide, and is closely connected with hypertension, high serum glucose and other cardiaccerebral vascular diseases. The high fat diet is the main pathogenic cause of obesity and dyslipidemia, how to regulate blood lipid and control body mass effectively has been hotspots on nutriology research nowadays. Agents from natural products that inhibit fat digestion and absorption are of theoretical benefit in the treatment of obesity and dyslipidemia. Among them, lotus (Nelumbo nucifera) leaf is a kind of natural plant material used both in food and medicine, which contains multiple useful components. In modern research, scientists found that alkaloids were one of the main bioactive ingredients in them, helping lowing the serum lipid level and controlling body mass. Thus, this study aims to investigate the effect of lotus leaf total alkaloids on the body mass and lipid regulation in in vivo and in vitro experiments, and to find a new potential food additive to prevent effectively obesity from abnormal fat accumulation. Lipidlowering efficacy in vivo of lotus leaf total alkaloids was determined by animal experiment. After adapting to the feeding environment for 10 days, 30 experimental rats were randomly divided into six experimental groups according to their serumlipid level and body mass, including general diet group (blank group), high fat diet with lotus leaf total alkaloid (high, moderate, low doses) groups, positive drug control group and high fat diet group (model group). Except for the general diet group feeding normal diet, the other five groups were fed high fat diet for 40 d to set up the model of hyperlipidemia rats. Then, the contents of TC (total cholesterol), TG (triglyceride), LDLC (low density lipoproteincholesterol), HDLC (high density lipoproteincholesterol), the levels of AST (aspartate aminotransferase) and ALT (alanine aminotransferase) in serum were assayed in hyperlipidemia rats which had been fed lotus leaf total alkaloids of 20, 40, 80 mg/(kg·d) and positive drug of 10 mg/(kg·d) simvastatin for another 40 days, respectively. In addition, the body mass and liver mass, body length, tail length were measured. Lipidlowering efficacy in vitro of lotus leaf total alkaloids was determined by measuring the inhibitory activity against pancrelipase by monitoring the hydrolysis of pNPb, which released the yellow chromogen pnitrophenol, and the buffer solution was adjusted to the condition of 37 ℃, pH=7.4. The results showed that, compared with high fat diet group (model group), the body mass decreased significantly in the high fat diet with lotus leaf total alkaloid (high, moderate, low doses) groups, as well as the Lee’s index, liver and spleen mass of rats; moreover, the contents of TC, TG, LDLC, the levels of AST and ALT in serum and atherosclerosis index (AI) were significantly decreased and the ratios of HDLC/TC and apoAI/apoB increased in the hyperlipidemia rat fed with lotus leaf total alkaloids. The maximum inhibitory rate of lotus leaf total alkaloids of 600 μg/mL against pancrelipase activity was 25.6%. Based on the LinweaverBurk plots, the lipase inhibitor demonstrated noncompetitive inhibition for pancrelipase. It is concluded that the lotus leaf total alkaloids are effective in significant reduction of body mass, body fat and obesityrelated body indicators, and can inhibit lipase activity. Also, it is sure that the alkaloids from lotus leaves have potential to prevent effectively obesity from abnormal fat accumulation.

The fruit shell of Camellia oleifera Abel is the byproduct in C. oleifera processing, which is greatly wasted by abandoned or burnt in actual production. In recent years, some studies indicated that the fruit shell of C. oleifera had some biological activity such as antioxidant activity, indicating that the fruit shell components can be further exploited as some food additive or taken as some raw material of industry. Insoluble dietary fiber (IDF) in the fruit shell of C. oleifera has various physiological functions against some diseases, such as diabete, cardiovascular disease, intestine cancer, constipation etc., which give it higher health care value and dual performance in economy and society. In order to turn the waste residue of C. oleifera into wealth and dietary fibercontaining functional food, IDF from the wasted part of the shell was extracted with alkali treatment, and the optimal extraction condition and physicochemical properties were studied. The results showed that: 1) After investigating the single factor influencing the extraction yield, the optimal point for each factor was as follows: 0.35 mol/L for the concentration of sodium hydroxide, 80 ℃ for extraction temperature, 2 hours for extraction duration and 1∶18 for the ratio of solid to liquid. 2) Through orthogonal experiment, the optimal extraction condition of IDF was as follows: the ratio of solid to liquid of 1∶14, sodium hydroxide of 0.35 mol/L with the reaction at 80 ℃ for 3 h. The influential order of each factor on the IDF extraction was alkali concentration > solidliquid ratio > temperature > extraction time. Under this optimal condition, the extraction yield of IDF from C. oleifera shell was 40.4% and the purity was 91.52%. 3) The water holding capacity of IDF from C. oleifera shell was 2.04 g/g and the degree of swell was 1.2 mL/g. Meanwhile, the physicochemical properties of IDF were quite stable in the normal food system with different conditions of pH, sugar, salt or preservative. In conclusion, the shell from C. oleifera contains rich dietary fiber, and the IDF is quite stable in normal food system. It is worth developing food additive products by adding IDF extraction in the future food production development.

Fatty acids are ubiquitous molecules, which are typically bounded to other compounds such as glycerol, sugar or phosphate head groups to form various derivatives. They have diverse and potent biological activities, such as antiinflammatory, antitumor, antiprotozoan, antiviral, antifungal and antibacterial activities. Although the antimicrobial activity of fatty acids and their derivatives has been well known for many years, the structural and functional features causing them to prevent microbial growth or survival are still unknown. The purpose of the present study was to find out the correlation between structural features and the antimicrobial activities. At first step, it was necessary to evaluate the efficacy of various fatty acids and their derivatives to control the growth of Escherichia coli O157:H7 and Candida albicans. The microorganisms used for screening studies were common in food (E. coil O157:H7) and clinical specimens (C. albicans) and were individually maintained through monthly transfers to the respective fresh medium (E. coil O157:H7 in trypticase soy broth at 37 ℃; C. albicans in SabouraudDextrose broth at 30 ℃) and were stored at 4 ℃. Stock cultures were inoculated and grown on respective broth for 1824 h to prepare active and working cultures. The tested samples included 10 kinds of saturated fatty acids, six different unsaturated fatty acids, nine kinds of monoacylglycerols, two types of fatty acid methyl esters, three kinds of fatty acid ethyl esters and four classes of fatty alcohols. They were dissolved in 95% ethanol, sterile Tween80 (final concentrations of ethanol and Tween80 were 1.5%, respectively) and then were filtersterilized using a 0.22 μm pore size membrane filter. Ethanol and Tween80 controls were used for each experiment to ensure that any observed inhibition was not due to them. Antibiotics were dissolved in 1.5% ethanol and tween80 used as positive controls. The respective broths which contained 0.015, 0.007 5 mol/L fatty acids and their derivatives were inoculated with approximately 1.0×1055.0×105 CFU/mL of active cultures, then they were incubated for 24 h at the optimum temperature. Samples (100 μL) were removed and immediately diluted 10fold in sterile physiological saline. The number of viable microorganisms was determined through a standard plate count technique. All the tested mediumchain fatty acids (C812) and their corresponding chain monoacylglycerols, longchain unsaturated fatty acids (>C12) showed more antimicrobial activities than fatty alcohols. Longchain fatty acids, fatty acid methyl esters and fatty acid ethyl esters had feeblish antimicrobial activity. Of the saturated fatty acids, The C13 fatty acids; as compared with their saturated counterparts, the C14:1, C16:1, C16:2 unsaturated fatty acids were more highly active, however, the linolenic acid (C18:3) was not very active. The fatty acid etherified with the alcohol and methanol showed no inhibition. The 1, 3dilaurin was less active than the fatty acid. The monoglyceride was proved to be more active. In sum, the chain length, substituent, numbers and position of double bond are the important factors influencing the antimicrobial activity of fatty acids and their derivatives.

Jumi, the flower bud of Chrysanthemum indicum L., is a special tea from Shilian, Zhejiang Province, and it is famous for its unique fragrance. Jumi essential oil has numerous efficacies and applications in the field of pharmacy and cosmetic industry, therefore developing effective oil extracting method which can keep both biological activity and fragrance is of considerable significance. At present, steam distillation has been reported as essential oil extract method, but this method can not keep the unique fragrance of Jumi due to the decomposition of fragrant component at high temperature. However, the supercritical fluid of CO2 (SFECO2) is a low temperature processing technique and can fully keep the unique fragrance of Jumi, which is an efficient, nontoxic, pollutionfree, and nonresidual method to extract and separate Jumi essential oil. The objective of this paper is to evaluate the quality of Jumi essential oil extracted by SFECO2and determine its biological activity. The chemical compositions of Jumi essential oil were determined by GCMS and their inhibitory activities against Staphylococcus aureus and Escherichia coli were analyzed by filter paper tablet bacteriostatic method and minimum inhibitory concentration (MIC). The results showed that the 28 types of components with the similarity of more than 70% were identified in Jumi essential oil by GCMS analysis, which were mainly terpenes and their derivatives, and the former mainly include monoterpene and sesquiterpene, and the latter was mainly alcohols, for example, the contents of hexamethylbenzene, 2(2, 4hexadiynylidene)1,6dioxaspiro［4, 4］ nonane3ene and (2RCis)1, 2, 3, 4, 4a, 5, 6, 7octahydro.alpha., .alpha., 4a, 8tetramethyl2naphthalenemethanol were higher than 9%, respectively. Bacterial inhibition tests indicated that Jumi essential oil could inhibit the growth of S. aureus and E. coli, and its MIC was 1.6 mg/mL. It is indicated that the Jumi essential oil extracted by SFECO2 is nontoxic and pollutionfree and it offers a scientific evidence about developing Jumi essential oil with high additional value and goodquality in cosmetic and food additive.

Capsicum annuum is one of the world largest consumptive vegetables. As the biggest producer, consumer and exporter of C. annuum in the world, the development of China capsicum industry has an important influence on the development of global C. annuum industry. The C. annuum industry mostly focuses on its fruit, while C. annuum leaves, as a kind of easily wasted vegetables, which are rich in resources and nutrients, have not yet been widely developed. In order to better develop C. annuum leaves, we explore the αglucosidase inhibitory activity using 70% ethanol extracts of C. annuum leaves. The pnitrophenylαDglucopyranoside (pNPG) method was adopted to determine the inhibitory activity of C. annuum leaf extracts against αglucosidase. The results showed that the αglucosidase inhibitory activity of C. annuum leaf extracts was up to 60%, which was 10 times more than its fruit, and there were significant differences in the αglucosidase inhibitory activity among different cultivars. As to different target enzymes, C. annuum leaf extract showed different activity, for example, it could inhibit the activity of sucrase and maltase from animal, but it had no effect for microbial enzyme. The C. annuum leaf extract were validated with a strong hypoglycemic activity by animal glucose load test. In conclusion, The C. annuum leaf extract has high inhibition against αglucosidase and can be used for the development of a new type of hypoglycemic food.

In 2002, the discovery of acrylamide contaminant in heat processing foods got a worldwide attention because of its genetic and reproductive toxicities as well as mutagenic and carcinogenic properties. Therefore, it is necessary to find effective additive agents to reduce the formation of acrylamide in Maillard reaction. We investigated the effects of flavonols on the reduction of acrylamide in an equimolar asparagineglucose Maillard reaction system using potato matrix and microwave heating. To investigate the doseresponse effect, six different levels of flavonols were added into the selfprepared Maillard reaction system and different levels of acrylamide were generated and observed. Meanwhile, the correlation between the inhibitory rate of acrylamide affected by flavonols and the change of Trolox equivalent antioxidant capacity (ΔTEAC) in Maillard reaction products was also evaluated. Acrylamide levels in Maillard reaction products were determined by ultrahigh performance liquid chromatography coupled with tandem mass spectrometry (UHPLCMS/MS) and quantified with multiple reaction monitoring (MRM) mode after the pretreatment of final products. ΔTEAC values were simultaneously measured by three representative antioxidant evaluation methods, i.e. DPPH (1,1diphenyl2picrylhydrazyl), ABTS (2,2azinobis3ethylbenzothiazoline6sulfonic acid) and FRAP (ferric reducing ability of plasma). The results indicated a nonlinear relationship between addition levels of flavonols and inhibitory rate of acrylamide. The optimal addition level of all six flavonols, i.e. kaempferol, kaempferol3oglucoside, quercetin, quercetin-3oglucoside, myricetin and rutin was 1×10-9 mol/L, which exerted their maximal inhibitory effect against the generation of acrylamide. The inhibitory rate ranged from 48.9% to 69.3% when different types of flavonols were used at the optimal addition level. Taking ΔTEAC as the antioxidant measurement index, the correlation coefficients between ΔTEAC determined by three different antioxidant evaluation methods (DPPH, ABTS and FRAP) and inhibitory rate of acrylamide were 0.885 3, 0.834 2 and 0.805 3 respectively, indicating a close correlation between the inhibitory effects of flavonols against the generation of acrylamide and the antioxidant capacity in Maillard reaction products. Based on microwave heating and Maillard reaction system, this study not only describes novel experimental system for investigating the inhibitory effect of phytochemicals (e.g. flavonols) against the generation of acrylamide, but also provides substantial evidence of the correlation between the inhibitory rate of acrylamide and antioxidant capacity in Maillard reaction products.

Only a small amount of vitamin B12 is absorbed and utilized by human body. The suitable vitamin B12 levels can improve the utilization efficiency of folic acid. Excessive intake of vitamin B12 may cause allergic reactions, while vitamin B12 deficiency in infancy can lead to anemia. At present, most of the infants are not breastfeeding. Formula food supplemented with a suitable level of vitamin B12 is an important nutrient guarantee for healthy growth and development of infants. Thus, it is very necessary and important to develop a sensitive, efficient and accurate method for determination of vitamin B12. Currently, the commonly used methods for the determination of vitamin B12 in infant formula foods are microbial analysis methods. But they usually lead to get higher results than the real amounts because they do not distinguish vitamin B12 from its analogues. In addition, liquid chromatography technologies are also applied to analyze vitamin B12 in infant formulas. However, they have some disadvantages including long sample preparation time and high analysis costs. In the present study, a new method using solid phase extraction (SPE) coupled to twodimensional liquid chromatography with ultraviolet detector was developed for the quantification of vitamin B12 in infant formulas. The different forms of vitamin B12 were transformed to cyanocobalamin by reacting with potassium cyanide solutions after the dissolved samples were digested with amylase. The extracts of samples were purified through the HLB SPE cartridges before they were injected into a dual gradient pump series liquid chromatography system in large volume injection. The samples were separated and enriched in ZORBAX GF250 column (9.4 mm × 250 mm, 4 μm) with the mobile phase of 7.5% acetonitrile in water. Afterwards they were switched into Agilent ZORBAX BonusRP column to analyze in gradient elution using 0.4% triethanolamine in water (pH=6) and 75% acetonitrile in water (including 0.4% triethanolamine, pH=6) as the mobile phases. The contents of vitamin B12 in samples were determined at the wavelength of 550 nm using external standard method. The developed method showed a good linearity (R2>0.999) when the calibration curve ranged from 2 to 40 ng/mL. The limit of detection and the limit of quantitation were evaluated as the signaltonoise 3∶1 and 10∶1, and they were 1.0 and 2.5 μg/kg, respectively. The accuracy of the method was evaluated by employing the standard addition method. The spiked samples with 5.0, 10.0 and 50.0 μg/kg vitamin B12 standard (six portions for each spike level) were prepared and analyzed. The results showed that the spike recoveries were 88.0%94.8% with the relative standard deviation (RSD) of 2.54%4.87% at the three spiked levels. The sample pretreatment of the established method involved enzymic hydrolysis using amylase, in order to maximize the extraction of vitamin B12. The transformation of vitamin B12 from different forms to cyanocobalamin with the best stability ensured the accuracy of quantitation. Most of the impurities were removed by employing HLB SPE cartridges during the cleanup of sample extracts. Compared with the purification technology using immunoaffinity cartridges, the experimental costs were significantly reduced and the time of sample preparation was shortened. After the sample extracts were further purified and enriched through the first dimensional liquid chromatography by large volume injection, they were analyzed at 550 nm by the dual column switching method. The wavelength of 550 nm minimized the signal interferences from impurities and further improved the accuracy of results. In conclusion, the twodimensional liquid chromatography method with online column switching shows better sensitivity and reproducibility than the traditional liquid chromatography methods, and it is verified to be suitable for the determination of vitamin B12 in infant formulas.

Aflatoxins (AFT), whose basic structure is composed of difuran and coumarin, have 17 kinds of derivatives including B1, B2, G1, G2, M1, M2, etc. AFT M1 was firstly separated from milk. AFT M1 and AFT M2 were the derivatives of AFT B1 and AFT B2 through the animal metabolism. Especially, AFT B1 and AFT M1 have been defined as a category A and 2B carcinogen by the International Agency for Research on Cancer (IARC) from World Health Organization (WHO) in 1993, respectively. Moreover, AFT B1 and AFT M1 are regarded as strong carcinogens, the carcinogenic mechanism of which is achieved via affecting the pericellular membrane, inhibiting the synthesis of RNA and interfering the inductive style of specific enzymes. In December 2011, the General Administration of Quality Supervision, Inspection and Quarantine of the People’s Republic of China (AQSIQ) announced the selective examination results of 200 kinds of liquid milk products. The aflatoxin M1 in parts of milk products were over ranging the maximum residue limits (MRLs) of M1 in milk and milk products, of which the maximum superscalar were 140% exceeded. Moreover, the contents of aflatoxin M1 were found exceeded in milk powder again in July 2012. The method used now was to first heat the milk and milk products in water bath, then samples were cleanup and concentrated by immunoaffinity column after filtered or centrifuged. In this method, no clear sample solutions were obtained, when passing through the immunoaffinity column, sometimes the immunoaffinity column would be blocked, and the recovery would not in expectation. So a better method was needed for determinating the aflatoxin M1 in milk and milk products. The present study developed an improved analytical method for the fast determination of aflatoxin M1 in milk and milk products by ultraperformance liquid chromatography (UPLC) combined with large volume flow cell fluorescence detection (FLD). The milk sample was extracted by acetonitrile, and the ratio of the milk sample to acetonitrile was 1 to 2.5 in mass to volume. Then, the sample was extracted by vortex and ultrasound assisted liquidliquid extraction (ULLE), and was cleanedup and concentrated by aflatoxin M1 immunoaffinity column. The analyte was separated by UPLC BEH C18 column (100 mm×2.1 mm, 1.7 μm), and was eluted with acetonitrilemethanol (50∶50) and pure water. The results showed that the limit of quantitation (LOQ) of aflatoxin M1 was 0.03 μg/kg, which was lower than the national criteria on determination of the minimum level of aflatoxin M1 in milk and milk products. Meanwhile, high correlation coefficient (R2>0.999) was obtained within linear range from 0.06 to 1.2 μg/kg, and reasonable recoveries (81.95%94.20%) were in different spike level. In addition, acetonitrile could effectively precipitate protein in milk during the pretreatment to obtain clear extraction which could rapidly pass through immunoaffinity column only by gravity. When using the large volume flow cell, the sensitivity was increased, which was three times than the standard flow cell. The results obtained from this method were similar to the classical method. In conclusion, this quantitative method has many advantages including simple pretreatment, rapid determination and high sensitivity, which can be applied to the determination and quantification of aflatoxin M1 in milk sample.

Sulforaphane (SFN) is an isothiocyanate (ITC) which exists as a precursor of glucosinolate (GS) in various cruciferous vegetables especially in broccoli. Sulforaphane has been regarded as a potential of anticancer agent derived from diet, mostly because of its powerful induction of phase Ⅱ enzymes, and it is also acted as an antagonist of injury factors including lipopolysaccharide (LPS) and homocysteine (HCY). The present study aims to explore whether broccoli extract, rich in sulforaphane, can protect the acute liver injury induced by ethanol in mice or not. The quantity of sulforaphane in broccoli extract was detected by high performance liquid chromatography (HPLC). Hematoxylineosin (HE) staining was used to determine the pathological changes in C57BL/6 mice model with the acute liver injury induced by ethanol. The activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) in serum of mice were measured using semiautomatic biochemical analyzer. The results showed that the quantity of sulforaphane in the broccoli extract was 1.26 mg/tablet. Liver masses in all sulforaphane treatment groups were obviously reduced which were presented as the sharply decreased ratio of liver mass/body mass. The seriously pathological evidence was observed in ethanol treatment group, while less changes were seen in the sulforaphane treatment groups. Sulforaphane could dramatically inhibit the activities of ALT, AST and ALP in serum of mice induced by ethanol, with more obvious suppression in the moderate (40 mg/kg) and highdose (80 mg/kg) groups, respectively. The result above indicates that the sulforaphane from broccoli extract tablets is able to protect the liver injury induced by ethanol.