Seafood as a Reservoir of Gram-negative Bacteria Carrying Integrons and Antimicrobial Resistance Genes in Japan

PCR and DNA sequencing were used to screen and characterize integrons and resistance genes in Gram-negative bacteria isolated from seafood products in Japan.A total of 215 Gram-negative bacteria were isolated from local and imported seafood samples collected from retail markets in Hiroshima Prefecture.Class 1 integrons containing gene cassettes encoding resistance to trimethoprim

Portage of Bacteria Responsible of Foodborne Illness in Scholarly Canteens (Republic of Benin)

This study has determined the portage of bacteria responsible for foodborne illness in the school canteen staff. 336 samples taken on the nose, mouth and hands were collected. Microbiological analyses were realized and several pathogenic bacterial strains were isolated from the vendors: Staphylococcus aureus (26/122), sulphite-reducing clostridia (14/122) and Escherichia coli (10/122). The food vendors in the school canteen may be the vectors of germs that cause food poisoning among young students.

Rapid biosorption and reduction removal of Cr(Ⅵ) from aqueous solution by dried seaweeds

Four types of common seaweeds(Laminaria japonica,Undaria pinnatifida,Porphyra haitanensis,and Gracilaria lemaneiformis) were examined to remove Cr(Ⅵ) ions from aqueous solution.The experimental parameters that affected the biosorption process including pH,biomass dosage,contact time and temperature were investigated via batch experiments.The surface characteristics of seaweeds before and after Cr(Ⅵ) adsorption were studied with scanning electron microscopy and Fourier transform infrared spectroscopy.The results show that an initial solution with the pH of 1.0 is most favorable for Cr(Ⅵ) adsorption.Rapid adsorption is observed in the initial stage and adsorption equilibrium state is reached within 1 h.The adsorption efficiency by Porphyra haitanensis is the maximum among four types of seaweed powders,followed by Laminaria japonica and Undaria pinnatifida with biosorption efficiency up to 90%.The removal rate of Gracilaria lemaneiformis is less than 60%.The kinetic data obtained using the seaweeds are found to follow pseudo-second order kinetic model.Experimental sorption data adequately correlate with the Langmuir model.FTIR indicates that amino and carboxyl groups play an important role in the process of Cr(Ⅵ) adsorption and a large percentage of Cr(Ⅵ) ions are reduced by reductive groups on the surface of seaweeds.

Overexpression and characterization of a thermostable β-agarase producing neoagarotetraose from a marine isolate Microbulbifer sp.AG1

An agarase gene containing 1 302 bp was cloned from Microbulbifer sp. AG1. It encoded a mature protein of 413 amino acids plus a 20-residue signal peptide. The recombinant enzyme without the signal peptide was expressed and purified from Escherichia coli BL21(DE3). When agarose was used as a substrate, the optimal temperature and pH for the enzyme were 60℃ and 7.5, respectively. The recombinant agarase showed excellent thermostability with 67% and 19% of residual activities after incubation at 50℃ and 60℃ for 1 h, respectively.Except SDS, the recombinant agarase had a relatively good resistance against the detected inhibitors, detergents and urea denaturant. Thin layer chromatography analysis and enzyme assay using p-nitrophenyl-α/β-Dgalactopyranoside revealed that the recombinant agarase was a β-agarase that degraded agarose into neoagarotetraose as the main end product. The enzymatic hydrolysis products with different degree of polymerization exhibited the antioxidant activities.

Biochemical and Microbial Properties of Palm Wine: Effect of Tapping Length and Varietal Differences

The effects of variety and tapping length on several physical, biochemical, nutritional and micro-biological parameters (viscosity, pH, Total Titratable Acidity (TTA), fermenting microorganisms, contaminants) of palm wine extracted from two varieties of palm oil tree (Dura and Tenera) were studied. Each variety presented its own palm wine specific characteristics regarding the chosen parameters. Microbiological and biochemical contents of palm wine were determined during the tapping of Dura and Tenera felled oil palm trees for 4 weeks. Some differences in chemical compositions between fresh palm wine samples of two palm trees varieties were observed. The exudates obtained during the first day of tapping of Dura palm wine were very sugary, less sour and did not contain substantial concentrations of alcohol but the highest loads of microorganisms were observed in Tenera palm wine. Throughout the tapping of palm wine, yeasts and lactic acid bacteria population changed with undoubtedly influence on the palm wine quality.

Transformation of coccolithophorid Emiliania huxleyi harboring a marine virus(Coccolithoviruses)serine palmitoyltransferase(SPT)gene by electroporation

Emiliania huxleyi is the most prominent modern coccolithophore,a group of marine unicellular eukaryotes that play a critical role in ocean biogeochemistry.Coccolithoviruses are large double stranded DNA viruses,which is responsible for the demise of large oceanic blooms formed by E.huxleyi.E.huxleyi virus(EhVs)acquired a series of enzyme-coding genes predicted to be involved in the sphingolipid biosynthesis by horizontal gene transfer between virus-host.Currently,there is limited experimental validation identifying the functions of these genes in EhV.Genetic transformation of eukaryotic cells is a powerful tool to get an insight into gene functions of the studied organisms.Serine palmitoyltransferase(SPT)catalyzes the first committed step in de novo sphingolipid biosynthetic pathway.Here,a novel vector system for the transformation of E.huxleyi was designed.It contained fragments of promoter and terminator sequences of E.huxleyi endogenic fucoxanthin chlorophyll a/c-binding protein gene“fcp”and harbored EhV-99B1 spt gene.The resultant recombinant transformation vectors pEhux-I-spt and pEhux-II were co-transferred into E.huxleyi BOF92 by electroporation.Transformants were obtained upon glufosinate-ammonium selection,and confirmed by Southern hybridization,genome PCR,qRT-PCR and Western blot screening of spt gene,which indicated that spt gene was integrated into the nuclear genome and was expressed at the mRNA and protein levels.The expression of the viral spt gene led to differences in lipid compositions analyzed using thin-layer chromatography(TLC).The results present the genetic transformation system for E.huxleyi,providing additional genetic resource with potential for exploring basic biological questions such as the virus-host interactions.

Substitution of His260 residue alters the thermostability of Pseudoalteromonas carrageenovora arylsulfatase

This study aimed to improve the thermostability of arylsulfatase from Pseudoalteromonas carrageenovora. A library of P. carrageenovora arylsulfatase mutants was constructed by introducing random mutagenesis using error-prone PCR. After screening, two mutants of H260L and D84A/H260L showed enhanced thermal stability than the wild-type predecessor (WT). Site-directed mutagenesis demonstrated that only amino acid residue at Position 260 plays an important role in the thermostability of P. carrageenovora arylsulfatase. Thermal inactivation analysis showed that the half-life (t1/2) values at 55°C for H260L, H260I, H260Q, H260F and H260R were 40.6, 48.4, 30.9, 29.1 and 34.5 min, respectively, while that of WT was 9.1 min. Structure modeling demonstrated that the additional hydrogen bonds and/or optimization of surface charge-charge interactions could be responsible for the increased thermostability imparted by H260L, H260I, H260Q, H260F and H260R.

Purification and biochemical characterization of manganesecontaining superoxide dismutase from deep-sea thermophile Geobacillus sp. EPT3

Thermostable SOD is a promising enzyme in biotechnological applications. In the present study, thermo-phileGeobacillussp.EPT3 was isolated from a deep-sea hydrothermal field in the East Pacific. A thermo-stable superoxide dismutase （SOD） from this strain was purified to homogeneity by steps of fractional am-monium sulfate precipitation, DEAE-Sepharose chromatography, and Phenyl-Sepharose chromatography. SOD was purified 13.4 fold to homogeneity with a specific activity of 3 354 U/mg and 11.1% recovery. SOD fromGeobacillussp. EPT3 was of the Mn-SOD type, judged by the insensitivity of the enzyme to both KCN and H2O2. SOD was determined to be a homodimer with monomeric molecular mass of 26.0 kDa. It had high thermostability at 50＆#176;C and 60＆#176;C. At tested conditions,SOD was relatively stable in the presence of some inhibitors and denaturants, such asβ-mercaptoethanol （β-ME）, dithiothreitol （DTT）, phenylmethylsulfonyl fluoride （PMSF）, urea, and guanidine hydrochloride.Geobacillussp. EPT3 SOD showed striking stability across a wide pH range from 5.0 to 11.0. It could withstand denaturants of extremely acidic and alkaline conditions, which makes it useful in the industrial applications.

New Ionic Liquids with Buffering and Chelating Abilities for Enzyme Engineering

Ionic liquids (ILs) with buffering and chelating abilities were designed and synthesized on the basis of ethylenediaminetetraacetic acid (EDTA) for the development of buffered enzymatic IL systems and for enzymatic reaction in heavy metal containing aqueous system. Transesterification activity of Candida antarctica lipase B dissolved in the hydroxyl-functionalized IL was buffer dependent. High activity and outstanding stability was obtained with the buffered enzymatic IL systems for the transesterification. In heavy metal containing aqueous system, EDTA IL buffers as Hg2+ chelators protected horseradish peroxidase (HRP) against Hg2+-induced denaturation and precipitation. Higher pH favored the protection, while at lower pH the protection diminished. We can conclude that the new ILs possess both buffering and chelating abilities.

Carposome productivity of Pleurotus ostreatus and Pleurotus eryngii growing on agro-industrial residues enriched with nitrogen,calcium salts and oils

The suitability of the abundant agro-industrial residues wheat straw(WS;control),barley and oats straw(BOS)and rice husk(RH),supplemented with various sources of oils(sunflower,corn oil),nitrogen(peptone,yeast extract)and calcium salts(CaSO_(4)⋅2H_(2)O,CaCl2),as novel substrates in solid-state fermentation of selected Pleurotus ostreatus and P.eryngii mushrooms was investigated.The possible effect of different additives on mycelial growth rate,biomass production and endoglucanase,laccase and lipase biosynthesis were evaluated.Moreover,their impact on essential cultivation aspects(earliness,total mushroom yield,biological efficiency)and carposome quality parameters(weight,morphological characteristics)was assessed.Both fungi showed their highest growth rates on BOS substrates and the most positive implementation was CaSO_(4)⋅2H_(2)O 6%w/w(Kr=9.58 mm/day;P.ostreatus,Kr=9.42 mm/day;P.eryngii),while different additives led to enhancement of biomass production.Pleurotus species demonstrated minimal levels of endoglucanase activity,with values ranging from 0.01 to 0.42 U/g of dry weight,regardless of the substrate and the stage of colonization.On the contrary,the maximum values of laccase activity were observed at 50%of colonization on BOS and RH,while supplementation with nitrogen and calcium sources positively affected its biosynthesis.P.ostreatus and P.eryngii cultivated on BOS supplemented with peptone at 2 and 5%w/w,synthesized significant laccase amounts,i.e.,12,165.78 and 8,624.55 U/g d.w.,respectively.Satisfactory amounts of lipase were produced,especially in substrates supplemented with sunflower 2%w/w,in quantities up to 1.42 U/g d.w.,whereas the highest lipase activity was achieved by P.eryngii on WS supplemented with corn oil at 2%w/w,with a value of 4.25 U/g d.w.being recorded.Regarding fermentation of Pleurotus species in polypropylene bags,WS and BOS supported faster colonization and shorter earliness period than RH substrates,whereas supplementation did not seem to affect these culture parameters.Furthermore,oils supplementation had a positive effect on BE of both species,with values up to 100%for P.ostreatus and 80%for P.eryngii on WS and BOS,whereas on RH the lowest BE values were detected.Morphological characteristics were not significantly affected by the additives.Results indicate the positive impact that certain additives have on mushroom productivity and production of enzymes with great financial and environmental importance.

Green synthesis of Au@WSe_(2) hybrid nanostructures with the enhanced peroxidase-like activity for sensitive colorimetric detection of glucose

We report a colorimetric method for glucose detection based on Au nanoparticle-decorated WSe_(2)(Au@WSe_(2))hybrid nanostructures.These hybrid structures are easily synthesized by simply stirring HAuCl_(4) precursor with WSe_(2) nanosheets in aqueous solution.Owing to strong synergistic catalytic effects of Au nanoparticles and WSe_(2) nanosheets,the Au@WSe_(2) hybrid nanostructures exhibit enhanced peroxidase-like activity(about 2-fold higher compared to WSe_(2) nanosheets alone)for 3,3',5,5'-tetramethylbenzidine oxidation by H_(2)O_(2).Based on the highly catalytical property,the colorimetric method for glucose detection is established by coupling glucose oxidase(GOx).The detection limit of glucose is 3.66 pM.Moreover,the proposed colorimetric method is applicable to glucose detection in serum samples and is promising for applications in biomedical fields.

Gold nanoparticle-decorated MoSe_(2) nanosheets as highly effective peroxidase-like nanozymes for total antioxidant capacity assay

A visual colorimetric detection strategy is reported for total antioxidant capacity(TAC)assay by using 3,3',5,5'-tetramethylbenzidine(TMB)oxidation as chromogenic substrate based on gold nanoparticle-decorated MoSe_(2) nanosheets(Au@MoSe_(2)).Au@MoSe_(2) nanostructures exhibit high peroxidase-like activity and can catalyze H_(2)O_(2)to oxidize TMB.Based on inhibition effect of ascorbic acid(AA)on TMB oxidation,a facile and sensitive colorimetric method was developed for AA detection.Under optimal conditions,the proposed method showed a sensitivity for AA in a concentration range from 2 to 120μM and limit of detection was 0.41μM.Furthermore,the method was employed for TAC assay in actual samples,including commercial beverages and vitamin C tablets.This work represents a model in nanostructure design and will lead to further development of TAC assay in evaluation of antioxidant food quality.

利用液体发酵提高灵芝三萜产量及HPLC-ESI-MS鉴定灵芝菌丝中灵芝酸成分(英文)

目的:通过优化液体发酵培养基提高灵芝三萜产量,并对其三萜成分进行初步鉴定。创新点:麦芽作为啤酒生产原料,含有丰富的糖类及其他营养物质,并且价格低廉,来源广泛。本文首次将麦芽汁作为碳源物质以提高灵芝三萜产量,并对其所含灵芝酸成分进行初步鉴定。方法:以灵芝菌丝生物量和胞内三萜产量为检测指标,对其液体发酵培养基中的碳(C)源和氮(N)源进行筛选,并利用单因素实验确定其相应浓度,再经响应面法优化液体发酵培养基(C源、N源、pH)(表1)。另一方面,酸碱转化法纯化灵芝三萜提取物后,利用高效液相色谱-电喷雾-质谱(HPLC-ESI-MS)法初步鉴定灵芝所含灵芝酸成分(表2)。结论:本实验响应面结果显示:当培养基含麦芽汁4.1%,酵母浸出粉1.89%,pH为5.40时,灵芝液体发酵菌丝生物量及胞内三萜产量均达到最优,分别为1.87g/100ml和93.21mg/100ml。经HPLCESI-MS鉴定,初步确定其含有10种灵芝酸成分。

高通量测序在探究中国传统发酵食品菌群多样性及动态变化中的应用（英文）

中国传统发酵食品有着上千年的历史,已经成为中国饮食文化中不可缺少的部分。微生物是发酵食品的灵魂,为了探究传统发酵食品中的微生物组成,多种依赖培养和非培养技术都已应用于不同发酵体系的微生物菌相分析中。高通量测序技术是近几年兴起的生物学技术,它极大地方便了环境微生物多样性的研究,促进了我们对复杂微生物环境的认知,其在发酵食品中的应用提升了我们对发酵食品品质与微生物关系的认识水平。本文综述了高通量测序技术在探究我国传统发酵食品菌相中的应用,总结分析了不同发酵体系中微生物的组成和动态变化。

传统酸面团制备的馒头特征风味物质的研究（英文）

目的:通过同时蒸馏萃取(SDE)、顶空固相微萃取(SPME)及吹扫捕集(P&T)技术结合气相色谱-嗅闻-质谱(GC-O-MS)技术对传统酸面团制备的馒头特征风味成分进行分析,并初步探讨特征风味形成机理。创新点:首次采用不同风味物质分析技术探索传统酸面团制备的馒头关键风味成分。方法:采用同时SDE、SPME及P&T方法,对不同传统酸面团制备的馒头特征风味物质进行分析。结论:SDE结果显示,反式-2-壬烯醛和反式-2,4-癸二烯醛为传统酸面团馒头中关键风味成分。SPME和P&T方法表明乙醇和醋酸是关键风味物质。因此,需要结合不同技术方法,全面分析传统酸面团制备的馒头中特征风味成分。

灵芝菌生物转化大豆异黄酮及其产物对结直肠癌细胞HTL-9的体外凋亡诱导研究（英文）

目的:通过灵芝菌生物转化大豆异黄酮,得到富含苷元及灵芝活性成分的多因子转化产物,并研究了转化产物对结直肠癌细胞HTL-9的体外凋亡诱导,初步探讨转化产物的抗癌活性及机理。创新点:灵芝是一种珍贵的药用真菌,大豆异黄酮的苷元物质也具有重要的药理活性,本文首次利用灵芝菌液体发酵的匀浆液生物转化大豆异黄酮,所得到的产物中大豆苷元与染料木素转化率高,同时还富集了灵芝菌的活性成分,并对转化产物的抗癌活性及机理进行了初步探讨。方法:首先利用灵芝菌液体发酵的匀浆体系生物转化大豆异黄酮(图1)。其次,对转化产物的抗癌活性进行研究,主要包括对癌细胞存活率(图2)、细胞凋亡(图3)及细胞周期分布(图4)的影响。最后,利用蛋白质印迹(Western-blot)与逆转录聚合酶链反应(RT-PCR)技术对凋亡相关的基因和蛋白进行检测(图5和表2),初步探讨转化产物的体外抗癌机理。结论:本实验结果显示,转化产物中大豆苷元及染料木素的转化率分别为96.63%和87.82%,其中染料木素的含量可达(703.21±4.35)mg/g,同时转化产物中还富含了灵芝菌的活性成分。其次,对转化产物抗癌活性研究发现,其能有效降低HTL-9细胞的存活率,可通过将细胞阻滞于G1期而诱导细胞晚期凋亡。此外,转化产物(100μg/ml)还可明显上调Bax、Caspase-3、Caspase-8、Cyto-c和p53的表达量,而Survivin和NF-κB表达量发生明显下调。结果表明,转化产物主要通过线粒体途径诱导细胞凋亡,但同时还调控多个与凋亡相关的基因。