Determination of egg and milk allergen in food products by liquid chromatography-tandem mass spectrometry based on signature peptides and isotope-labeled internal standard

The aim of this work was to develop a liquid chromatography-tandem mass spectrometry method for the determination of milk allergen and egg allergen in food products.Signature peptides GGLEPINFQTAADQAR,VGINYWLAHK,VLVLDTDYK,FFVAPFPEVFGK,and NAVPITPTLNR were confirmed and synthesized as the quantitative peptide of ovalbumin,α-lactalbumin,β-lactoglobulin,α_(S1)-casein andα_(S2)-casein,the relative isotope-labeled internal standards were used in the quantitative analysis.Linear range was in the range of0.5-5000.0 nmol/L for egg and milk allergen in bread,cake,cookie,rice crust and wheat flour samples with free from egg and milk,the limits of detection of milk allergens and egg allergen were in the range between0.94 mg/100 g and 56.71 mg/100 g,limits of quantification of milk allergens and egg allergen were in the range between 2.36 mg/100 g and 141.78 mg/100 g.The recoveries ranged from 76.7%to 122.8%,the relative standard deviations were in the range of 1.60%-15.60%.The developed method has been successfully used for the detection of egg and milk allergen in various food samples.

Leaf Morphology Genes SRL1 and RENL1 Co-Regulate Cellulose Synthesis and Affect Rice Drought Tolerance

The morphological development of rice(Oryza sativa L.)leaves is closely related to plant architecture,physiological activities,and resistance.However,it is unclear whether there is a co-regulatory relationship between the morphological development of leaves and adaptation to drought environment.In this study,a drought-sensitive,roll-enhanced,and narrow-leaf mutant(renl1)was induced from a semi-rolled leaf mutant(srl1)by ethyl methane sulfonate(EMS),which was obtained from Nipponbare(NPB)through EMS.Map-based cloning and functional validation showed that RENL1 encodes a cellulose synthase,allelic to NRL1/OsCLSD4.The RENL1 mutation resulted in reduced vascular bundles,vesicular cells,cellulose,and hemicellulose contents in cell walls,diminishing the water-holding capacity of leaves.In addition,the root system of the renl1 mutant was poorly developed and its ability to scavenge reactive oxygen species(ROS)was decreased,leading to an increase in ROS after drought stress.Meanwhile,genetic results showed that RENL1 and SRL1 synergistically regulated cell wall components.Our results revealed a theoretical basis for further elucidating the molecular regulation mechanism of cellulose on rice drought tolerance,and provided a new genetic resource for enhancing the synergistic regulation network of plant type and stress resistance,thereby realizing simultaneous improvement of multiple traits in rice.

Changes in Antioxidant Enzyme Activities and Malondialdehyde(MDA) Content of Rice with Blast Resistance Induced by Ag-antibiotic 702

The paper was to explore the effect of rice blast resistance induced by Ag-antibiotic 702 on antioxidant enzyme activities and malondialdehyde( MDA)content. At three-leaf and one-heart stage of Luliangyou 996,15 μg/mL Ag-antibiotic 702 was sprayed,while Validamycin and distilled water were sprayed as positive control and negative control,respectively. The antioxidant enzyme( SOD,POD,PPO,CAT and PAL) activities and malondialdehyde( MDA) content in rice leaves were determined every 24 h within 168 h post spraying. Compared with CK,the MDA content and activities of the five antioxidant enzymes increased in Agantibiotic 702 treatment,Validamycin treatment and M. grisea inoculation treatment. The MDA content and SOD,CAT and POD activities in Ag-antibiotic 702 treatment were significantly higher than those in M. grisea inoculation treatment in early stage,and the APX and LOX activities increased more remarkably and rapidly. The induction effect of Ag-antibiotic 702 could reach or exceed that of Validamycin treatment. Ag-antibiotic 702 enhanced the resistance to rice blast because MDA content and antioxidant enzymes activities were improved in rice leaves. Therefore,the increase of rice blast resistance induced by Ag-antibiotic 702 in rice was positively correlated with the increase of MDA content and antioxidant enzymes activities in rice leaves.

Construction and analysis of a plant transformation binary vector pBDGG harboring a bi-directional promoter fusing dual visible reporter genes

The constitutive promoter of cauliflower mosaic virus 35S (CaMV 35S) is a polar unidirectional promoter and is widely used in plant genetic engineering. In the present study, the unidirectional CaMV 35S promoter has been modified to a bi-directional promoter by fusing its minimal promoter element to the 5′end of CaMV 35S promoter in the opposite orientation. To qualitatively and quantitatively esti-mate its bi-directional transcriptional function and activity, two visible reporter genes, gusA (β-glucuronidase, GUS) and gfp (green fluo-rescent protein, GFP), were fused to the two ends of the promoter in bi-orientations ending with NOS terminator sequences, respectively. Stable expression of gusA and gfp genes in transgenic tobacco (Nicotiana tabacum L.) was visulized by histochemically staining for GUS and fluorescence microscopic observation under UV for GFP in transgenic plants. The expression of two reporter genes showed that the constructed bi-directional promoter did have the bi-directional transcriptional function in both expected orientations. The quantitative estimation of GUS and GFP were determined on a HITACHI F1000 Fluorescence Spectrophotometer with various wavelengths of excita-tion and emission. The GUS activity varied from 8 to 250 pmol 4-MU / min / mg protein and the GFP content varied from 0.9 to 1.8 μg / mg protein in various lines of transgenic tobacco plants. Higher GUS activity generally coupled with lower GFP content, and vice versa.

Identification and Characterization of Tonoplast Sugar Transporter (TST) Gene Family in Cucumber

Tonoplast sugar transporters(TSTs)play essential roles in regulating plant growth,development,and response to various biotic and abiotic stresses.In this study,a total of three TST genes were identified by a genome-wide analysis in cucumber.Phylogenetic analysis showed that TST proteins from cucumber and other plant species can be classified into five groups,and nearly all TST members in the same groups displayed similarmotif distributions,transmembrane(TM)domains,and gene structures.All of the three CsTST genes possess a number of development-,stress-,and hormone-related cis-elements in the promoter sequences.Meanwhile,qRT-PCR assays revealed that the CsTST1 was expressed in fruits,flowers,leaves,and other tissues,and its expression varied significantly under various abiotic stresses such as cold,salt,drought(PEG),and abscisic acid(ABA).Finally,functional analysis of CsTST1 in yeast revealed that it was able to complement the deficiency in galactose,mannose and sucrose transport.These results revealed that CsTST1 can act as a functional sugar transporter to play important roles in cucumber growth and response to abiotic stress probably through affecting carbohydrate distribution.

Changes in Protein Content and Chitinase and β-1,3- glucanase Activities of Rice with Blast Resistance Induced by Ag-antibiotic 702

[Objective] The paper was to explore the effect of rice blast resistance induced by Ag-antibiotics 702 on protein content and chitinase and β-1,3-glucanase activities in rice. [Method]At the fourth leaf stage of Luliangyou 996,15 μg/m L Ag-antibiotic 702 was sprayed,while Validamycin and distilled water were sprayed as positive control and negative control,respectively. All treatments were inoculated with spore fluid of Magnaporthe grisea at 48 h post spraying,and the rice inoculated with only distilled water was used as blank control. The enzymes activities( endochitinase,exochitinase and β-1,3-glucanase) and total protein content in rice leaves were determined every 24 h within 168 h post spraying. [Result] Compared with the blank control,the rice inoculated with spore fluid of M. grisea could significantly increase the total protein content and the activities of β-1,3-glucanase and chitinase. The induction effect of Ag-antibiotics 702 exceeded that of Validamycin treatment. And the changes in activities of β-1,3-glucanase and chitinase had obvious synchronicity. [Conclusion] Ag-antibiotic 702 can significantly improve the total protein content and the activities of β-1,3-glucanase and chitinase,thus enhancing the resistance to rice blast.

The Study of Food-Grade Induced Expression and Enzymatic Properties of L-Arabinose Isomerase from Lactobacillus plantarum WU14 with High D-Tagatose Yield

L-arabinose isomerase (L-AI) is the key enzyme for D-galactose isomerization of D-tagatose by biological method. In this research, Lactobacillus plantarum WU14 with high D-tagatose yield was identified as Lactobacillus plantarum was isolated from the number of lactic acid bacteria from pickled vegetables. The crude L-arabinose isomerase activity of Lactobacillus plantarum WU14 with high D-tagatose yield was 13.95 U/mL under the optimal temperature 60&degC, pH 7.17 and substrate concentration 0.8 mol/L, and the conversion rate of 56.12% could be gained after 28 hours. Protein structure and specific of L-Arabinose Isomerase of Lactobacillus plantarum WU14 were researched. The results showed that L-arabinose isomerase is mainly composed of alpha helix and random coil. Then the recombinant L-AI gene was inserted into the food-grade expression vector pRNA48 and expressed in L. lactis NZ9000 successfully. The target protein expression reached the maximum amount when the induced concentration of nisin reaches 30 ng/mL after 12 h. And the crude enzyme activity of recombinant bacteria reached 6.21 U/mL under 60&degC. Otherwise the optimal conversion rate recombinant of L. lactis NZ9000/pRNA48-L-AI can reach 39.21% under the temperature of 50&degC, pH 7.17 and D-galactose concentration was 0.6 mol/L.

Preparation of Gold Nanorods of High Quality and High Aspect Ratio

We report the synthesis of gold nanorods (NRs) by seed-mediated growth method. A small amount of different shapes such as triangles, hexagons and a large amount of rods are obtained by varying the proportion of seed to metal salt, adding NaOH to growth solution as well as using the seed solution of CTAB-capped agent. The gold nanorod (NR) formation yield is improved. Meanwhile, the growth mechanism of high yield gold NRs is discussed. The high quality single size NRs can be separated from polydisperse samples using surfactant-assisted nanorod self-assembly. The gold NRs synthesized were characterized by transmission electron microscopy (TEM) and UV-vis spectroscopy.

The infection kinetics and transmission potential of two Guaico Culex viruses in Culex quinquefasciatus mosquitoes

Guaico Culex virus(GCXV)is a newly identified segmented Jingmenvirus from Culex spp.mosquitoes in Central and South America.The genome of GCXV is composed of four or five single-stranded positive RNA segments.However,the infection kinetics and transmission capability of GCXV in mosquitoes remain unknown.In this study,we used reverse genetics to rescue two GCXVs(4S and 5S)that contained four and five RNA segments,respectively,in C6/36 cells.Further in vitro characterization revealed that the two GCXVs exhibited comparable replication kinetics,protein expression and viral titers.Importantly,GCXV RNAs were detected in the bodies,salivary glands,midguts and ovaries of Culex quinquefasciatus at 4–10 days after oral infection.In addition,two GCXVs can colonize Cx.quinquefasciatus eggs,resulting in positive rates of 15%–35%for the second gonotrophic cycle.In conclusion,our results demonstrated that GCXVs with four or five RNA segments can be detected in Cx.quinquefasciatus eggs during the first and second gonotrophic cycles after oral infection.

Microbial Remediation of Heavy Metal(loid)Contaminated Soil: A Review

Because of the rapid development of industrial processes, increased urban pollution and agricultural chemicals applied in recent years, heavy metal(loid) pollution in soil has been very serious, and there is an urgent need for fast and efficient removal of heavy metal(loid) pollution. Currently, environmental microorganisms are always used to perform biological alteration or improvement of soils and sewage. Using functional microorganisms that are resistant to toxic heavy metal(loid) ions for alteration and transformation of heavy metal(loid)s in ionic form is an effective measure for microbial remediation of heavy metal(loid)contaminated soil. This paper reviewed the microbial remediation mechanism of heavy metal(loid) contaminated soils, and the approaches for breeding bacteria those can be used for highly efficient removal of heavy metal(loid)s, as well as the application examples of microbial remediation and transformation of heavy metal(loid) contaminated soil, and finally described the future trends and further research work of heavy metal(loid) contaminated soils by microbial remediation.

Damage to DNA of effective microorganisms by heavy metals:Impact on wastewater treatment

The research is to test the damage to DNA of effective microorganisms(EMs)by heavy metal ions As3+,Cd2+,Cr3+,Cu2+,Hg2+, Pb2+,and Zn2+,as well as the effects of EM bacteria on wastewater treatment capability when their DNA is damaged.The approach applied in this study is to test with COMET assay the damage of EM DNA in wastewater with different concentrations of heavy metal ions As3+,Cd2+,Cr3+,Cu2+,Hg2+,Pb2+,Zn2+,as well as the effects of EM treated with As3+,Cd2+,Cr3+,Cu2+,Hg2+,Pb2+,and Zn2+ on COD degrading capability in wastewater.The results showed that the damage of the DNA of EM were negatively correlated with their treatment capability and that EM bacteria maximum tolerant concentrations of these heavy metal ions was at 0.05 mg/L for As3+, 0.2 mg/L for Hg2+,0.5 mg/L for Cd2+,Cr3+,and Cu2+,and 1 mg/L for Pb2+and Zn2+.

ALM1, encoding a Fe-superoxide dismutase, is critical for rice chloroplast biogenesis and drought stress response

Chloroplasts are the center of plant life activities including photosynthesis,growth and development,and abiotic stress response.Chloroplast development and biogenesis in rice have been studied in detail,but how does abiotic stress affect chloroplasts is less studied.We obtained an albino mutant,alm1,whose chlorophyll content was greatly decreased.Transmission electron microscopy showed that chloroplast development in alm1 was blocked,especially in thylakoid-like structures,which could not form normally.The ALM1 gene encodes a chloroplast-localized superoxide dismutase.Full-length ALM1 successfully restored the non-albino phenotype,and in knockout lines,the albino phenotype reappeared.The ALM1gene is expressed mainly in young leaves.alm1 plants died as a consequence of excessive reactive oxygen accumulation after the third-leaf stage.A series of biochemical assays verified that ALM1 interacted with the OsTrxz protein,which is one of the components of plastid-encoded RNA polymerase (PEP) complexes.A western blot experiment indicated that ALM1 played an important role in stabilizing OsTrxz in rice.An overexpression test of ALM1 revealed that ALM1 can increase drought resistance by removing excess reactive oxygen in rice seedlings.This study suggests that ALM1 not only participates in rice chloroplast biogenesis,but also increases rice stress resistance by scavenging excess reactive oxygen.

Preparation of Ag+/TiO2 Composite Coating Film for Preservation of Nanfeng Citrus

Using tetrabutyl titanate as the precursor,silver nitrate as the load agent,silver-doped titanium dioxide coating film was prepared by sol-gel method.Three agent samples were characterized by X-ray diffraction( XRD) analysis to investigate the crystal structure,which confirmed a synergistic antibacterial effect.The antibacterial and preservative effects of silver-doped titanium dioxide coating film on Nanfeng citrus were investigated preliminarily. Nutritional indicators of ripe Nanfeng citrus before and after preservation were detected to investigate the preservative properties of silver-doped titanium dioxide coating film and compare the effects of different components of composite coating film. The results showed that Ag^+/TiO_2 composite coating film exhibited high antibacterial and preservative effects on Nanfeng citrus under visible light illumination condition.

Activation of macrophages by an exopolysaccharide isolated from Antarctic P sychrobacter sp.B-3

An exopolysaccharide(EPS) was isolated and purified from an Antarctic psychrophilic bacterium B-3,identified as Psychrobacter sp.,and the activation of RAW264.7 cells by B-3 EPS was investigated.The results show that B-3 EPS,over a certain concentration range,promoted cell viability,nitric oxide production,tumor necrosis factor(TNF)a secretion,and phagocytic ability.Furthermore,TAK-242,an inhibitor of the toll-like receptor 4(TLR4) significantly reduced nitric oxide production by these cells after stimulation with B-3 EPS.Moreover,B-3 EPS induced p65 phosphorylation and IκBα degradation in these cells.In conclusion,B-3 EPS might have activated RAW264.7 cells by combining with TLR4 on cell surface and triggering activation of NF-κB signaling pathways,implying that this EPS could activate macrophages and regulate initial immune response.

Blast Resistance of Rice Induced by Ag-antibiotic 702

To explore rice blast resistance induced by Ag-antibiotic 702,different concentrations of Ag-antibiotic 702 were spayed on susceptible variety Luliangyou 996 at three-leaf and one-heart stage,to screen the optimal concentration for inducing rice blast resistance. Ag-antibiotic 702 was sprayed at seven different growth stages of rice,to determine the best growth stage for induced blast resistance and duration of blast resistance. Various treatments were inoculated with spore fluid of Magnaporthe grisea at 4 d post spraying,and disease index and incidence rate as well as induction effect of different treatments were investigated after 7 d.The results showed that six concentrations of Ag-antibiotic 702 could induce rice resistance against blast,and 15 μg/mL led to the highest blast resistance; spraying15 μg/mL Ag-antibiotic 702 at seven different growth stages could induce rice resistance against blast; the three-leaf and one-heart stage was the best growth stage for inducing rice blast resistance,and the relative induction effect reached 56. 56%; rice blast resistance was the highest at 48-96 h post spraying,and duration of induced rice blast resistance exceeded 144 h. The study will provide useful experimental data for further development of Ag-antibiotic 702 and application of prevention and control methods against rice blast.

Inhibition Mechanism of Antifungal Monomer DZP8 against Rhizoctonia solani

In order to study the inhibition mechanism of antifungal monomers DZP8 produced by streptomyces 702 strain against Rhizoctonia solani, the effects of DZP8 on mycelial morphology were investigated under light microscope, and the effects of DZP8 on mycelial inclusion leakage and cell membrane damage of mycelium were determined. The results showed that DZP8 caused a series of changes in mycelial morphology of R. solani in liquid culture condition. DZP8 treatments with concentrations of 1.81 and 3.35 μg/mL for 24 h caused big vacuole, rough surface and more inclusions of mycelium. With the treatment time prolonging, the mycelium distorted and appeared irregular constriction. DZP8 treatment with concentration of 20.10 μg/mL led to the increase of conductivity of mycelium culture liquid, leakage of soluble sugar and protein, and the lipid peroxidation of mycelium membrane. It was found that DZP8 at a very low concentration could cause changes of mycelial morphology of R. solani, while only a certain concentration could cause significant damage to cell membrane. This indicated that cell membrane might be one of the action sites of DZP8, and it might have other action sites or mechanism.

Evaluation of 0.3% Agricultural Antibiotic 702 AS against Three Pathogenic Fungi of Rice

[Objective] The paper was to study control effect and bio-control potential of 0.3% agricultural antibiotic 702 aqueous solution(AS) against Rhizoctonia solani,Magnaporthe grisea and Ustilaginoidea virens. [Method] The toxicity effect of 0.3% agricultural antibiotic 702 AS against R. solani,M. grisea and U. virens were studied by mycelial growth rate method and mycelial wet weight method. [Result] The EC50 and EC90 values of agricultural antibiotics 702 AS to R.solani,M. grisea and U. virens in vitro condition were 4.16 and 16.17 mg/L,16.05 and 41.85 mg/L,111.2 and 389.0 mg/L,respectively. Agricultural antibiotics 702 AS had good inhibition effect against R. solani and M. grisea and could basically achieve the effects of commercially available Jinggangmycin and kasugamycin,but the inhibition effect against U. virens was relatively weak. The preventive activity of aqueous solution was obviously higher than treatment activity.[Conclusion] 0.3% Agricultural antibiotic 702 AS is considered to be a potential biological control agent against R. solani,M. grisea and U. virens,and can be used as the potential alternative pesticide of Jinggangmycin.

Effects of Exogenous Nutrient Additions on Azodye Wastewater Treatment

Since the azo dyes widely used in textile-dyeing industry are toxic,highly persistent,and ubiquitously distributed in the environment,their treatment efficiencies and fade in ecosystems have received worldwide attention.Due to the microbes play important roles in azo-dye degradation,exogenous nutrient addition is used as a promising biostimulation strategy to improve the treatment efficiencies of azo-dye wastewater.However,little is known about the effects of different kinds of exogenous nutrients on the azo-dye wastewater treatment directly.Here,three kinds of common nutrients,glucose,starch and yeast extract,were added to the sequencing batch reactors(SBR)for the treatment of wastewater containing acid red 73 to compare the biostimulation efficiencies by investigating the dye's removal efficiencies and the changes of related water qualities.Our results showed that the reactors added yeast extract had highest removal efficiencies and chemical oxygen demand(COD)as well,followed by glucose and starch.The removal rates of acid red 73 and COD were 90.63%and 87.3%in the reactors added yeast extract,respectively,while 86.49%and 78.4%in those with glucose and 85.38%and 75.2%in those with starch.This study provided some useful information for the biostimulation strategy of azo-dye wastewater treatment and preliminarily suggested that yeast extract would be the optimal choice.

Triglycerides Isolated from <i>Streptomyces</i>sp. ZZ035 and Their Nuclear Magnetic Resonance Spectroscopic Characters

Streptomyces sp. ZZ035 isolated from a folk medicinal soil sample in China showed remarkable antimicrobial activities. During the isolation of secondary metabolites, a white crystal powder (1) was isolated from the broth of this strain. Its nuclear magnetic resonance (NMR) and infrared (IR) spectra indicated that it was a complex composed of triglycerides. Next, six C15-17 long- chain fatty acids derived from these triglycerides were respectively identified as n-pentadecanoyl, 12-methyltetradecanoyl, 14-methyl pentadecanoyl, palmitoyl, 15-methyl hexadecanoyl and 14-methyl hexadecanoyl using the gas chromatography-mass spectroscopy (GC-MS) technology. Finally, the 13C and 1H assignments of 1 were achieved through the analyses of NMR data. Based on above, their detailed NMR spectroscopic elucidation and meticulous 13C, 1H assignments, especially the split peaks and coupling correlation of protons attached on the glycerol carbons, were performed for distinguishing triglycerides from other glycerides and for the identification of the long-chain fatty acids, and which would be helpful to the qualitative and quantitative analyses of tri-, di-and mono-glycerides.

Screening of Extraction Methods for Protein from Potato Tubers

[Objectives]This study was conducted to screen different extraction methods for protein from potato tubers.[Methods]The bud eye,lateral and pith parts of Qingshu 168 and Kexin 1 were used as test materials,and were extracted by acetone extraction,trichloroacetic acid(TCA)extraction,salt extraction,alcohol extraction and phenol extraction,respectively.The protein contents of different parts were determined by spectrophotometry.A 2-factor randomized block design was used to study the effects of varieties,extraction methods,sampling parts and combinations of different factors on protein content.[Results]The protein contents of different varieties were different,and Qingshu 168 was significantly higher than Kexin 1(P<0.05),reaching 128.0 mg/L.The protein contents obtained by different extraction methods were different.Acetone extraction,salt extraction and TCA extraction showed no significant differences in the protein content,but they were significantly higher than phenol extraction and alcohol extraction(P<0.05).The protein contents obtained by the former three were in the range of153.3-159.7 mg/L.The protein content in the bud eye part of potato tubers was significantly higher than those in the lateral and pith parts(P<0.05),reaching140.7 mg/L.There were significant differences among different combinations of varieties,extraction methods and sampling parts(P<0.05).The comprehensive comparison showed that the salt extraction method was suitable for the bud eye part of Qingshu 168,and the TCA extraction method was suitable for the bud eye part of Kexin No.1.[Conclusions]This study provides a reference for potato protein extraction and further research.