Characterization of a novel bacteriocin produced by Lactobacillus sakei LSJ618
英国文学史名词解释
名词解释Heroic Couplet: a couplet consisting of two rhymed lines of iambic pentameter, and written in an elevated style.1．Renaissance: a revival or rebirth of the artistic and scientific revival which originated in Italy in the 14th century and gradually spread all over Europe. It has two features: a thirsting curiosity for the classical literature and keen interest in activities of humanity.2．Sonnet: 14-line lyric poem, usually written in rhymed iambic pentameter.3．Blank verse: poetry written in unrhymed iambic pentameter.4．Neoclassicism: the Enlightenment brought about a revival of interest in Greek and Roman works. This tendency is known as Neoclassicism.5．Sentimentalism: it was one of the important trends in English literature of the later decades of the 18th century. It concentrated on the free expression of thoughts and emotions, and presented a new view of human nature which prized feeling over thinking, passion over reason. 6．Romanticism: imagination, emotion and freedom are certainly the focal points of romanticism. The particular characteristics of the literature of romanticism include: subjectivity and an emphasis on individualism; freedom from rules; solitary life rather then life in society; the beliefs that imagination is superior to reason; and love of and worship of nature. 7．LakePoets: the English poets who lived in and drew inspiration from the Lake District at the beginning of the 19th century.8．Byronic Heroes: a variant of the Romantic heroes as a type of character( enthusiasm, persistence, pursuing freedom), named after the English Romantic Poet Gordon Byron. 9．Realism: seeks to portray familiar characters, situations, and settings in a realistic manner. This is done primarily by using an objective narrative point of view and through the buildup of accurate detail.10．Aestheticism: an art movement supporting the emphasis of aesthetic values more than socio-political themes for literature, fine art, music and other arts.11．Stream-of-Consciousness: it is a literary technique that presents the thoughts and feelings of a character as they occur without any clarification by the author. It is a narrative mode. 12．Dramatic Monologue: a kind of narrative poem in which one character speaks to one or more listeners whose replies are not given in the poem.13．Iambic Pentameter: a poetic line consisting of five verse feet, with each foot an unstressed syllable followed by a stressed syllable, that is, with each foot an iamb.14．Epic: a long narrative poem telling about the deeds of a great hero and reflecting the values of the society from which it originated.15．Elegy: a poem of mourning, usually over the death of an individual; may also be a lament over the passing of life and beauty or a meditation of the nature of death; a type of lyric poem. 16．Canto: a section of a long poem. The cantos can be a great poem17．Ode: a complex and often lengthy lyric poem, written in a dignified formal style on some lofty or serious subjects. Odes are written for a special occasion, to honor a person or a season or to commemorate an event.Spenserian Stanza: a nine-line stanza made up of 8 lines of iambic pentameter ending with an Alexandrine. Its thyme scheme is ababbcbcc. This stanza was common to travel literature. 18．Metrical Pattern: a lyric poem of five 14-lined stanzas containing four tercets and a closing couplet. The rhyme scheme is aba bcb cdc ded ee.文学史中古时期1.Beowulf?贝奥武甫?: the natural epic of the English people; Denmark story, alliteration, metaphor, understatements2.Sir Gawain and Green Knight?高文爵士和绿衣骑士?3.Geoffrey Chaucer(杰弗里乔叟〕：the Father of English Poetry; The Canterbury Tales?埃特伯雷故事集?〔24stories)文艺复兴时期1.Thomas More: Utopia?乌托邦?- the communication between more and the traveler which just came back from Utopia.2.Francis Bacon: the first English Essayist; Essays?随笔集?- Of Studies, Of Truth (philosophical and literary works)3.Thus Wyatt: first to introduce the sonnet into English literature.4.Edmund Spenser: Poet's poet; The Fairy Queen?仙后?(to Queen Elizabeth I)5.William Shakespeare:Sonnet 18(Shall I compare thee to a summer's day)17世纪英国文学1.John Donne: the leading poet of Metaphysical school of poetry; A Valediction: Forbidding Mourning?别离：莫忧伤?2.John Milton: Paradise Lost?失乐园?〔a revolt against God's authority), Paradise Regained?复乐园?〔how Christ overcame Santa) ——stories were taken from Bible3.John Bunyan: the son of Renaissance; Pilgrim's Progress?天路历程?(imagination, shadowing, realistic religious allegory)18世纪英国文学Novel：1.the age of reason, classicism, sentimentalism and romanticism (novels, prose, dramas, poetry)2.Daniel Defoe: representative of English realistic novel; Robinson Crusoe?鲁滨逊漂流记?(the development of a young man from a naive and artless youth to a clever and hardened man)3.Jonathan Swift: Gulliver's Travels?格列佛游记?(fictional, satirical- human nature, the European Government, the differences between religions, whole English state system)4.Henry Fielding: the Father of English novel; The History of Tome Jones, a Foundling ?汤姆琼斯?，satiricPoetry：5.Thomas Gray: Elegy Written in a Country Churchyard?墓园挽歌?6.Alexander Pope: perfected in heroic couplet; An Essay on Criticism?论批评?7.William Blake: pre-romantic; Songs of Innonce?天真之歌?，Songs of Experience?经历之歌?-London, The Tiger8.Robert Burns: A Red Red Rose?一朵红红的玫瑰?Drama:9.Richard Brinsley Sheridan:Master of Comedy of manners;The School for Scandal?造谣学校?浪漫主义时期1798-18321.William Wordsworth: the Lake Poets; The Prelude?序曲?；I Wondered Lonely as a Cloud?我似流云天自游?；The Solitary Reaper?孤寂的割麦女?；features: poet of nature and human heart2.Samuel Taylor Coleridge: the first critic of the Romantic school; The Rime of the Ancient Mariner?古舟子咏?3.George Gordon Byron: vigorous, strong and beautiful; Childe Harold's Pilgrimage?恰尔德哈罗尔德游记?(spenserian stanza, fights for liberty); Don Juan?唐璜?(a broad critical picture of European life); When We Two Parted?昔日依依别?；She Walks in Beauty?她走在美的光影中?；The Isles of Greece?哀希腊?4.Percy Bysshe Shelley: Ode to the West Wind?西风颂?-赞颂西风，希望与其严密相连; Prometheus Unbound?解放了的普罗米修斯?(the victory for man's struggle against tyranny and oppression)5.John Keats: sensuous, colorful and rich in imagery; Ode to a Nightingale?夜莺颂?；Ode on a Grecian Urn?希腊古瓮颂?6.Walter Scott: Father of Historical Novel; combine historical fact and romantic imagination7.Jane Austen: wit, dry humour, subtle irony,realistic; Pride and Prejudice?傲慢与偏见?(Elizabeth and Darcy)；Sense and Sensibility?理智与情感?；Emma?爱玛?8.Charles Lamb: Poor Relations?穷亲戚?; Dream-children?童年梦幻?; A Reverie?梦想曲?维多利亚时期1.summit：realistic novel2.Charles Dickens: critical realist writer; humour, wit, happy endings; A Tale of Two Cities?双城记?(London & Paris, where there is oppression, there is revolution); David Copperfield?大卫科波菲尔?；Oliver Twist?雾都孤儿?；Hard Time?困难时世?；Great Expectations?远大前程?；Dombey and Son?董贝父子?；Pickwick Papers?匹克威克外传?3.William Makepeace Thackeray: Vanity Fair?名利场?(to satirize the social more, decadence and corruption of his time; to criticize the values measured by wealth)4.George Eliot: novelist, her novels are celebrated for their realism and psychological insights; Adam Bede?亚当比德?(social inequality)；The Mill on the Floss?弗洛斯河上的磨坊?；Silas Marner?织工马南?5.Alfred Tennyson: succeeded Wordsworth as Poet Laureate in 1850; Break, Break, Break?拍吧，拍吧，拍吧?；Crossing the Bar?过沙洲?6.Robert Browning: dramatic monologues; My Last Duchess?我已故的公爵夫人?7.The Bronte Sisters:Charlotte: Jane Eyre?简爱?：简·爱是一个心地纯洁、善于思考的女性，她生活在社会底层，受尽磨难。
刺杀小说家英语作文摘抄
In the realm of literature,the concept of a fictional assassination is a fascinating and complex theme that has been explored in various forms.Here are some excerpts from English essays that delve into the intricacies of this subject:1.The Power of Words:In the world of fiction,a writer wields the power to create and destroy.The act of assassinating a character is a demonstration of this power,often serving as a pivotal moment in the narrative that can reshape the entire story.2.Moral Dilemmas in Storytelling:Assassinating a character in a novel is not merely a plot device it is a moral decision that the author must grapple with.It raises questions about the ethics of storytelling and the responsibility of the writer towards their creations.3.The Impact on the Reader:When a beloved character is assassinated in a novel,it can elicit strong emotional responses from the reader.This emotional connection is a testament to the power of storytelling and the deep bonds that readers form with fictional characters.4.Symbolism and Metaphor:The act of assassination in fiction often carries symbolic weight.It can represent the end of an era,the triumph of evil over good,or the inevitable consequences of certain actions.It serves as a metaphor for larger themes within the story.5.The Role of Foreshadowing:Skillful writers use foreshadowing to prepare the reader for the eventual assassination of a character.This technique can heighten the tension and anticipation,making the moment of the assassination all the more impactful.6.The Aftermath of Assassination:The death of a character is not the end of their story. The aftermath of an assassination can be just as significant as the event itself.It can lead to character growth,plot twists,and a deeper exploration of the storys themes.7.The Assassin as a Character:In some narratives,the assassin is a character in their own right.Their motivations,backstory,and moral compass add another layer of complexity to the story,inviting the reader to question the nature of justice and the blurred lines between right and wrong.8.Cultural and Historical Context:Assassination in literature can also reflect cultural and historical contexts.It can be a commentary on political assassinations,a critique of power structures,or an exploration of the human capacity for violence.9.The Unpredictability of Life:The unpredictability of life is mirrored in the unpredictability of fiction.An assassination can serve as a reminder that life is fragile andthat the threads of our existence can be severed at any moment.10.The Rebirth of a Story:In some cases,the assassination of a character can lead to the rebirth of a story.It can open up new narrative paths,introduce new characters,and provide a fresh perspective on the world the author has created.These excerpts highlight the multifaceted nature of character assassination in literature, touching on themes of power,morality,emotional impact,and narrative structure.They provide a glimpse into the rich tapestry of storytelling and the profound effects that such a dramatic event can have on a work of fiction.。
英美文学名词解释
Metaphysical Poetry玄学派诗歌: The term "metaphysical poetry" is commonly used to name the work of the 17th century writers who wrote under the influence of John Donne. Metaphysical poetry is characterized by verbal wit and excess, ingenious structure, irregular meter, colloquial language, elaborate imagery, and a drawing together of dissimilar ideas. The metaphysical poets tried to break away from the conventional fashion of the Elizabethan love poetry.Blank Verse素体诗：V erse written in unrhymed iambic pentameter. Blank verse has been the dominant verse form of English drama and narrative poetry since the mid-sixteenth century. It was introduced by Henry Howard from Italy, and then Shakespeare transformed blank verse into a supple instrument, uniquely capable of conveying speech rhymes and emotional overtones.Romance传奇文学: it was a long composition ,sometimes in verse, sometimes in prose, describing the life and adventure of a noble hero. //Any imagination literature that is set in an idealized world and that deals with a heroic adventures and battles between good characters and villains or monsters.Heroic Couplet英雄双韵体: Heroic couplet is a rhymed couplet of iambic pentameter. It is Chaucer who used it for the first time in English in his work The Legend of Good Woman.Epic史诗: An epic is a long oral narrative poem that operates on a grand scale and deals with legendary or historical events of national or universal significance. Many epics were drawn from an oral tradition and were transmitted by song and recitation before they were written down.Sonnet十四行诗: abab cdcd efef gg A fourteen-line lyric poem, usually written in rhymed iambic pentameter. It was introduced by Wyatt and developed by Surrey and was thereafter widely used, notably in the sonnet sequences of Shakespeare. A sonnet generally expresses a single theme or idea.Puritanism清教主义: Puritanism was the religious doctrine of the revolutionary bourgeoisie during the English Revolution. It stressed the virtue of self-discipline, thrift, hard work and unceasing labour. It advocated a strict moral code which prohibited many worldly pleasures.Naturalism自然主义: naturalism is a literary trend prevailing in Europe, especially in France and Germany, in the second half of the 19th century. According to the theory of naturalism, literature must be “true to life”and exactly reproduce real life, including all its details without any selection. Naturalism writers usually write life of the poor and oppressed, or the “slum life”, but by giving all the details of life without discrimination, they can only present the external appearance instead of the inner essence of real life. Humanism人文主义:Humanism is the key-note of the Renaissance. According the humanists, both man and world are hindered only by external checks from infinite improvement. They emphasizing the dignity of human beings and the importance of the present life. Man could mould the world according his desire, and attain happiness by removing all the external checks by the exercise of reason.Aestheticism唯美主义: prevailing at the middle of the 19th century. Aestheticism places art above life, and holds that life should imitate art, not art imitate life. All art creation is absolutely subjective and should be free from any influence of egoism. Only when art is for art’s sake, can it be immortal. They believe art should be unconcerned with controversial issues.Sentimentalism感伤主义: Sentimentalism came into being as a result of a bitter discontent among the enlighteners with social reality. The representatives of sentimentalism continued to struggle against feudalism, but they sensed the contradictions in the process of capitalist development at the same time. Dissatisfied with reason, which classicists appealed to, they appealed to sentiment, “the human heart”.Critical Realism批判现实主义：critical realism is one of the literary genres that flourished mainly in the 19th century. The English critical realists of the 19th century not only gave a satirical portrayal of the bourgeoisie and all the ruling classes, but also showed profound sympathy for the common people. Hence the use of humor and satire in the realistic novels. But the trend of their works is not of revolution but rather of reformism.Neo-Romanticism新浪漫主义:a literary trend prevailing at the end of the 19th century. Dissatisfied with the drab and the ugly social reality and yet trying to avoid the positive solution of the acute social contradictions, the neo-romanticists laid emphasis upon the invention of exciting adventures and fascinating stories to entertain the readers.Modernist现代主义：prevailing during the 20s and 30s of the 20th century. It was a movement of experiments in new technique of writing. Modernist fiction put emphasis on the description of character s’psychological activities, and so has sometimes been called modern psychological fiction.Stream of Conscience意识流:a psychological term indicating “the flux of conscious and subconscious thoughts and impressions moving in the mind at any given time independently of the person’s will”. In the 20th century, under the influence of Freud’s theory of psychological analysis, a number of writers adopted the “stream of conscious” method of novel writing. The striking feature of these novelists is their giving precedence to the depiction of the characters’ mental and emotional reaction to external events, rather than the events themselves.。
characterization of two GAD cDNA clones from arabidopsis
Characterization of Two Glutamate Decarboxylase cDNAClones from ArabidopsisFrank J.Turano*and Tung K.FangUnited States Department of Agriculture,Agricultural Research Service,Climate Stress Laboratory,Beltsville,Maryland20705Two distinct cDNA clones encoding for the glutamate decarbox-ylase(GAD)isoenzymes GAD1and GAD2from Arabidopsis(L.) Heynh.were characterized.The open reading frames for GAD1and GAD2were expressed in Escherichia coli and the recombinant proteins were purified by affinity chromatography.Analysis of the recombinant proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis suggest that GAD1and GAD2encode for58-and56-kD peptides,respectively.The enzy-matic activities of the pure recombinant GAD1and GAD2proteins were stimulated35-and13-fold,respectively,by Ca2؉/calmodulin but not by Ca2؉or calmodulin alone.Southern-blot analysis of genomic DNA suggests that there is only one copy of each gene in Arabidopsis.The GAD1transcript and a corresponding58-kD pep-tide were detected in roots only.Conversely,the GAD2transcript and a corresponding56-kD peptide were detected in all organs tested.The specific activity,GAD2transcript,and56-kD peptide increased in leaves of plants treated with10m M NH4Cl,5m M NH4NO3,5m M glutamic acid,or5m M glutamine as the sole nitrogen source compared with samples from plants treated with10 m M KNO3.The results from these experiments suggest that in leaves GAD activity is partially controlled by gene expression or RNA stability.Results from preliminary analyses of different tissues imply that these tendencies were not the same in flower stalks and flow-ers,suggesting that other factors may control GAD activity in these organs.The results from this investigation demonstrate that GAD activity in leaves is altered by different nitrogen treatments,sug-gesting that GAD2may play a unique role in nitrogen metabolism. GAD(EC4.1.1.15)catalyzes the conversion of Glu to GABA in the presence of the cofactor PLP.GAD is present in Escherichia coli(Smith et al.,1992),mammals(Erlander and Tobin,1991),and plants(Satyanarayan and Nair, 1990).In plants the enzyme has a unique feature,a CaM-binding domain at the carboxy terminus(Baum et al.,1993; Arazi et al.,1995;Gallego et al.,1995).CaM binding has been demonstrated in GAD isolated from petunia(Baum et al.,1993)and fava bean(Ling et al.,1994).In addition,the last30amino acids of the GAD1gene product from Ara-bidopsis has been shown to bind CaM(Arazi et al.,1995). In vitro analyses have shown that Ca2ϩand CaM stimulate GAD activity1-to9-fold(Ling et al.,1994;Snedden et al., 1995;Cholewa et al.,1997;Johnson et al.,1997)in partially purified protein preparations,and nearly20-fold in puri-fied preparations(Snedden et al.,1996).These findings suggest that GAD may be stimulated in vivo by Ca2ϩsignal pathways.This hypothesis is consistent with data collected from studies demonstrating the rapid increase in cytoplasmic Ca2ϩconcentrations(Knight et al.,1991,1992; Price et al.,1994;Cholewa et al.,1997)and GABA titers (Wallace et al.,1984;Mayer et al.,1990;Cholewa et al., 1997)in plant cells upon exposure to various environmen-tal stimuli.Despite a better understanding of the cellular factors that may stimulate GAD activity,the physiological roles of the enzyme or the product,GABA,have not been clearly es-tablished in plants.Since elevated GAD activity is usually seen in tissues with low cytoplasmic pH(Satyanarayan and Nair,1990),and the synthesis of GABA consumes a proton, GABA metabolism has been proposed to regulate cytoplas-mic pH in plant tissues subjected to various stress condi-tions(Streeter and Thompson,1972;Davies,1980).How-ever,Cholewa et al.(1997)demonstrated that GABA accumulation may be stimulated by Ca2ϩand not by de-creased cytoplasmic pH when plants are subjected to an abrupt cold-shock treatment.But several other physiolog-ical roles for GABA have been proposed.Selman and Coo-per(1978)suggested that GABA may provide a direct temporary reserve of carbon and nitrogen for Glu or an indirect reserve for protein synthesis.Since GABA is an inhibitor of neuron transmission in animals,Wallace et al. (1984)suggested that increased levels of GABA could alter the eating habits of insects.Recently,Ramputh and Bown (1996)demonstrated that elevated levels of GABA in the diet of oblique-banded leaf-roller larvae decreased their growth,development,and survival.In addition,Chen et al. (1994)questioned whether GABA in plants was involved in the control of ion channels,as in animal neurons.Baum et al.(1996)overexpressed a truncated version of a petunia GAD gene,which lacked the CaM-binding site,in transgenic tobacco plants and demonstrated that the CaM-binding domain was required for normal plant develop-ment and for the maintenance of GABA and Glu levels. These results provide some evidence that GAD is involved in nitrogen metabolism.Other investigators demonstrated that GAD may not be solely involved in the maintenance of cytoplasmic pH.Robinson et al.(1991)and Carroll et al. (1994)showed that GABA was a major nitrogen source for*Corresponding author;e-mail fturano@;fax 1–301–504–7521.Abbreviations:CaM,calmodulin;GABA,␥-aminobutyric acid; GAD,glutamate decarboxylase;PLP,pyridoxal5-phosphate; rGAD,recombinant GAD;TFP,trifluoperazine.Plant Physiol.(1998)117:1411–14211411freshly assimilated ammonium in nonstressed cell suspen-sions.Tuin and Shelp(1994)demonstrated that the in situ synthesis of GABA in developing soybean cotyledons was via GAD,but that GABA was rapidly metabolized to pro-vide tricarboxylic acid cycle intermediates for amino acid metabolism,possibly via the GABA shunt.In addition, Cholewa et al.(1997)reported a Ca2ϩ/CaM-independent increase in GABA when cells were treated with Glu or butyrate.Together,these results suggest not only that GABA synthesis may be a response to stress but that GAD may perform a unique physiological role in nitrogen and/or carbon metabolism via the GABA shunt. Although several of the factors that stimulate GAD ac-tivity have been identified,there is still a gap in the knowl-edge of the role(s)of transcriptional and/or posttransla-tional events in the control of GAD activity.Chen et al. (1994)demonstrated that a58-kD CaM-binding GAD was expressed in various floral parts,seeds,stems,roots,and leaves of petunia.In addition,their data suggested that there was a correlation between in vitro GAD activity, abundance of the58-kD peptide,and levels of GAD tran-script in developing leaves and germinating seeds.How-ever,this phenomenon was not observed in developing flowers.In open flowers the58-kD peptide was abundant and in vitro GABA synthesis was high,but the level of GAD transcript was almost undetectable.The authors sug-gested that posttranslational regulation in leaves and seeds may differ from that of flowers,and the process may play a role in controlling GAD activity in flowers.Chen et al.(1994)used immunoblot analysis to monitor the accumulation of GAD in germinating petunia seeds. They identified three peptides of48,58,and66kD that cross-reacted with the antiserum to a recombinant petunia GAD(58kD).There appeared to be a synchronous increase and decrease of the48-and66-kD proteins,which was inversely proportional to levels of the58-kD GAD through-out germination.These data suggest that there may be several GAD-like peptides or isoenzymes in plants.Molec-ular mass determinations of GAD from potato(45.5kD) (Satyanarayan and Nair,1985),and fava bean(62kD)(Ling et al.,1994)also suggest that there may be distinct GAD isoenzymes.The presence of several GAD isoenzymes could explain the discrepancies in apparent molecular masses of GAD from different plant species and could explain the apparent contradictions concerning physiolog-ical role(s)of GAD in plants.The existence of several isoenzymes could be due to posttranslational modifications of a single peptide or to the existence of multiple loci or genes encoding GAD.To gain a greater understanding of the function and role of the GAD isoenzymes in plant nitrogen metabolism,we initiated a study of the isoen-zymes and genes in Arabidopsis.In this study we com-bined molecular biological approaches with biochemical and immunological techniques to identify the cDNA clones and the protein/peptide components that they encode,and to demonstrate that the gene products bind CaM.In addi-tion,we determined the effects of different nitrogen sources on GAD activity in Arabidopsis.MATERIALS AND METHODSPlant MaterialArabidopsis(L.)Heynh.ecotype Columbia(Col-0)seeds were obtained from the Arabidopsis Biological Resource Center(Ohio State University,Columbus).Plants were grown in(20ϫ10ϫ6cm[lengthϫwidthϫdepth])plastic pots,either in a peat-vermiculite mixture(Jiffy Mix,Jiffy Products of America,Batavia,IL)1,or in vermiculite.Plants grown in soil were watered as needed by subirrigation throughout the experiment.Plants grown in vermiculite were subirrigated with a complete mineral nutrient solu-tion(Cammaerts and Jacob,1985)with nitrate(10m m KNO3)as a sole nitrogen source.After15d,the pots containing vermiculite were flushed daily for5d with25 mL of sterile distilled water,and the plants were subirri-gated for4d with different nitrogen treatments(10m m KNO3[control],10m m NH4Cl,5m m NH4NO3,5m m Glu, or5m m Gln),as described by Turano et al.(1997).Plants were maintained at20°C to21°C and60%to70%RH under cool-white lights(120–140mol PPFD mϪ2sϪ1)in a16-h light/8-h dark cycle.Crude Protein ExtractionsProteins were extracted from frozen or fresh samples. Frozen samples(approximately500mg)were ground to a fine powder with a mortar and pestle.The powder was transferred to1mL of extraction buffer(50m m Tes,pH5.8, 5m m EDTA,1m m MgCl2,0.1m m PLP,1m m DDT,0.5% [w/v]PVP-40,and4m m Cys).Fresh PMSF was added to a final concentration of1m m in all extracts.The fresh sam-ples(200mg)were ground in400L of extraction buffer as described above.The samples were incubated on ice for15 to30min.Debris were removed from the sample by cen-trifugation at13,000g for10min.The supernatant contain-ing the crude protein extract was used to determine the protein concentration and GAD activity.Protein Determinations and Enzymatic Activity Assays Protein concentrations were determined using a modifi-cation of the Lowry method as described by Markwell et al. (1978)or by NanoOrange as described by the manufacturer (Molecular Probes,Inc.,Eugene,OR).GAD activity was determined using a method similar to that of Snedden et al. (1992).Assays were conducted in17-ϫ52-mm glass vials plugged with a soft rubber stopper.An18-gauge syringe needle was used to pierce the stopper and a folded piece of Whatman paper no.3(7ϫ75mm)held in place by a small piece of rubber tubing.The Whatman paper was saturated with180L of1n KOH and suspended in the vial when the rubber stopper was put in place to trap the CO2that evolved from the GAD reaction.A small stopper was 1Mention of trademark,proprietary product,or vendor does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may be suitable.1412Turano and Fang Plant Physiol.Vol.117,1998placed in the end of the syringe needle to prevent theescape of CO2from the reaction.The vials were maintainedon ice prior to the initiation of the reaction.Reactions fromcrude protein extracts from plant tissues were conducted in500L with100m m pyridine-HCl,pH5.8,10m m NaCl,0.1 m m PLP,and20m m Glu containing0.02Ci/mol l-(1-14C)Glu with25to50L of enzyme extract.Crude protein extracts were assayed at pH5.8and not7.3because therewere problems associated with CaM binding and the ef-fects of proteases on the CaM-binding domain of GAD inthese extracts.Pure rGAD1and rGAD2were assayed in500L with100m m1,3-bis-Tris-propane-HCl,pH7.3,10 m m NaCl,0.1m m PLP,and20m m Glu containing0.02Ci/mol l-(1-14C)Glu with25to50L of enzyme extract. To test Ca2ϩand/or CaM stimulation,CaCl2and CaM were added alone or in combination to a final concentra-tion of1m m and0.4m,respectively.The CaM antagonist TFP was dissolved in water and added to a final concen-tration of100m.Assays were initiated with the addition of the substrate.The vials were incubated in a gently shaking water bath at30°C for1h.Vials were immediately placed on ice and100L of2n H2SO4was added to the vessels through the18-gauge syringe to terminate the re-action.The reactions were incubated on ice for30to45min to allow complete absorption of the CO2.The filter paper for each vial was placed into a scintillation vial containing 4.5mL of Formula A-989high-flash-point cocktail(Packard Instrument Co.,Inc.,Meriden,CT)and counted in a scintil-lation counter(model LS2800,Beckman).In each experi-ment,enzyme determinations were performed in triplicate.Gel Electrophoresis and Immunoblot AnalysisProteins were separated by SDS-PAGE(8.0%or9.0% polyacrylamide)using a Mini-Protean II(Bio-Rad)system. Silver staining and immunoblot analysis were conducted as described by Turano et al.(1990).Rabbit serum raised against petunia rGAD was kindly provided by Dr.Hillel Fromm(Baum et al.,1993).For the identification of GAD isoenzymes in different organs,an equal amount of protein (150g)from roots,leaves,flower stalks,flowers,and siliques of42-d-old plants was added per lane.Similarly, 150g of protein was added per lane to determine the effects of different nitrogen sources on GAD isoenzymes in leaves of24-d-old plants.To determine the effects of dif-ferent nitrogen sources on GAD isoenzymes in roots of 24-d-old plants,75g of protein was added per lane.GAD ClonesTwo expressed sequence tag clones encoding GAD1 (35B3T7)and GAD2(5B2T7P)were obtained from the Ara-bidopsis Biological Resource Center.The clones were se-quenced by the dideoxy chain-termination method using modified T7DNA polymerase(Sequenase 2.0,United States Biochemical),as described by the manufacturer.The data were analyzed with the IntelliGenetics Suite(Intelli-Gentics,Inc.,Mountain View,CA)on a Sun system.DNA and RNA Isolation and Gel ElectrophoresisTotal DNA was isolated,digested with restriction en-zymes,and blotted to nitrocellulose as described by Turano et al.(1992).For the determination of organ-specific expres-sion,total RNA was isolated from roots,leaves,flower stalks,flowers,and siliques of42-d-old Arabidopsis plants. To determine the effects of nitrogen on GAD,total RNA was isolated from roots and leaves of24-d-old plants.Total RNA was isolated from each organ,separated by gel elec-trophoresis using formaldehyde-formamide gels,blotted to nitrocellulose,and hybridized as described by Turano et al. (1997).Construction of rGAD1and rGAD2forExpression in Escherichia coliThe open reading frames for GAD1and GAD2were amplified and cloned in the E.coli expression vector pKK223-3(Pharmacia).Oligonucleotide primers were syn-thesized with Hin dIII sites at the5Ј(5Ј-GCCCAAGCTTAGG AAACAGAAATGGTGCTCTCCCACGCCG-3Ј)and3Ј(5Ј-GCCCAAGCTTAGCAGATACCACTCG-3Ј)for rGAD1and the5Ј(5Ј-GCCCAAGCTTAGGAAACAGAAATG-GTTTTGA CAAAAACCG-3Ј)and3Ј(5Ј-GCCCAAGC-TTTAGCAC ACACCATTCA-3Ј)for rGAD2.Separate amplification reac-tions were conducted with5Ј-and3Ј-specific primers with the appropriate cDNA clone as a template using a gene-amplification kit(PanVera Corp.,Madison,WI).Amplifica-tion reactions were conducted as follows:94°C for30s,55°C for30s,and72°C for4min,for25cycles.The amplified fragments were digested with Hin dIII,ligated into the vec-tor,and transformed into XL1-Blue MRFЈ(Stratagene)com-petent cells.The correct orientation was verified by restric-tion endonuclease analysis.E.coli cultures containing rGAD1or rGAD2were grown overnight,approximately16h,at37°C in Luria-Bertani medium with100g/mL ampicillin.Overnight cultures were diluted1:100in fresh Luria-Bertani medium with100g/mL ampicillin and incubated at25°C with shaking at200rpm.After4h,isopropylthio--galactoside was added to a final concentration of100m to induce expression of the recombinant protein,and incubation was continued for 3to4h for rGAD1and20h for rGAD2.Bacterial cells were collected by centrifugation at6000g for10min.Pellets were immediately frozen in liquid nitrogen and stored atϪ80°C. The E.coli pellets could be stored atϪ80°C for2months without a significant decrease in GAD activity.Pellets con-taining cells were resuspended in5to7mL of extraction buffer(50m m Tris-HCl,pH7.5,0.2m m EDTA,2.5%[v/v] glycerol,2m m DTT,0.05m m PLP,0.05%[v/v]Triton,10m leupeptin,20g/mL lysozyme,and1m m PMSF).The cells were lysed by sonication with122-s bursts at150W. Cellular debris were removed by centrifugation at12,000 rpm for20min.The supernatant was filtered through a 0.45-m filter.CaCl2,DTT,and PMSF were added to final concentrations of1,2,and1m m,respectively.The super-natant was loaded onto an approximately1-mL bed vol-ume CaM-Sepharose column(Pharmacia)and preequili-brated in binding buffer(50m m Tris-HCl,pH7.5,150m mGlutamate Decarboxylase cDNA Clones from Arabidopsis1413NaCl,2.5%[v/v]glycerol,1m m CaCl 2,2m m DTT,10m leupeptin,and 1m m PMSF).The column was washed with 20bed volumes of wash buffer minus calcium (25m m Tris-HCl,pH 7.5,150m m NaCl,2.5%[v/v]glycerol,2m m DTT,10m leupeptin,and 1m m PMSF).CaM-binding proteins were eluted with 50m m Tris-HCl,pH 7.5,2m m EDTA,150m m NaCl,2.5%[v/v]glycerol,and 1m m PMSF.Samples were immediately assayed as described above.Amplification and Radioactive Labeling of GAD Probes and HybridizationOne oligoprimer (17-mer)was synthesized (Bio-Synthesis,Inc.,Lewisville,TX)to the 3Јregions of each of the GAD genes.The primers,designated 3primGAD1(5Ј-GATCGATATAGAGAAAG-3Ј)and 3primGAD2(5Ј-TAGATACCTTGCCTTCC-3Ј),were used in separatePCRsFigure 1.Nucleotide sequence of Arabidopsis (At)GAD cDNA clones.The nucleotide sequence of the 3Јnoncoding region of the cDNA clone corresponding to GAD1(A)and the full-length cDNA clone corresponding to GAD2(B)are shown in the sense strand.A,Nucleotides are numbered from the putative stop codon of GAD1.B,The amino acids are numbered from the putative start codon of GAD2.C,Schematic representation of the 3Јgene-specific probes.Thin lines represent either the 5Јor 3Јnoncoding regions,the open boxes represent the coding regions,and the thick bars represent the gene-specific probe for either GAD1or GAD2.1414Turano and Fang Plant Physiol.Vol.117,1998with the M13-forward primer and their corresponding cDNA clones as the templates for use in a gene-amplification kit(Perkin-Elmer).Reactions were conducted as follows:94°C for30s,45°C for30s,and72°C for2min, for25cycles.Each of the amplified DNA fragments,404 and332bp,unique to the3Јregions of GAD1and GAD2 (Fig.1),respectively,were gel purified,amplified a second time,and used as gene-specific probes in genomic South-ern and RNA-blot analyses.Amplification of the DNA probes,total RNA isolation,blotting,prehybridizations, and hybridizations were performed as described by Turano et al.(1997).A probe encoding for a26S rRNA gene (F.J.Turano,unpublished data)was used as an internal control on RNA blots to ensure equal loading of RNA per lane.Data were quantified on a beta scanner(Betagen, IntelliGentics,Inc.).RESULTSSequence Analysis of GAD ClonesThe nucleotide sequence(1509bp)for the coding region of35B3T7(GAD1)was available in GenBank(accession no. U10034),and an additional250bp of sequence was deter-mined for the5Ј(data not shown)and3Јnoncoding(Fig. 1A)regions.The full-length GAD1clone was1760bp.A partial nucleotide sequence(465bp)for5B2T7P(GAD2) was available in GenBank(accession no.T04804).The re-mainder of the cDNA was sequenced;the full-length clone was1676bp long and encoded a494-amino acid peptide (Fig.1B).The nucleotide identity between GAD1and GAD2 was74%and28%in the coding and3Јnoncoding regions, respectively.Oligoprimers(17-mer)specific for GAD1and GAD2,designated3primGAD1and3primGAD2(see“Ma-terials and Methods”),were synthesized to the3Јregions of each of the cDNA clones.The primers were used to make 404-and332-bp DNA probes unique to the3Јregions of GAD1and GAD2(Fig.1C),respectively.GAD1and GAD2have open reading frames that encode for proteins containing502-and494-amino acid residues, respectively.Theoretically(estimated using PROSITE[ver-sion14.0,Medical Biochemistry Department,Geneva,Swit-zerland]),GAD1encodes for a57.1-kD peptide and GAD2encodes for a56.1-kD peptide.The deduced amino acid sequences of the two Arabidopsis GAD cDNA clones were aligned with the deduced amino acid sequences of petunia (Baum et al.,1993)and tomato(Gallego et al.,1995)GAD cDNA clones(Fig.2).The GAD1and GAD2gene products had82%amino acid identity.The gene product for GAD1 had85%and76%amino acid identity with the petunia and tomato GAD sequences,respectively.The gene product for GAD2had79%and74%amino acid identity with the petunia and tomato GAD sequences,respectively.Neither of the Arabidopsis peptides contained putative organellar target sequences.The amino acid residues Ser-X-X-Lys are conserved at amino acid residues274to277and273to276in the GAD1 and GAD2peptide sequences,respectively.This motif is common among PLP-requiring enzymes(Tanase et al., 1979).The identity and position of the Ser-X-X-Lys motif in the Arabidopsis GAD peptides are conserved with the motif in the products of the gadA and gadB genes from E. coli(Smith et al.,1992),which have been shown to require PLP for GAD activity,and the putative PLP sites of petunia GAD(Baum et al.,1993)and tomato GAD(Gallego et al., 1995).A putative CaM-binding region,located at the car-boxy termini of the two peptides,was variable among the sequences.Both peptides contained Trp residues in this region,Trp-488(GAD1)and Trp-480(GAD2),which has been shown to be essential for CaM binding in petunia GAD(Arazi et al.,1995).Arazi et al.(1995)demonstrated that the last30amino acids of GAD1were sufficient for CaM binding(Arazi et al.,1995).parison of the deduced amino acid sequences for Arabidopsis(At)GAD1and GAD2with GAD from tomato(tomGAD; Gallego et al.,1995;accession no.X80840)and petunia(petGAD; Baum et al.,1993;accession no.L16797).The Lys associated with the putative PLP-binding motif and the Trp associated with the putative CaM-binding domains are indicated in bold type.Glutamate Decarboxylase cDNA Clones from Arabidopsis1415CaM Binding and Ca 2؉/CaM Stimulation of rGAD1and rGAD2To demonstrate CaM binding and/or Ca 2ϩ/CaM stimu-lation,the open reading frame of GAD1or GAD2was overexpressed in E .coli .The recombinant proteins were purified by affinity chromatography (Fig.3)and tested for Ca 2ϩ/CaM stimulation (Table I).E .coli extracts overex-pressing the open reading frames for GAD1or GAD2were loaded onto CaM-Sepharose columns.The columns were washed extensively to remove nonspecific proteins (see “Materials and Methods”for details).CaM-binding pro-teins were eluted with 2m m EGTA.Purified CaM-binding proteins were analyzed by silver staining SDS-polyacrylamide gels and by immunoblot analysis (Fig.3).The estimated molecular masses for rGAD1and rGAD2were 58and 56kD,respectively.These findings are similar to the estimated molecular masses derived from the de-duced amino acid sequences of the two cDNA clones de-scribed above.In crude and purified extracts,the 58and 56-kD peptides cross-reacted with rabbit serum raised against petunia rGAD.Furthermore,neither the 58-nor the 56-kD peptides was observed in crude E.coli extracts con-taining only the vector pKK223-3.These results confirm that the recombinant proteins were expressed in E.coli ,were purified to homogeneity,and were CaM-binding pro-teins.The purified recombinant proteins were assayed for GAD activity in the presence of Ca 2ϩ,CaM,Ca 2ϩ/CaM,or Ca 2ϩ/CaM/TFP (Table I).Neither rGAD1nor rGAD2was stimulated by Ca 2ϩor CaM alone.However,both rGAD1and rGAD2were stimulated 35-and 13-fold,respectively,by Ca 2ϩ/CaM.In both cases,the Ca 2ϩ/CaM stimulation was significantly reduced by the CaM antagonist TFP.These data confirm that both rGAD1and rGAD2encode for CaM-binding proteins and that both are enzymatically stimulated by Ca 2ϩ/CaM.Southern-Blot AnalysisSouthern-blot analysis was used to determine the num-ber of copies of GAD1and GAD2genes in Arabidopsis (Fig.4).Specific 3Јprobes for the cDNA corresponding to GAD1and GAD2were hybridized under stringent conditions to Southern blots of Arabidopsis genomic DNA digested with Bam HI,Eco RI,or Hin dIII.It was evident that under the hybridization conditions used in these experiments,the probes were gene specific,since there were different hy-bridization patterns for each probe.The hybridization pat-terns for both GAD1and GAD2were simple;one band was apparent in each digest.Based on the simplicity of the hybridization patterns,comparisons of the intensities of these Southern blots with those of GDH1(data not shown),a single-copy gene (Melo-Oliveira et al.,1996),and the initial results from the identification of genomic clones (data not shown),it appears that both GAD1and GAD2represent single-copy an SpecificitySpecific activity,immuno-,and RNA-blot analyses were used to determine the level of activity,peptide,and/or transcript(s)for each GAD isoenzyme in different plant organs (Fig.5).The specific activity of GAD was highest in flower stalks and lowest in siliques of 42-d-old plants.Protein extracts from different organs were separated by SDS-PAGE (9.0%polyacrylamide),and immunoblot anal-ysis was used to identify the GAD peptides (Fig.5A).A 58-kD peptide was identified only in root samples,and based on the migration of the peptide in the gel,this subunit was designated GAD1.A 56-kD peptide,desig-nated GAD2,was identified in protein samples from all organs.The transcript corresponding to GAD1was de-tected only in root samples.The transcript corresponding to GAD2was readily detected in all tissues tested,except for siliques,but was visible in samples from siliques upon longer exposure (data not shown).The GAD2transcript was abundant in samples from roots,young leaves,and flowers,but the level of transcript was relatively lower in flower stalks and old leaves.The results from this experi-ment suggest that GAD1encodes for a 58-kD peptide and that GAD2encodes for a 56-kD peptide.These findings are consistent with the estimated sizes of the peptides deduced from the amino acid sequences and the results obtained from SDS-PAGE and immunoblot analysis of the recombi-nant proteins described above.Effect of Different Nitrogen Sources on GADPlants were maintained in soil or vermiculite with com-plete nutrient solution and 10m m KNO 3as a sole nitrogen source.After 20d the pots containing vermiculite were treated with complete mineral nutrient solutioncontainingFigure 3.Characterization of rGAD1and rGAD2by SDS-PAGE and immunoblot analysis.Crude protein extracts (20g/lane)from E.coli containing the expression vector pKK223-3(lanes 1and 7)or the open reading frame for GAD1(lanes 2and 8)or GAD2(lanes 4and 10)cloned into pKK223-3were resolved in a SDS 8.0%polyacryl-amide gel.Protein extracts from E.coli expressing the open reading frame for GAD1or GAD2were purified by affinity chromatography using CaM-Sepharose.The eluted peptides (0.075g/lane)rGAD1(lanes 3and 9)and rGAD2(lanes 5and 11)were resolved by ne 6contains a molecular mass marker (10-kD ladder,Life Technologies).The proteins in lanes 1through 6were stained with silver;the proteins in lanes 7through 11were blotted onto nitrocellulose and incubated with antiserum raised against petunia rGAD.The positions of the rGAD1(58kD)and rGAD2(56kD)peptides are indicated.1416Turano and Fang Plant Physiol.Vol.117,1998。
Preparation and Characterization of a Novel
ORIGINAL ARTICLEPreparation and Characterization of a NovelExtracellular Polysaccharide with Antioxidant Activity,from the Mangrove-Associated Fungus Fusarium oxysporumYan-Li Chen &Wen-Jun Mao &Hong-Wen Tao &Wei-Ming Zhu &Meng-Xia Yan &Xue Liu &Tian-Tian Guo &Tao GuoReceived:1August 2013/Accepted:7January 2015/Published online:28January 2015#Springer Science+Business Media New York 2015Abstract Marine fungi are recognized as an abundant source of extracellular polysaccharides with novel structures.Mangrove fungi constitute the second largest ecological group of the marine fungi,and many of them are new or inadequate-ly described species and may produce extracellular polysac-charides with novel functions and structures that could be explored as a source of useful polymers.The mangrove-associated fungus Fusarium oxysporum produces an extracel-lular polysaccharide,Fw-1,when grown in potato dextrose-agar medium.The homogeneous Fw-1was isolated from the fermented broth by a combination of ethanol precipitation,ion-exchange,and gel filtration chromatography.Chemical and spectroscopic analyses,including one-and two-dimensional nuclear magnetic resonance spectroscopies showed that Fw-1consisted of galactose,glucose,and man-nose in a molar ratio of 1.33:1.33:1.00,and its molecular weight was about 61.2kDa.The structure of Fw-1contains a backbone of (1→6)-linked β-D -galactofuranose residues with multiple side chains.The branches consist of terminal α-D -glucopyranose residues,or short chains containing (1→2)-linked α-D -glucopyranose,(1→2)-linked β-D -mannopyranose,and terminal β-D -mannopyranose residues.The side chains are connected to C-2of galactofuranose res-idues of backbone.The antioxidant activity of Fw-1was eval-uated with the scavenging abilities on hydroxyl,superoxide,and 1,1-diphenyl-2-picrylhydrazyl radicals in vitro,and the results indicated that Fw-1possessed good antioxidant activ-ity,especially the scavenging ability on hydroxyl radicals.Theinvestigation demonstrated that Fw-1is a novel galactofuranose-containing polysaccharide with different structural characteristics from extracellular polysaccharides from other marine microorganisms and could be a potential source of antioxidant.Keywords Mangrove-associated fungus .Fusarium oxysporum .Extracellular polysaccharide .Preparation .Characterization .Antioxidant activityIntroductionMangroves grow in saline coastal sediment habitats in the tropics and subtropics harboring a great diversity of marine fungi (Shearer et al.2007).Mangrove fungi constitute the second largest ecological group of the marine fungi and may produce chemicals with novel functions and structures (Kobayashi and Tsuda 2004).Fungi often produce extracellu-lar polysaccharides that are secreted into the growth media or remain tightly attached to the cell surface (Seviour et al.1992).The research on extracellular polysaccharides from marine fungi is attempted for providing polysaccharide with novel functions and structures (Chen et al.2012;Sun et al.2011).The extracellular polysaccharides produced by marine fungi become an important research area in new drug discovery and show enormous development prospects (Kanekiyo et al.2005).Polysaccharides with hexofuranose units are of interest be-cause of their unique structures and specific properties (Leal et al.2010).The investigations showed that galactose is the most widespread hexose in furanose form in naturally occur-ring polysaccharides (Pedersen and Turco 2003;Peltier et al.2008).The galactofuranose-containing extracellularY .<L.Chen :W.<J.Mao (*):H.<W.Tao :W.<M.Zhu :M.<X.Yan :X.Liu :T.<T.Guo :T.GuoKey Laboratory of Marine Drugs,Ministry of Education,Institute of Marine Drugs and Foods,Ocean University of China,5Yushan Road,Qingdao 266003,People ’s Republic of China e-mail:wenjunmqd@Mar Biotechnol (2015)17:219–228DOI 10.1007/s10126-015-9611-6polysaccharides with novel structural characteristics have been isolated from the fermented broth or cell walls of some microorganisms(Gander et al.1974;Ikuta et al.1997;Latgéet al.1994;Unkefer and Gander1990).With today’s interest in new renewable sources of polymers,the galactofuranose-containing extracellular polysaccharides represent potential source to be explored.However,the galactofuranose-containing extracellular polysaccharides from marine fungi have not yet been fully studied.In the current study,a novel galactofuranose-containing extracellular polysaccharide was isolated from the fermented broth of the mangrove-associated fungus Fusarium oxysporum by a combination of ethanol precipitation,ion-exchange,and gel filtration chroma-tography,and its structural characterization was investigated using chemical and spectroscopic methods,including one-and two-dimensional nuclear magnetic resonance(1D and 2D NMR)spectroscopic analyses.The antioxidant activity of the extracellular polysaccharide was also evaluated by scavenging assays involving hydroxyl,superoxide,and1,1-diphenyl-2-picrylhydrazyl(DPPH)radicals.Materials and MethodsMaterialsMonosaccharides(D-glucose,L-rhamnose,D-xylose,L-arabi-nose,D-mannose,L-fucose,D-galactose,D-glucuronic acid,D-galacturonic acid,D-mannuronic acid,N-acetyl-β-D-glucos-amine),1,1-diphenyl-2-picrylhydrazyl,trifluoroacetic acid, thiobarbituric acid,trichloroacetic acid,and1-phenyl-3-meth-yl-5-pyrazolone were from Sigma-Aldrich(St.Louis,MO, USA).Pullulan standards(Mw=344,200,107,47.1,21.2, and9.6kDa)were from the Showa Denko K.K.(Tokyo, Japan).Q Sepharose Fast Flow and Sephacryl S-100were from GE healthcare(Piscataway,NJ,USA).Dialysis mem-branes(flat width,44mm;molecular weight cut-off,3500) were from Lvniao(Yantai,China).Microbial Strain and Culture ConditionsThe marine fungus F.oxysporum was isolated from fresh leaves of Ipomoea pes-caprae(Linn.)collected from South Sea,China.It was identified according to its morphological characteristics and18S rRNA sequences,and the accession number of Genbank was JN604549.Briefly,the fungus was cultivated in the liquid medium containing yeast extract(3g/ L),peptone(5g/L),glucose(20g/L),malt extract(3g/L),sea salt(24.4g/L),KH2PO4(0.5g/L),NH4Cl(0.5g/L),pH6.0–6.5,at25°C for40days,and50L of fermented broth was obtained.Preparation of the Extracellular PolysaccharideThe fermented broth was filtered through cheese cloth,the filtrate was concentrated to1/15of its original volume under reduced pressure at40°C,and a threefold of the volume of 95%(v/v)ethanol was added.The resulting precipitate was recovered by centrifugation at3600×g for10min,dialyzed in cellulose membrane tubing against distilled water for72h. The retained fraction was dried,and the protein in the fraction was removed as described by Matthaei et al.(1962).The crude polysaccharide was fractionated by anion-exchange chroma-tography using a Q Sepharose Fast Flow column(30×3cm) coupled to an AKTA FPLC system and elution with a step-wise gradient of0,0.2,and1.0M NaCl.The fractions were assayed for carbohydrate content by the phenol–sulfuric acid method.The fractions eluted with distilled water were pooled, dialyzed,and further purified on a Sephacryl S-100column (70×2cm)eluted with0.2M NH4HCO3at a flow rate of 0.3mL/min.The major polysaccharide fractions were pooled, freeze–dried,and designated as Fw-1.Determination of Purity and Molecular WeightPurity and molecular weight were determined by high-performance gel permeation chromatography(HPGPC)with a Shodex Ohpak SB804(7.8×300mm,Tokyo,Japan)column and a refractive index detector(Agilent RID-10A Series),and elution with0.1M Na2SO4at a flow rate of0.5mL/min(Li et al.2012).Of1%sample solutions in0.2M Na2SO4,20μL was injected.The molecular weight was estimated by refer-ence to a calibration curve made by pullulan standards.General AnalysisTotal sugar content was measured by the phenol–sulfuric acid method using galactose as the standard(Dubois et al.1956). Protein content was assayed according to the modified Lowry method(Bensadoun and Weinstein1976).Sulfate content was measured according to Silvestri et al.(1982).Uronic acid con-tent was determined by the carbazole–sulfuric acid method (Bitter and Muir1962).Analysis of Monosaccharide CompositionFive milligrams of polysaccharide was hydrolyzed with2M trifluoroacetic acid at100°C for6h.Excess acid was re-moved by co-distillation with methanol after the hydrolysis was completed.Sample was subjected to reversed-phase high-performance liquid chromatography(HPLC)after pre-column derivatization and UV detection(Li et al.2011). Sugar identification was done by comparison with reference sugars(D-glucose,L-rhamnose,D-xylose,L-arabinose,D-man-nose,L-fucose,D-galactose,D-glucuronic acid,D-galacturonicacid,D-mannuronic acid,N-acetyl-β-D-glucosamine). Calculation of the molar ratio of the monosaccharide was car-ried out on the basis of the peak area of the monosaccharide. Methylation AnalysisMethylation analysis was performed by the method of Hakomori(1964)with some modifications.In brief, 2mg of polysaccharide in dimethyl sulfoxide was meth-ylated using NaH and iodomethane,and the completion of methylation was confirmed by Fourier transform infrared (FTIR)spectroscopy by the disappearance of OH bands. After hydrolysis with2M trifluoroacetic acid at105°C for6h,the methylated sugar residues were converted to partially methylated alditol acetates by reduction with NaBH4,followed by acetylation with acetic anhydride. The derivatised sugar residues were extracted into dichlo-romethane and evaporated to dryness,and dissolved again in100μL of dichloromethane.The products were ana-lyzed by gas chromatography–mass spectrometry(GC-MS)on a DB225using a temperature gradient of100–220°C with heating at a rate of5°C/min and mainte-nance of a temperature at220°C for15min.GC-MS was performed on an HP6890II instrument.Identification of partially methylated alditol acetates was carried out on the basis of retention time and mass fragmentation patterns.IR Spectroscopy AnalysisFTIR spectra were measured on a Nicolet Nexus470spec-trometer.The polysaccharide was mixed with KBr powder, ground up,and then pressed into1-mm pellets for FTIR mea-surements in the frequency range of4000–500cm−1with a resolution of4.0cm−1and320scans co-addition.NMR Spectroscopy Analysis1H nuclear magnetic resonance(NMR)and13C NMR spectra were measured at23°C using a JEOL JNM-ECP600MHz spectrometer.60mg of polysaccharide was deuterium ex-changed by two successive freeze–drying steps in99%D2O and then dissolved in0.5mL of99.98%D2O.1H–1H corre-lated spectroscopy(COSY),1H–1H total correlation spectros-copy(TOCSY),1H–1H nuclear overhauser effect spectrosco-py(NOESY),1H–13C heteronuclear multiple quantum coher-ence spectroscopy(HMQC)and1H–13C heteronuclear multi-ple bond correlation spectroscopy(HMBC)experiments were also carried out.Chemical shifts are expressed in ppm using acetone as internal standard at2.225ppm for1H and 31.07ppm for13C.Analysis of Antioxidant ActivityScavenging ability of hydroxyl radicals was determined ac-cording to the method of Smirnoff and Cumbes(1989). Scavenging ability of superoxide radicals was assessed ac-cording to the method reported by Marklund and Marklund (1974).Scavenging ability of DPPH radicals was measured according to the method described by Shimada et al.(1992). The scavenging ability was calculated according to the equa-tion below:scavenging ability(%)=(1–A sample/A control)×100, where A control is the absorbance of control without the tested samples,and A sample is the absorbance in the presence of the tested samples.The EC50value(mg/mL)was the effective concentration at which the tested radicals were scavenged by 50%.Ascorbic acid was used as positive control in all anti-oxidant assays.All bioassay results were expressed as means ±standard deviation(SD).The experimental data were sub-jected to an analysis of variance for a completely random design,and three samples were prepared for assays of every antioxidant attribute.ResultsPreparation and Chemical Composition of the Extracellular PolysaccharideProcedures used for the preparation of the extracellular poly-saccharides from the fermented broth of the mangrove-associated fungus F.oxysporum were shown in Fig.1.Crude extracellular polysaccharide(0.59g/L)was obtained from the fermented broth,and fractionated using a Q Sepharose Fast Flow column(Fig.2a).The polysaccharide fraction,eluted with distilled water,was a major component of the crude polysaccharides.The fraction was further purified by a Sephacryl S-100column(Fig.2b),and a polysaccharide frac-tion Fw-1was obtained.The yield of Fw-1from crude polysaccharide was about 42.86%.Fw-1gave a single and symmetrical peak in the HPGPC chromatogram(Fig.2c),thus Fw-1could be a homo-geneous polysaccharide.The linear relationship between the logarithm of molecular weight of pullulan standards and re-tention time was obtained.The retention time in HPGPC chro-matogram of Fw-1was used to calculate its molecular weight by the obtained regression equation.Thus,the molecular weight of Fw-1was estimated to be about61.2kDa.Fw-1 contained91.3%total carbohydrate and minor amounts of protein(0.79%)and did not have any sulfate ester. Monosaccharide composition analysis by reversed-phase HPLC showed that Fw-1consisted of galactose,glucose, and mannose with a molar ratio of1.33:1.33:1.00.No acidic sugar and amino sugar were detected in Fw-1.Thepolysaccharide fraction Fs,eluted at 0.2M NaCl,was not further investigated due to the limit of sample amount.It is possible that fraction Fs contains an acidic polysaccharide,such as a polysaccharide with phosphate ester (Chen et al.2013).IR SpectroscopyFrom the FTIR spectrum of Fw-1,the broad and intense band at 3416cm −1was the result of valent vibrations OH groups.The signal at 2931cm −1was attributed to the stretch vibration of the C –H bond.The band at 1649cm −1was assigned to the bending vibrations of HOH,and the band at 1416cm −1originated from the bend-ing vibrations of O –H bond.The band at 1241cm −1was due to the stretch vibration of C –O –C linkages.The signal at 1032cm −1was assigned to the stretch vibration of C –O and change angle vibration of O –H.The characteristic ab-sorption bands at 876and 809cm −1suggested the pres-ences of furan ring and mannopyranose units,respectively (Ahrazem et al.2000;Mathlouthi and Koenig 1986).Methylation AnalysisIn order to determine the linkage pattern of the sugar residues,Fw-1was subjected to methylation analysis (Table 1).A large amount of 1,2,4,6-tetra-O -acetyl-3,5-di-O -methyl-galactitol,which originated from the (1→2,6)-linked galactofuranoseresidue,was detected in Fw-1,suggesting that Fw-1was a highly branched polysaccharide.1,5-di-O -acetyl-2,3,4,6-tet-ra-O -methyl-glucitol,1,2,5-tri-O -acetyl-3,4,6-tri-O -methyl-mannitol,and 1,2,5-tri-O -acetyl-3,4,6-tri-O -methyl-glucitol were also detected,indicating the presence of (1→)-linked glucopyranose,(1→2)-linked mannopyranose and (1→2)-linked glucopyranose residues.In addition,1,5-di-O -acetyl-2,3,4,6-tetra-O -methyl-mannitol,which originated from the (1→)-linked mannopyranose residue,was also found in Fw-1.The results suggested that the structure of Fw-1is com-posed of (1→2,6)-linked galactofuranose,(1→2)-linked glu-copyranose,(1→2)-linked mannopyranose,terminal gluco-pyranose,and mannopyranose residues.NMR SpectroscopyThe 1H NMR spectrum (Fig.3a )of Fw-1showed anomeric proton signals at 5.20,5.10,5.09,4.91,4.75,and 4.65ppm,which were labeled A –F from low to high field.The anomeric signals B and C almost overlapped.The anomeric proton sig-nals A –F had relative integrals of 1.0:0.5:0.5:0.25:0.25:0.25.A might be signal of β-galactofuranose residue.B and C were attributed to the signals of α-configuration pyranose units,and D –F were likely the signals of β-configuration pyranose units.The chemical shifts from 3.42to 4.26ppm were assigned to H2–H6of glycosidic ring.In the anomeric region of the 13C NMR spectrum (Fig.3b )of Fw-1,there were six main anomeric carbon signals that occurred at 107.8,102.4,101.8,101.3,99.6,and 99.5ppm.The anomeric carbon signal at 107.8ppm was due to signal of β-galactofuranose residue because of extremely low field shifts (Ahrazem et al.2006).As shown in the DEPT spectrum,the signal at 70.8ppm was assigned to the substituted C-6of β-galactofuranose units.The result confirmed the presence of the substituted C-6linkage patterns,which was in accordance to the methylation results.The 1H NMR spin systems chemical shifts of the polysac-charide were assigned by means of the 1H –1H COSY spec-trum (Fig.3c )and the 1H –1H TOCSY spectrum (Fig.3d ).Combined with the analysis of the 1H –13C HMQC spectrum of Fw-1(Fig.3e ),the observed 1H and 13C chemical shifts and the assignment of the sugar residues were given (Table 2).A was assigned to →2,6)-β-D -Gal f (1→because of the down-field chemical shifts of the C-2(88.1ppm)and C-6(70.8ppm).B and C were suggested to be Glc p because of the high field chemical shift of H-2(3.59and 3.69ppm).In the 1H –1H TOCSY spectrum,H-1of B and C showed the correlation peaks with H-2,H-3,H-4,and H-5,which con-firmed this speculation.The 1H –13C HMQC spectrum re-vealed the substitution of C at C-2due to the downfield chem-ical shift (77.0ppm)of C-2compared with the parent α-D -Glc p .Thus,B was attributed to α-D -Glc p (1→,and C was due to →2)-α-D -Glc p (1→.Combined with methylationanalysisFig.1Scheme for the preparation of the extracellular polysaccharide produced by the mangrove-associated fungus F .oxysporumand NMR spectra data (Takegawa et al.1997),E was assigned to →2)-β-D -Man p (1→because of C-2(78.0ppm)of E had a relative downfield chemical shifts.D and F were assigned to be β-D -Man p (1→,the different glycosidic bond and sugar rings,which linked with D and F,had different chemical en-vironments and chemical shifts.The sequence of glycosyl residues was determined from the 1H –1H NOESY spectrum,followed by confirmation with 1H –13C correlations obtained from the 1H –13C HMBC spec-trum.In the 1H –1H NOESY spectrum (Fig.3f )of Fw-1,A had a strong NOE contact of its H-1with the H-2of C,indicating C linked to the C-2position of A.B and C had a strongcontactFig.2Isolation and HPGPC chromatogram of the extracellular polysaccharide from the fermented broth of the mangrove-associated fun-gus F .oxysporum .a The crude polysaccharides were fractionated using a Q Sepharose Fast Flow column.The fraction eluted with distill water was pooled and named as Fw.b Fw was purified on a Sephacryl S-100column and eluted with 0.2M NH 4HCO 3.The peak fractions containing the polysaccharides were pooled and named as Fw-1.c HPGPC chro-matogram of Fw-1on a Shodex Ohpak SB-804column and the standard curve of molecular weightof its H-1with the H-2of A,suggesting B and C linked to theC-2position of A.D had a strong inter-residue contact be-tween its H-1and the H-2of E,indicating D linked to theC-2position of E.From the1H–13C HMBC spectrum ofFw-1(Fig.3g),the presence of strong cross peak H-1/C-4,C-6of A confirmed that A wasβ-galactofuranose configura-tion and→6)-β-D-Gal f(1→was the main pattern of linkage.The cross-peak H-1B,C/C-2A,and H-2A/C-1B,C indicatedthat B and C linked to the C-2of→6)-β-D-Gal f(1→.The 1H–13C HMBC spectrum of Fw-1also showed H-1F/C-2 C,H-1E/C-2C,H-1D/C-2E,H-2E/C-1D,B H-1/C-5crosspeaks,which further proved the existence ofβ-D-Man p(1→2)-β-D-Man p(1→2)-α-D-Glc p(1→andβ-D-Man p(1→2)-α-D-Glc p(1→.The results also revealed both the furanoid char-acter of A and the pyranoid structure of B–F.The NMR resultswere thus in agreement with methylation results.These anal-yses allowed the identification of most of the1H and13Csignals of the sugar residues.Thus,structure of Fw-1couldbe characterized to consist of the backbone of(1→6)-linked β-D-galactofuranose residues,with multiple branches at C-2 consisting of theα-D-Glc p(1→,β-D-Man p(1→2)-β-D-Man p(1→2)-α-D-Glc p(1→andβ-D-Man p(1→2)-α-D-Glc p(1→.The hypothetical structure of Fw-1was shown in Fig.4.Analysis of Antioxidant ActivityAs shown in Table3,the scavenging abilities of Fw-1on hydroxyl,DPPH,and superoxide radicals were in a concentration-dependent manner.Less scavenging of hydrox-yl radicals was observed with Fw-1at2mg/mL,but the scav-enging ability of Fw-1on hydroxyl radicals at10.0mg/mL was up to90.2%.Fw-1showed strong scavenging ability on hydroxyl radicals as evidenced by its low EC50value(1.1mg/ mL).The scavenging ability of Fw-1on superoxide radicals was50.2%at2.0mg/mL,and the scavenging ability of Fw-1 was up to89.2%at10.0mg/mL.The EC50value of scaveng-ing ability of Fw-1on superoxide radicals was2.0mg/mL. The scavenging ability of Fw-1on DPPH radicals was up to 88.2%at10.0mg/mL,and its EC50value was2.1mg/mL, indicating that Fw-1was also good effectiveness in the anti-oxidant attribute.The scavenging abilities of Fw-1on hydroxyl,superoxide and DPPH radicals were all relativelylower than that of ascorbic acid at the same concentrations. DiscussionA novel extracellular polysaccharide Fw-1is successfullyobtained from the mangrove-associated fungus F.oxysporum.Fw-1is an extracellular polysaccharidewith different structural characteristics from other extra-cellular polysaccharides produced by Fusarium sp.Thecell wall polysaccharides from F.oxysporum are com-posed of glucosamine and N-acetylglucosamine(Fukamizo et al.1992,1996),and the polysaccharidefrom Fusarium sp.M7-1consists of mannose,glucose,galactose,and glucuronic acid(Iwahara et al.1992).However,a small amount of→2)-β-D-Gal f(1→and→6)-α-D-Glc p(1→residues present in the cell wall polysac-charide of Fusarium sp.M7-1(Iwahara et al.1996).Somealkali-extractable and water-soluble extracellular polysac-charides from Fusarium species contain a backbone of β-(1→6)-linked galactofuranose residues almost fully branched at O-2by single residues of glucopyranose oracidic chains containing glucuronic acid and mannose.The extracellular polysaccharide from F.oxysporumY24-2is composed of→2)-β-D-Gal f(1→6)-α-D-Glc p(1→units(Guo et al.2013).The structure of Fw1also differs from othergalactofuranose-containing extracellular polysaccharides re-ported previously(Gómez-Miranda et al.2003;Leal et al.2010).The galactofuranans from Aspergillus niger, A.fumigatus,Trichophyton species and Penicillium charlesii,have been characterized as linear chains of(1→5)-linkedβ-D-galactofuranose units(Gander et al.1974;Latgéet al.1994; Unkefer and Gander1990;Ikuta et al.1997).For the extracel-lular polysaccharide from the deep-sea fungus P.griseofulvum,its galactofuranan chain consists of(1→5)-linkedβ-D-galactofuranose,with additional branches at C-6 consisting of(1→)-linkedβ-D-galactofuranose residues and phosphate esters(Chen et al.2013).Fw-1contains a backbone of(1→6)-linkedβ-D-galactofuranose residues with multipleTable1Results of methylation analysis of Fw-1Methylated sugar Primary mass fragments(m/z)Molar ratio Deduced linkage1,5-Di-O-acetyl-2,3,4,6-tetra-O-methyl-mannitol101,117,129,145,161,205 2.0Man p(→1,5-Di-O-acetyl-2,3,4,6-tetra-O-methyl-glucitol101,117,129,145,161,205 2.0Glc p(1→1,2,5-Tri-O-acetyl-3,4,6-tri-O-methyl-mannitol87,101,129,161,189 1.0→2)Man p(1→1,2,5-Tri-O-acetyl-3,4,6-tri-O-methyl-glucitol101,117,129,161,201,233,277 2.0→2)Glc p(1→1,2,4,6-Tetra-O-acetyl-3,5-di-O-methyl-galactitol87,101,117,129,173,189,201,233 4.0→2,6)Gal f(1→Fig.3NMR spectra of Fw-1.Spectra were performed at23°C on a JEOL ECP600MHz spectrometer Chemical shifts are expressed in ppm using acetone as internal standard at2.225ppm for1H and 31.07ppm for13C.a1H NMR spectrum.b13C NMR and DEPT spectra.c1H–1H COSY spectrum.d1H–1H TOCOSY spectrum.e 1H–13C HMQC spectrum.f1H–1H NOESY spectrum.g1H–13C HMBC spectrum.A→2,6)-β-D-Gal f(1→.Bα-D-Glc p(1→.C→2)-α-D-Glc p(1→.Dβ-D-Man p(1→,linked to→2)-β-D-Man p(l→.E→2)-β-D-Man p(l→.Fβ-D-Man p(1→,linked to→2)-α-D-Glc p(l→.Glcpglucopyranose,Manp mannopyranose,Galf galactofuranosebranches at C-2consisting of terminal α-glucopyranose resi-dues,or short chains containing (1→2)-linked α-D -glucopy-ranose,(1→2)-linked β-D -mannopyranose,and terminal β-D -mannopyranose residues.To the best of our knowledge,this is the first report of such kind of galactofuranose-containing mannoglucogalactan isolated from fermented broth of micro-organism.The present result suggested that mangrove-associated fungi could be a potential source of extracellular polysaccharides with unique structures to be worth being fur-ther studied.In order to investigate the antioxidant activity of Fw-1,the assays based on scavenging abilities of hydroxyl,superoxide,and DPPH radicals were carried out and compared with that of ascorbic acid,one standard antioxidant.Hydroxyl radical is considered to be a highly potent oxidant,which can react with most biomacromolecules functioning in living cells and in-duce severe damage to the adjacent biomolecules.In cellular oxidation reactions,superoxide radical is normally formed first,and its effects can be magnified because it produces hydrogen peroxide and hydroxyl radical through dismutationTable 21H and 13C chemical shifts for the extracellular polysaccharide Fw-1Sugar residuesChemical shifts (ppm)a H1/C1H2/C2H3/C3H4/C4H5/C5H6/C6A b 5.20/107.8 4.21/88.1 4.26/76.9 4.05/83.9 4.02/71.0 3.94,3.69/70.8B c 5.10/99.5 3.59/72.6 3.77/73.1 3.47/71.0 3.79/73.8 3.91,3.73/62.1C d 5.09/99.6 3.69/77.0 3.81/73.1 3.45/71.0 3.76/72.6 4.12,3.79/62.3D e 4.91/102.4 4.18/72.6 3.73/72.4 3.61/72.6 3.45/71.9 3.79,3.90/62.6E f 4.75/101.3 4.24/78.0 3.68/68.3 3.95/71.2 3.76/73.5 3.96,3.45/62.4F g4.65/101.84.02/71.93.73/72.43.96/71.13.80/73.63.47,3.86/62.3Glcp glucopyranose,Manp mannopyranose,Galf galactofuranoseaThe spectra were recorded using a JEOL JNM-ECP 600MHz spectrometer.Chemical shifts are referenced to internal acetone at 2.225ppm for 1H and 31.07ppm for 13C b →2,6)-β-D -Gal f (→c α-D -Glc p (1→d →2)-α-D -Glc p (1→e β-D -Man p (1→,linked to →2)-β-D -Man p (l →f →2)-β-D -Man p (l →gβ-D -Man p (1→,linked to →2)-α-D -Glc p (l→Fig.4One of the possible structures of Fw-1(Glcp gluco-pyranose,Manp ,mannopyranose,Galf ,galactofuranose,n ≈16)and other types of reaction and was the source of free radicals formed in vivo.DPPH is a useful reagent to evaluate the free radical scavenging ability of the hy-drogen donating antioxidant,which can transfer hydro-gen atoms or electrons to DPPH radicals.It was found that Fw-1had a more noticeable scavenging ability on hydroxyl radicals than the extracellular polysaccharide AVP produced by coral-associated fungus Aspergillus versicolor LCJ-5-4,and the EC50value of AVP was 4.0mg/mL(Chen et al.2012).Moreover,the scaveng-ing ability of Fw-1on superoxide radicals appears to be higher than that of the extracellular polysaccharide As1-1produced by marine fungi Aspergillus sp.Y16,and the EC50value of As1-1was 3.4mg/mL(Chen et al. 2011).Scavenging ability of Fw-1on DPPH radicals was similar to that of extracellular polysaccharide AVP produced by coral-associated fungus,A.versicolor LCJ-5-4,and its EC50value was2.05mg/mL(Chen et al. 2012).Fw-1had a higher scavenging ability on DPPH radicals than the extracellular polysaccharides PS2-1, PS1-2,and PS1-1isolated from marine fungus Penicillium sp.F23-2(EC50value 2.53–6.81mg/mL) (Sun et al.2009).The present result suggested that the extracellular polysaccharide Fw-1could be a potential antioxidant.The antioxidant activity of Fw-1may be attributed to the extracellular polysaccharide can connect with radicals,and terminate the radical chain reaction. However,the antioxidant mechanisms of polysaccha-rides are complex.Further study on antioxidant property of extracellular polysaccharides with different structural characterization will play an important role in the un-derstanding of the mechanism of antioxidant activity.In conclusion,the extracellular polysaccharide Fw-1pro-duced by the mangrove-associated fungus F.oxysporum is a galactofuranose-containing mannoglucogalactan differing from previously described extracellular polysaccharides.Fw-1exhibits good antioxidant activity in vitro.An in-depth investigation of the antioxidant activity of Fw-1will be re-quired to determine if the extracellular polysaccharide will be useful in the food and pharmaceutical industry. Acknowledgments This work was supported by the Science and Tech-nology Development Program of Shandong Province,China (2014GHY115015),NSFC-Shandong Joint Fund for Marine Science Re-search Centers(U1406402),and the National Oceanographic Center of Qingdao of China.ReferencesAhrazem O,Gómez-Miranda B,Prieto A,Barasoaín I,BernabéM,Leal JA(2000)An acidic water-soluble cell wall polysaccharide:a che-motaxonomic marker for Fusarium and Gibberella.Microbiol Res 104:603–610Ahrazem O,Prieto A,Giménez-Abián MI,Leal JA,Jiménez-Barberoa J, Bernabe M(2006)Structural elucidation of fungal polysaccharides isolated from the cell wall of Plectosphaerella cucumerina and Verticillium spp.Carbohydr Res341:246–252Bensadoun A,Weinstein D(1976)Assay of proteins in presence of in-terfering materials.Anal Chem70:241–256Bitter T,Muir HM(1962)A modified uronic acid carbazole reaction.Anal Biochem4:330–334Chen Y,Mao WJ,Tao HW,Zhu WM,Qi XH,Chen YL,Li HY,Zhao CQ, Yang YP,Hou YJ,Wang CY,Li N(2011)Structural characterization and antioxidant properties of an exopolysaccharide produced by the mangrove endophytic fungus Aspergillus sp.Y16.Bioresour Technol102:8179–8184Chen Y,Mao WJ,Yang YP,Teng XC,Zhu WM,Qi XH,Chen YL,Zhao CQ,Hou YJ,Wang CY,Li N(2012)Structure and antioxidant activity of an extracellular polysaccharide from coral-associated fun-gus,Aspergillus versicolor LCJ-5-4.Carbohydr Polym87:218–226 Chen Y,Mao WJ,Wang BF,Zhou LN,Gu QQ,Chen YL,Zhao CQ,Li N, Wang CY,Shan JM,Yan MX,Lin C(2013)Preparation and char-acterization of an extracellular polysaccharide produced by the deep-sea fungus Penicillium griseofulvum.Bioresour Technol132: 178–181Dubois C,Gilles KA,Hamilton JK,Rebers PA,Smith F(1956) Colorimetric method for determination of sugars and related sub-stances.Anal Chem28:350–356Table3Antioxidant activity of the extracellular polysaccharide Fw-1in vitroa The results were expressed as means±standard deviation(SD). The experimental data were subjected to an analysis of variance for a completely random design,and three samples were prepared for assays of every antioxidant attribute Sample Concentration(mg/mL)a0 2.0 4.0 6.08.010.0Scavenging ability on hydroxyl radicals(%)Fw-10.059.5±1.482.5±2.885.6±2.486.8±3.590.2±2.3 Ascorbic acid0.097.2±2.497.2±2.697.4±2.697.5±1.997.7±2.1 Scavenging ability on superoxide radicals(%)Fw-10.050.2±1.868.3±3.179.1±2.385.7±3.289.2±2.8 Ascorbic acid0.097.2±1.997.3±2.297.4±2.797.5±2.897.8±2.4 Scavenging ability on DPPH radicals(%)Fw-10.049.1±1.766.9±2.475.0±2.585.2±2.388.2±2.6 Ascorbic acid0.097.2±2.297.3±1.797.4±2.097.5±2.597.7±2.8。
美国文学毕业论文选题
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Tragedy15.浅析《野性的呼唤》中的自然主义The Brief Analysis of Naturalism in The Call of the Wild16.从《老人与海》看海明威作品中的硬汉形象—桑提亚哥On the Image of a Tough Guy in The Old Man and the Sea —Santiago17.试析《S.》中厄普代克对女权运动的态度On Updike ' s Attitude towards Feminism in S.18.《飘》中女主角斯嘉丽的性格分析An Analysis of the Characters of Scarlett in Gone with the wind19.从变态心理学的视角分析电影《沉默的羔羊》An Analysis of the Silence of the Lambs —From the Perspective of Abnormal Psychology 20.《紫颜色》中艾伯特人物分析An Character Analysis of Albert in The Color Purpl21.浅析弗罗斯特的诗歌特色On the Features of Robert Frost ' s Poetry22. 论海明威作品《太阳照样升起》中的女性形象On the Images of Women in Hemingway`s The Sun Also Rises23. 美国自然文学在斯蒂芬 ?克莱恩的作品中体现American Naturalism Reflected in Stephen Crane' s Works24. 浅谈海明威的作品《太阳照常升起》中 “迷惘的一代 ”‘ The Lost Generation ' in Hemingway ' s The Sun Also Rises25. 《小妇人》中四姐妹的人物塑造 On the Characterization of the Four Sisters in Little Woman26. 美国黑人文化身份的困境：评托妮 ?莫里森的《柏油娃》The Dilemma of Black American Cultural Identity: on Toni Morrison' 27. 《看不见的人》的爵士乐风格Jazz Style in Invisible Man28. 论《一个干净明亮的地方》的写作技巧On the Techniques of A Clean,Well-Lighted Place29. 托妮 ?莫里森笔下的微笑意象 The Smile Image in Toni Morison' s Writing30. 成长的艰辛 — 《麦田里的守望者》的主题分析 Difficulties in Growing Up: A Thematic Analysis of The Catcher in the Rye31. 《小镇畸人》中的怪人形象The Image of Grotesques in Winesburg, Ohio32. 《赫索格》的艺术表现手法Techniques of Artistic Expression in Herzog33. 浅谈纳博科夫的《洛丽塔》中的病态心理On Insanity in Lolita by Nabokov34. 《汤姆叔叔的小屋》中女性意识力量The Female Consciousness in Uncle Tom's Cabin35. 论《愤怒的葡萄》中体现的《圣经》元素On the Elements of the Bible in the Grapes of Wrath36. 书信体叙述模式在《紫色》中的运用The Epistolary Narration in The Color Purple37. 托尼莫里森作品中人物名字的意义The Meaning of Names in Toni Morisson's Novels38. 论小说《在路上》中垮掉的一代A Survey on the Beat Generation from On the Road39. 论《永别了 ,武器》中的悲剧策略Analysis on the Tragic Strategy of A Farewell to Arms The Tragic Spirit in Death Salesman 40. 孤独与失落的守望 — 析《麦田里的守望者》Waiting in Perplexity and Degradation —— Analysis of The Catcher in The Rye 41. 论海明威《一个干净明亮的地方》中的虚无主义The Analysis on Nihilism in Hemingway' s Short Story A C-lleigahnt,e Wd Pelllace42. 试析《哈克贝利 ?费恩历险记》中的人性刻画 On the Humanity Reflected in The Adventures of Huckleberry Finn43. 唐人街文化分析 —— 以水仙花与朱路易作品为例Analysis of Chinatown Culture — Taking the Works of Sui Sin Far and Louis Chu as thes Tar Boyof aExample44.意象派诗歌中东方审美因素的分析On the Analysis of Oriental Aesthetic Elements in the Imagist Poetry45.《这个杀手不太冷》的主人公性格分析Character Analysis of “ LEON”46.论马丁?伊登和杰伊?盖茨比的比较研究A Comparative Study between Martin Eden and Jay Gatesby47.浅析《鸡蛋的胜利》中运用象征手法表现美国梦On the American Dream Expressed by Symbolism by The Triumph of the Egg48.从《教父》看两代人不同的家庭观The Discrepancy of Family Values between Two Generations in The Godfather49.《推销员之死》中的矛盾与冲突Conflict and Contradiction —On Death of a Salesman50.论霍桑作品中的象征手法-以《年轻人古德曼?布朗》为例On the Symbolism in Hawthorne's Works---taking Young Goodman Brown as a example 51.浅谈《厄舍古屋的倒塌》的叙事视角On the Narrative Point of View in The Fall of the House of Usher52.论《厄舍古屋倒塌》中的哥特元素On Gothic Elements in “ The Fall of the House of Usher ”53.解析《第二十二条军规》中的黑色幽默On the Black Humor in Catch-2254.论《所罗门之歌》的主题55.论《推销员之死》的悲剧观56.从休斯到莫里森浅析美国黑人文学的嬗变57.浅析爱伦?坡的侦探小说58.浅析《汤姆叔叔的小屋》主人公性格59.《嚎叫》——垮掉的一代的预言60.从愤怒的葡萄中看美国大萧条61.杰克伦敦的自然主义——通过作品《野性的呼唤》和《白牙》分析其自然主义倾向62.《哈克贝利?费恩历险记》的写作特点分析63.《愤怒的葡萄》中《圣经》的象征意义64.海勒斯与卡米拉的爱情对比分析65.浅析王熙凤与斯嘉丽的异同66.从生态女权主义角度来解读托妮?莫里森的《宠儿》67.《最蓝的眼睛》中非裔美国人的自我憎恨68.“心之罪”与“魂之恶”——比较研究《红字》与《厄榭尔府的倒塌》69.杰克?伦敦的《野性的呼唤》中的自然主义元素70.论简爱中的女性意识71.汤姆.索亚，哈克.贝丽芬和马克吐温的时代观72.海明威的女性意识73.论嘉莉妹妹成功的原因74.从《喧哗与骚动》中凯莉的悲剧看女性的社会地位75.透视《宠儿》中美国黑人女性的悲剧与成长76.浅析《心是孤独的猎手》中人物异化的生存状态77.论小男孩在《老人与海》中的作用78.浅析《论自助》中人生自主的源泉79.浅析马克?吐温小说的地方色彩主义特点On the Characteristics of Dickinson 's Poems80.哈克贝利?费恩的性格分析An Analysis of the Characteristics of Huckleberry Finn81.浅析《欲望号街车》的主题An Analysis of the Theme of A Streetcar Named Desire82.狄金森诗歌的特点之浅析On the Characteristics of Dickinson 's Poems83.浅析狄金森诗歌中的死亡主题On the Death Theme of Dickinson 's Poems84.从凯蒂的悲剧中看20世纪初女性的社会地位From Caddy 's Tragedy to View Women 's Social Status in the Early 20th Century 85.《乱世佳人》对21世纪女性的启示An Analysis of the Inspiration of Gone with the Wind to the 21st Century Women 86.解读《献给艾米丽的一朵玫瑰》中的悲剧元素On Tragic Elements in A Rose for Emily87.浅析《麦田里的守望者》中霍顿的性格特点An Analysis of Holden 's Characteristics in The Catcher in the Rye88.斯嘉丽：“旧”时代的“新”女性Scarlett O ' Hara, A “New” Woman PIne riod“ Old”89.“无形”困境——对《看不见的人》的主题分析“ Invisible Plight An” A nalysis of the theme of Invisible Man90.情感与理智——浅析《飘》中的婚姻观Emotion and Intellect -- An Analysis of View of Marriage in Gone with the Wind91.浅析《欲望号街车》中布兰奇的悲剧根源On the Origin of Blanche 's Tragedy eintc Aa rS Ntraemed Desire92.论对《哈克贝利?费恩历险记》的种族主义误读On Racist Misperception of The Adventures of Huckleberry Finn93.从《喜福会》透视中美文化冲突与融合On the Cultural Conflicts and Blending Embodied in The Joy Luck Club94.浅析惠特曼的写作技巧A Brief Analysis of the Writing Techniques of Whitman95.对《宠儿》中叙事方法的分析An Analysis of the Narrative Tactics in Beloved96.浅析《赫索格》中的犹太情结A Brief Analysis of the Jewish Complex in Herzog97.抗争与守望：论《小镇畸人》中的畸形人物Struggle and Watch: A Study of the Grotesques in Winesburg, Ohio98.浅析《嘉莉妹妹》中的自然主义特征A Brief Analysis of Naturalistic Features in Sister Carrie99.浅析《看不见的人》中黑人的被漠视境遇An Analysis of Blacks ' Invisible Situation in Invisible Man100.浅析欧?亨利短篇小说的结尾艺术与人文关怀A Brief Analysis of the Twist Ending and the Humanity Cares in O. Henrys Short Stories 101.浅析《看不见的人》中的布鲁斯神韵An Analysis on the Spirit of the Blues in Invisible Man102.哈克贝利?费恩的性格分析An Analysis of the Characteristics of Huckleberry Finn103.伊迪斯?华顿《纯真年代》中的女性意识Feminine Consciousness in Edith Wharton ' s The Age of Innocence104.《嘉莉妹妹》中的早期自然主义Dreiser ' s Early Naturalism in Sister Carrie105.论《夜色温柔》中美国梦的破灭On the Collapse of American Dream in Tender is the Night106.艾米莉?狄更森诗歌之主题研究On the Themes of Emily Dickinson ' s Poems107.对《推销员之死》对话的语用分析A Pragmatic Analysis of the Dialogues in Death of a Salesman108.《红字》中的象征意义The Symbolism in The Scarlet Letter109.浅析《美国悲剧》中罗贝塔的悲剧性On the Tragedy of Roberta in An American Tragedy110.论《洛丽塔》中的彼岸世界On the “ Otherworld ” of Lolita111.通过《飘》看女人的觉醒Gone with the Wind and the Awakening of Women112.浅析托尼?莫里森《宠儿》中的魔幻现实主义的应用Magic Realism in Tony Morrison ' s Beloved113.《看不见的人》中的社会问题分析Analysis of Social Problems in Invisible Man114.论《秀拉》中女性的自我寻找Self-realization of the Females in Sula115.浅析《了不起的盖茨比》比中颜色的象征意义The Symbolic Meaning of Color Words in The Great Gatsby116.从《嘉莉妹妹》看女性价值观变迁Changes of Female ' s Values in Sister Carrie117.浅析多斯? 帕索斯在“美国”三部曲中的写作手法Techniques Employed in U.S.A by John Dos Passos118.爱伦?坡小说中的恐怖因素Horror Elements in Edgar Allen Poe ' s Fiction119.浅析《我有一个梦想》的修辞手法与主题表现An Analysis of Rhetoric Method and Theme of I Have a Dream120.浅析《芒果街上的小屋》中的女性形象Analysis on the Female Images in The House on Mango Street121.浅析欧?亨利的黑色幽默On O. Henry ' s Black Humor122.欧?亨利短篇小说中的反衬艺术The Art of Making Contrasts in O? Henry's Short Stories123.论《了不起的盖茨比》中的美国梦On the American Dream in The Great Gatsby124.圣经对美国小说的影响The Influence of Bible in American Novel125.《白鲸》的生态解读Ecological Analysis of Moby-Dick126.论《汤姆?索亚历险记》的魅力所在The Charms of the Adventures of Tom Sawyer127.《老人与海》的象征主义Symbolism in The Old Man and the Sea128.《汤姆叔叔的小屋》主要人物性格分析Analysis of the Protagonists ' Character in Uncle Tom ' s Cabin129.基督教在《汤姆叔叔的小屋》中的体现Christianity in Uncle Tom's Cabin130.论T.S.艾略特《荒原》中的宗教思想A Discussion of the Religious Ideas in The Waste Land of T. S. Eliot131.论《推销员之死》的悲剧精神The Tragic Spirit in Death of a Salesman132.《秘密花园》现代主义主题分析On the Modernist Themes in The Secret Garden133.简析艾伦?金斯堡《嚎叫》中的“垮掉的一代”Brief Analysis of the Beat Generation in Allen Ginsber g' s Howl134.“嘉莉妹妹”—西方女性的缩影Sister Carrie —the Miniature of Western Women135.伊迪丝?华顿的《纯真年代》中的女性意识Feminine Consciousness in Edith Wharton ' s The Age of Innocence136.解析《红字》中A 的象征意义On the Symbolic Meaning of A in The Scarlet Letter137.《小妇人》—一曲新女性的赞歌Little Women, a Celebration of New Women138.梭罗的《瓦尔登湖》中蕴含的深层生态学思想Deep Ecological Thoughts Contained in Thoreau ' s Walden139.从《喧哗与骚动》中浅析20 世纪初女性的社会地位Analysis of Women ' s Social Position in Early 20th Century from The Sound and the Fury 140.浅析海明威的《一个干净明亮的地方》A Brief Discussion of Hemingway Cle'ans a And Well-lighted Place141.论《美国悲剧》中萝贝塔的悲剧性The Tragedy of Roberta in American Tragedy142.马克?吐温短篇小说的幽默与讽刺Humor and Irony in Mark Twain ' s Short Story143.论马克?吐温小说的讽刺144.浅析马克吐温的《败坏了哈得莱堡的人》中的讽刺艺术145.论《觉醒》的主题和特点146.透过《嘉莉妹妹》看世纪之交的美国消费文化147.从女性主义视角解读《紫色》148.论《麦田里的守望者》中读者对艺术真实的认同与重建149.《宠儿》中黑人女性的社会地位150.浅析艾米莉?狄金森的爱情诗151.《白鲸》所反映出的生活态度152.浅析《嘉莉妹妹》中的新女性形象153.论《土生子》中的种族主义154.《喜福会》中母爱主题的阐释155.从庞德的作品品读意象派风格156.从超验主义重新解读《小妇人》157.评莫里森《最蓝的眼睛》的艺术特色158.论《老人与海》的悲剧色彩159.小男孩在《老人与海》中的作用160.浅谈尼采思想对杰克?伦敦及其小说《马丁? 伊登》的影响161.论《白象似的群山》中海明威独特的写作风格162.对海明威短篇小说艺术特色的研究163.从《嘉莉妹妹》看美国梦的幻灭。
explicit characterization 语言学
explicit characterization 语言学Explicit Characterization in LinguisticsIntroductionCharacterization is a fundamental aspect of linguistics that aims to describe and understand the various features of language. In this article, we will explore the concept of explicit characterization in linguistics, its significance, and its application in language analysis and research. By delving into this topic, we can gain valuable insights into the nature of language and how it is studied.Defining Explicit CharacterizationExplicit characterization refers to the process of clearly and directly describing specific linguistic features or phenomena without ambiguity. It involves providing precise details and definitions that leave no room for misinterpretation. This form of characterization plays a crucial role in linguistic analysis as it allows researchers to examine language in a systematic and structured manner.Significance of Explicit CharacterizationThe use of explicit characterization is essential in linguistics due to the following reasons:1. Accuracy and Precision: By explicitly characterizing linguistic phenomena, researchers can ensure accurate and precise descriptions, minimizing the risk of confusion or miscommunication.2. Objectivity: Explicit characterization helps maintain objectivity in linguistic research by providing clear guidelines and criteria for analysis, reducing the potential for subjective interpretations.3. Replicability: In scientific studies, replication is crucial for validating research findings. Explicit characterization enables other researchers to replicate and verify linguistic analyses, contributing to the overall credibility of the field.Applications of Explicit Characterization in Linguistics1. Phonetics and PhonologyExplicit characterization is commonly used in the study of phonetics and phonology to describe and differentiate various speech sounds and phonological patterns. For example, in describing the English "th" sound, explicit characterization involves specifying the voiceless interdental fricative (/θ/) and voiced interdental fricative (/ð/) sounds, accompanied by their unique articulatory properties.2. MorphologyMorphological analysis often requires explicit characterization to identify and define morphemes, the smallest meaningful units in a language. For instance, in the analysis of the English word "unhappiness," explicit characterization would involve breaking it down into the prefix "un-" (meaning "not"), the root "happy," and the suffix "-ness" (denoting a state or quality).3. SyntaxIn the study of syntax, explicit characterization is crucial for describing sentence structures, grammatical rules, and word order patterns. For example, explicit characterization is employed in defining the passive voice as a grammatical construction where the subject of a sentence undergoes an action rather than performing it.4. SemanticsExplicit characterization is also used in semantic analysis to explore the meanings of words, phrases, and sentences. It helps linguists identify and define semantic features, such as polysemy (multiple meanings) and synonymy (similar meanings). Explicit characterization allows for clear and unambiguous descriptions of semantic relationships.ConclusionExplicit characterization is a fundamental tool in linguistics, enabling researchers to accurately describe and analyze various linguistic features and phenomena. Its importance lies in its ability to provide objective, replicable, and precise descriptions of language. By employing explicit characterization in areas such as phonetics, phonology, morphology, syntax, and semantics, linguists can deepen their understanding of language structure and usage. Through continued research and application, explicit characterization will further contribute to advancements in linguistic theory and practice.。
巴赫金.陀思妥耶夫斯基诗学问题 英文文献
巴赫金.陀思妥耶夫斯基诗学问题英文文献Mikhail Bakhtin's Poetics and the Works of Fyodor DostoevskyMikhail Bakhtin, the renowned Russian literary critic and philosopher, is widely recognized for his groundbreaking contributions to the understanding of literary works, particularly the writings of Fyodor Dostoevsky. Bakhtin's analysis of Dostoevsky's poetics has had a profound impact on the way we interpret and appreciate the complexities of the renowned Russian author's literary oeuvre.At the heart of Bakhtin's exploration of Dostoevsky's poetics lies the concept of the polyphonic novel. Bakhtin argued that Dostoevsky's works depart from the traditional monologic structure of the novel, where a single authorial voice dominates the narrative. Instead, Dostoevsky's novels are characterized by a plurality of independent and fully developed voices, each with its own distinct perspective and worldview. This polyphonic approach, Bakhtin contends, allows for a genuine dialogue between the characters, where no single voice or ideology is privileged over the others.One of the key aspects of Bakhtin's analysis is his understanding of the role of the hero in Dostoevsky's novels. Bakhtin asserts thatDostoevsky's heroes are not passive objects of the author's design but rather autonomous individuals with the capacity to challenge and even contradict the author's own views. This notion of the hero as a fully autonomous and self-conscious being is central to Bakhtin's conception of the polyphonic novel.Furthermore, Bakhtin explores the concept of the "unfinalized" hero in Dostoevsky's works. He argues that Dostoevsky's characters are not static, predetermined entities but rather dynamic, ever-evolving individuals who resist being reduced to a single, fixed identity. This idea of the "unfinalized" hero is closely tied to Bakhtin's understanding of the polyphonic novel, where the characters are not merely vehicles for the author's ideas but active participants in the ongoing dialogue that constitutes the literary work.Bakhtin's analysis also delves into the role of language and discourse in Dostoevsky's novels. He suggests that Dostoevsky's works are characterized by a multiplicity of social and ideological languages, each with its own distinct perspective and mode of expression. This heteroglossia, as Bakhtin terms it, serves to undermine the notion of a single, authoritative voice and instead presents a cacophony of competing voices and worldviews.Moreover, Bakhtin's exploration of Dostoevsky's poetics extends to the notion of the carnivalesque, a concept that he sees as central tothe author's literary approach. The carnivalesque, in Bakhtin's view, represents a subversive and liberating force that challenges traditional hierarchies and social norms. He argues that Dostoevsky's novels often incorporate elements of the carnival, such as the inversion of power structures, the blurring of boundaries between the sacred and the profane, and the celebration of the body and its functions.Bakhtin's insights into Dostoevsky's poetics have had a lasting impact on the field of literary criticism and have influenced generations of scholars and readers. By illuminating the complex and multifaceted nature of Dostoevsky's literary works, Bakhtin has helped us to better understand the profound philosophical and existential questions that lie at the heart of the Russian author's oeuvre.In conclusion, Mikhail Bakhtin's exploration of Dostoevsky's poetics has been instrumental in shaping our understanding of the Russian author's literary genius. Through concepts such as the polyphonic novel, the "unfinalized" hero, and the carnivalesque, Bakhtin has provided a nuanced and insightful perspective on the rich tapestry of Dostoevsky's literary universe. His work continues to inspire and challenge scholars and readers alike, inviting us to engage with the complexities and contradictions that lie at the core of Dostoevsky's enduring literary legacy.。
我对文学性质的理解作文
我对文学性质的理解作文英文回答：Literature, to me, is the essence of human expression and imagination. It is a reflection of our thoughts, emotions, and experiences, presented in a creative and artistic manner. Literature can take various forms, such as novels, poems, plays, and essays, and each form has its own unique way of conveying ideas and evoking emotions.One of the key aspects of literature is its ability to transport us to different worlds and perspectives. Through literature, we can explore different cultures, time periods, and even fantastical realms. For example, when I read J.R.R. Tolkien's "The Lord of the Rings," I am transported to the magical world of Middle-earth, where I can experience epic adventures alongside the characters. This ability to immerse ourselves in a different reality is what makes literature so captivating and engaging.Literature also has the power to inspire and provoke deep thoughts and emotions. It can make us laugh, cry, or feel a sense of awe. Take the works of William Shakespeare, for instance. His plays, such as "Romeo and Juliet" and "Hamlet," are filled with profound insights about love, tragedy, and the human condition. When I read these plays, I am not only entertained, but also prompted to contemplate the complexities of life and relationships.Furthermore, literature serves as a means of communication and connection between individuals. It allows us to understand and empathize with the experiences of others. For instance, when I read Maya Angelou's autobiography, "I Know Why the Caged Bird Sings," I gain a deeper understanding of the struggles and triumphs of African Americans during the Civil Rights Movement. Through literature, we can bridge the gaps between different cultures, generations, and perspectives, fostering empathy and promoting dialogue.In addition, literature has the ability to challenge societal norms and provoke critical thinking. Many literaryworks have tackled controversial topics and shed light on social issues. For example, George Orwell's "1984" is a dystopian novel that warns against the dangers of totalitarianism and the erosion of individual freedom. By presenting alternative perspectives and questioning established beliefs, literature encourages us to think critically and question the status quo.中文回答：文学对我来说，是人类表达和想象力的精华。
17century
Robert Herrick 罗勃特·赫瑞克 （1591-1674）
骑士派诗人 Gather Ye Rose Buds while Ye May 趁早采摘 玫瑰花蕾：Rightly reflects the flitting joys of the dying class； To Daffodils 致水仙
Andrew Marvell 安德鲁·马维尔(1621-1678)
Metaphysical poet, who wrote poems on nature. To his Coy Mistress 致她娇羞的女友 诗人将激情与理智并置，极力表达了文 艺复兴时期古典主义的一个重大主题： 在不可避免的死亡到来之前抓住性爱的 快乐。
Metaphysical Poets (玄学派诗人）
十七世纪上半叶的一群宗教诗人，他们作 品的特点在于机智、富有想象力的画面、 简略而含义模糊的表达，对悖论词语的玩 弄以及比喻的使用。他们运用当代的科学 发现和理论、对人文主义、信仰和永生的 争论、通俗的口语化韵律和创新的诗歌形 式来考察个人、上帝和宇宙的关系。
The Seventeend of the English Bourgeoise Revolution
17世纪是英国历史上最为动荡的时期，由于社会生活 及社会结构的巨大改变，最终贵族王室与英国资产阶 级之间的强烈冲突导致1642-1649的内战。这就是英 国历史上的资产阶级革命。新兴的资产阶级倡导清教 主义信念（Puritan），它主张勤俭节约，辛勤工作， 反对享乐。清教精神成为资产阶级的信条，所以又称 为”清教革命”。 其后，在1668年新教派的政治领袖发动了“光荣革命 ”
主要内容
故事的原型源于《圣经》中的《创世纪3 ：1-24》。其主题是“人类的堕落”，也 即人类的违抗和乐园的丧失，其根本原因 是撒旦。在天堂，魔鬼撒旦领导了一场反 抗上帝的叛乱。失败后，他和造反天使们 一起被扔进了地狱。然而，撒旦拒绝接受 失败，他发誓说“并不是所有的都已失去 ”，并且要为他的垮台寻找复仇机会。这 首诗继续讲述撒旦如何通过引诱亚当和夏 娃来复仇。
9Character
The story starts with a dream in which the author sees Christian the Pilgrim, with a heavy burden on his back, reading the Bible. When he learns from the book that the city in which he and his family live shall be burnt down in a fire, Christian tries to convince his family and his neighbors of the oncoming disaster and asks them to go with him in search of salvation, but most of they simply ignore him.
Dynamic characters & Static characters
Antihero (a character who undermines the reader’ reader’s expectation about heroes) Foil (a supporting role to reveal by contrast the distinctive qualities of the protagonist) Stock character (the stereotyped figure popular in formula fiction, television programs and comic books; ready-made readytypes)
Protagonist & Antagonist (no moral judgments)
Identification and characterization of a novel alp
专利名称：Identification and characterization of a novel alpha-amylase from maize endosperm发明人：Martha G. James,Alan M. Myers,Christophe Colleoni,Kevin D. Stokes申请号：US10952551申请日：20040927公开号：US07270988B2公开日：20070918专利内容由知识产权出版社提供摘要：SHE, a Starch Hydrolytic Enzyme active in maize endosperm (), and the cDNA sequence encoding SHE are disclosed. The specificity of native, purified SHE is similar, in general terms, to previously known alpha-amylases. However, the activity of SHE toward amylopectin results in hydrolysis products that are distinctly different from those of other alpha-amylases. SHE, and its homologous equivalents in other plants such as rice, , apple and potato, can be used in starch processing for generating different, e.g., larger sized, alpha-limit dextrins for industrial use, as compared to those generated by previously known alpha-amylases or other starch hydrolytic enzymes. In addition, modification of the expression of this enzyme in transgenic maize plants or in other transgenic organisms (including bacteria, yeast, and other plant species) can be useful for the generation of novel starch forms or altered starch metabolism.申请人：Martha G. James,Alan M. Myers,Christophe Colleoni,Kevin D. Stokes地址：Des Moines IA US,Ames IA US,Arques FR,Ames IA US国籍：US,US,FR,US代理机构：Weingarten, Schurgin, Gagnebin & Lebovici LLP 更多信息请下载全文后查看。
picaresquenovel名词解释
Picaresque NovelIntroductionA picaresque novel is a genre of fiction that originated in Spain during the 16th century. It is characterized by its episodic structure,satirical tone, and the adventures of a roguish protagonist, known as a picaro. This article aims to explore the concept of a picaresque novel, its key characteristics, notable examples, and its impact on literature.Characteristics of a Picaresque NovelA picaresque novel typically exhibits the following characteristics:1. Roguish ProtagonistThe central character in a picaresque novel is often a lower-class, cunning, and morally ambiguous individual. The picaro, through their various misadventures, provides a social critique of the society they navigate.2. Episodic StructureThe narrative of a picaresque novel is divided into a series of loosely connected episodes or adventures. These episodes often depict the picaro’s encounters with different social classes, institutions, and settings.3. Satirical TonePicaresque novels employ satire to criticize societal norms, customs, and institutions. Through humor and irony, authors expose the flaws and hypocrisies of the society in which the picaro operates.4. Social CritiquePicaresque novels serve as a commentary on the social and economic inequalities prevalent in society. T he picaro’s experiences shed light on the corruption, greed, and injustices that exist within the social hierarchy.5. RealismPicaresque novels often incorporate realistic and vivid descriptions of the settings and characters encountered by the picaro. This attention to detail enhances the sense of authenticity and provides a social commentary grounded in reality.Notable ExamplesSeveral notable picaresque novels have made significant contributions to the genre. Some of these include:1. “Don Quixote” by Miguel de CervantesConsidered one of the earliest and most influential picaresque novels, “Don Quixote” follows the adventures of the delusional knight-errant, Don Quixote, and his loyal squire, Sancho Panza. Through their exploits, Cervantes satirizes the chivalric romances popular during his time.2. “The Life and Opinions of Tristram Shandy, Gentleman” by Laurence SterneSterne’s novel is a unique and experimental picaresque work that challenges the traditional narrative structure. It humorously explores the life and thoughts of the eccentric Tristram Shandy, employing satire to critique various aspects of society.3. “Moll Flanders” by Daniel Defoe“Moll Flanders” follows the life of the titular character, who engages in a series of deceptions, crimes, and relationships in her quest for social advancement. Defoe’s novel offers a vivid portrayal of 18th-century England and highlights the struggles faced by women of that era.4. “The Adventures of Huckleberry Finn” by Mark TwainAlthough often categorized as an adventure novel, Twain’s “Huckleberry Finn” exhibits many picaresque elements. The story follows the escapades of Huck and Jim, an escaped slave, as they navigate the Mississippi River. Twain’s satire targets racism, slavery, and social hypocrisy.Impact on LiteratureThe picaresque novel has had a profound impact on literature,influencing subsequent works in various ways:1. Development of the BildungsromanThe picaresque novel played a significant role in the development of the Bildungsroman, a genre that focuses on the moral and psychological growth of the protagonist. The episodic structure and social critique seen in picaresque novels provided a foundation for later coming-of-age stories.2. Satirical TraditionPicaresque novels established a tradition of using satire as a means of social critique. This tradition can be seen in later works, such as Jonathan Swift’s “Gulliver’s Travels” and Voltaire’s “Candide,” both of which employ satire to criticize society and institutions.3. Influence on Modern FictionThe picaresque novel’s episodic structure and focus on theindividual’s experiences have influenced modern fiction. Authors like Jack Kerouac, in his no vel “On the Road,” and John Kennedy Toole, in “A Confederacy of Dunces,” have drawn inspiration from the picaresque tradition.4. Exploration of Marginalized PerspectivesPicaresque novels often give voice to marginalized individuals and shed light on their experiences. This exploration of the lower classes, women, and people on the fringes of society has influenced the representationof diverse perspectives in literature.ConclusionThe picaresque novel is a genre that continues to captivate readers with its roguish protagonists, satirical tone, and social critique. From its origins in Spain to its influence on modern fiction, this genre has left an indelible mark on literature. Whether it is Cervantes’ “Don Quixote” or Twain’s “Huckleberry Finn,” picaresqu e novels entertain, challenge, and offer valuable insights into the human condition.。
英国文学——精选推荐
英国⽂学●What are the characteristics of Anglo-Saxon literature?1.Anglo-Saxon literature is almost exclusively a verse literature in oral form.2.Two groups of poetry are found in Anglo-Saxon period. Before Christianity wasintroduced, there was pagan poetry. The representative is Beowulf After the introduction of Christianity, Christian poetry appearedIn what aspects did the Norman Conquest affect English language and literature?1.After the Norman Conquest, chivalry was introduced into England.2.French became the leading language used by the ruling class and a large numberof French words entered the English language. Latin was used by the scholars and clergymen.Give an introduction of Chaucer’s masterpiece The Canterbury Tales?1.The Canterbury Tales is a collection of 24 tales, with a long prologue at thebeginning. The propologue is better known than the tales because Chaucer reveals his plan of writing the book in it. He tells his reader how he meets some 29pilgrims at a small in of London and joins them to go to Canterbury,to make the long journey full of fun, each pilgrim, according to the suggestion of the host of the inn, will tell four tales,2.The pilgrims represent all the social classes of feudal England.hese tales deal with all aspects of medieval literature; romances of knights and ladies, folk tales, animal fables, stories of travels and adventures, legends, allegories and so +-●What is the social significance of The Canterbury Tales?1.In The Canterbury Tales, Chaucer draws a true-to-life picture of English feudal society of his day2. he praises man’s energy, intellect, quick wit and love of life. His tales expose and satires the social evils of his day. They criticise the degeneration of the noble, the heartlessness of the judge, and the corruption of the church.●Discuss Chaucer’s writing features.1. Chaucer’s language is vivid and exact.2. His poetry is full of vigour and swiftness.3. His style is flexible. His prose is easy and informal.4. He uses mild satire and the rhyming couplet5. He is the first great writer to use the dialect of London in writing.Metaphysical poetry （⽞学派诗歌）John Donne is generally regarded as the leading figure of this school. Enlightenment （启蒙运动）It was an expression of struggle of the bourgeoisie against feudalism.Sentimentalism（感情主义）a s a kind of mild protest against the social injustice.●What are the characteristic of English literature in the 18th century?1.The main literary stream of the 18th century was realism.2.The main characters of their works were usually common me n.3. Most of the writers concentrated their attention on daily life4. prose literature which include the book, the newspaper and the magazine,5.Novel writing made a big advance in this century6. In this age satire was much used in writing., some excellent satirists, such as Pope, Swift and Fielding.What are Jonathan Swift’s writing features?1.Jonathan Swift is one of the realist writers.2.all of Swift’s plots come fromimagination3.This makes his satire all the more powerful.4.Swift expresses democratic ideas in his works5.His language is simple, clear and vigorous.6.wift is one of the greatest masters of English prose.How are the poems in Songs of Innocence contrasted with the poems in Songs of Experience?1Songs of innocence contains short lyrical poems with little children as the speakers.2.the poet expresses his own love for the beauty of the world3.Blake expresses his delight in the sun, the hills4. The best-known poems in the collection are “The Lamb”,3. Songs of Experience is t a much maturer and Blakes’s most important work4. The poems in this collection show that the poet’s eyes are open to t he evils and vices of the world5. He points out that the earth is unhappy and lacks love and gaiety.6. The miserable living conditions of the poor are reflected.7.The best-known poems in the collection are “The Tyger”, “The Fly”, “London”, and “The Chimney-Sweeper”.What are the features of realistic novels and poetry?1.18th century realistic novel, they were all concerned about the fate of the common people2. and they were angry with the inhuman social institution.3.The poetry of this period was mainly characterized by experiments with new styles and new ways of expression.Why is Jane Eyre a successful novel?1. The work is one of the most popular and important novels of the Victorian age2. It is noted for its sharp criticism of the existing society, e.g. the religious hypocrisy of charity institutions, the social discrimination and the false social convention as concerning love and marriage3. The success of the novel is also due to its introduction to the English novel the first governess heroine.4.Jane Eyre is a completely new woman image. She represents those middle-class working women who are struggling for recognition of their rights and equality as a human being.5. The vivid description of her intense feelings and her thought and inner conflicts brings her to the heart of the audience. How do you understand that Dickens is the greatest critical realist writer of the Victorian Age?1.It is his serious intension to expose and criticize in his works all the poverty, injustice, hypocrisy and corruptness he sees around him.2.With his first sentence, Dickens engages the reader’s attention and holds it to the end.3The settings of his stories have an extraordinary vividness.4.In language, he is often compared with Shakespeare for his adeptness with the vernacular and large vocabulary.5.Character-portrayal is the most distinguishing feature of his works6..Dickens’ works are also characterized by a mingling of humor and pathos. Comment on Tess of the D’Urbervilles by Thomas Hardy1.As a pure woman brought up with the traditional idea of womanly virtue,2. Tess is abused and destroyed by both Alex and Ange2. naturalistic tendency: led to Tess’final destruction step by step by fate 3. conventions and morals which strangle the individual will and destroy natural human emotions and relationships.Comment on the characteristics of Modernist literature.● 1. Modernism takes the irrational philosophy and the theory of psychoanalysis asits theoretical base. 2. The major themes of the modernist literature are the distorted, alienated and ill relationships between man and nature, man and society, man and man, and man and himself.3.The modernist writers concentrate more on the private than on the public,more on the subjective than on the objectivity. They are mainly concerned with the inner world of an individual. 4.In their writings,the past, the present and the future are mixed together and exist at the same time in the consciousness of an individual.●What are the general literary characteristics of the Romantic Age?1.At first the literature reflected the political turmoil of the age stirred by French Revolution,When the turmoil was over, it suddenly developed a new2.he glory of the age is seen in the poetry of W ordsworth, Coleridge, Byron, Shelley and Keats.3. W omen novelists appeared in this age●What are the differences between the two groups of Romantic poets in theirpolitical attitude?1.The romantic poets split into two groups because of their different attitudes toward the capitalist society.2.The first group of romantic poets, that is, the older generation, reflected the thinking of those classes3. t hey turned their attention to describing nature and in the natural world they found the ideal form of human life4.When they were young they were politically enthusiastic,and later they became conservative.These romantic poets are represented by W ordsworth, Coleridge and Southey.4. The second group of romantic poets, that is , the younger generation,expressed the aspiration and ideas of the labouring classes. They held out the ideal picture of the future society free from oppression and exploitation. They were firm supporters of French Revolution. These romantic poets are represented by Byron, Shelley and Keats.●Features of Jane Austen’s Writing1.She draws vivid and realistic pictures of everyday life of the country society in her novels.2.Austen’s works have a v ery narrow literary field.3. Her novels show a wealth of humour, wit and delicate satire.●Comments on Byron1.Byron is one of the excellent representatives of English Romanticism.2.His poems show energy and vigour, romantic daring and powerful passion.3.And some of his poems show his individual heroism and pessimism.Comments on Shelley1.He advocates this social and political ideal all his life.2. Byron are justifiably regarded as the two great poets of revolutionary romanticism in England●Comments on John Keats1.Keats learned the art of poetry mainly from the poets of the Renaissanc e2. Theartistic aim in his poetry was always to create a beautiful world of imagination as opposed to the sordid reality of hisday3..His poetry is distinguished by sensuousness and the perfection of form.4.He expressed his dissatisfaction with the society and showed his sympathy with the sufferings of the poor people.。
Mr Knowall文章解析
Characterization :
Characterization is the creation of imaginary characters so that they exist for the reader as lifelike persons.
Characterization :
Three fundamental methods of characterization in fiction:
The Character:
• • • • •
character hero and heroine anti-hero protagonist / main character antagonist
The Character:
•
•
•
A round character: usually has more than one quality, and grows in the course of the story development, as he reacts to events and to other characters. A flat character: is constructed around a single idea or quality. Such a character does not develop in the course of narration and is easily recognized and remembered. A foil: is a supporting character whose role in the story is to highlight the protagonist by presenting a comparison or a contrast to him.
Character 文学原理教程 ppt课件
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Character 文学原理教程 ppt课件
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Hero / Heroine Carrying Connotation
Protagonist 主人公 Sounding Neutral
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Protagonist Antagonist 反面角色，对立面 ---- The rival or opponent against whom the protagonist / hero is contending.
the beginning of a work to the
end.
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Character 文学原理教程
• A character who experiences an essential change in personality or attitude. Protagonists are almost always dynamic.
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1. Dynamic Character
---- Exhibiting some kind of change as the story progresses.
2. Static Character
---- Remaining the same from
8
Character 文学原理教程
• A character who is complex and has many sides or traits with unpredictable behavior and a fully developed personality. Antagonists are usually a round characterization.
植物亚细胞定位（范文2篇）
植物亚细胞定位（范文2篇）以下是网友分享的关于植物亚细胞定位的资料2篇，希望对您有所帮助，就爱阅读感谢您的支持。
植物亚细胞定位（1）第42卷第4期东北农业大学学报42(4):83~87植物亚细胞定位载体卡盒pCEG的构建及验证朱丹，王希，朱延明*，陈超，李勇，柏锡，才华，纪（东北农业大学生命科学学院，哈尔滨150030）巍摘要：GFP基因被广泛地应用于植物转基因以及基因功能验证研究，然而构建与GFP融合的植物表达载体，常会因酶切位点难以选择，而使得载体构建过程复杂，周期长。
以含GFP的质粒pCAMBIA-1302为基础，消除此质粒本身的多克隆位点（MCS），并在此质粒GFP基因序列前插入原核表达载体pET-32b的多克隆位点，构建了植物GFP亚细胞定位载体卡盒pCEG；采用农杆菌介导的转化方法，将此载体卡盒pCEG导入模式植物烟草叶片中进行瞬时表达，用激光共聚焦显微镜观察GFP蛋白的亚细胞定位情况，发现GFP蛋白均匀的在细胞质和细胞核中表达，证明此载体卡盒可用于植物基因的亚细胞定位分析，并为构建目的基因与GFP融合的植物表达载体提供了很多可用的酶切位点，对植物基因功能验证提供了分子操作简便的植物表达载体卡盒，具有重要的利用价值。
关键词：载体卡盒构建；亚细胞定位；GFP；烟草中图分类号：Q782文献标志码：A文章编号：1005-9369（2011）04-0083-05 Constructionandapplicationofaplantsubcellularlocalization vectorboxofpCEG/ZHUDan,WANGXi,ZHUYanming,CHEN Chao,LIYong,BAIXi,CAIHua,JIWei(CollegeofLifeSciences,NortheastAgriculturalUniversit y,Harbin150030,China)Abstract:GFPgenewaswidelyappliedinthetransgenicplants andgenesfunction’sprovingstudy.However,theconstructionofGFPfusedplantexpressionvector swasalwayscomplicatedandtimeconsumingduetothehardchoo eplasmidpCAMBIA-130 2whichincludedGFPgeneasaoriginalvector,andeliminateditso wnmultipleclonesite(MCS),andtheninsertedpET-32b’smultip leclonesiteinfrontofGFPgenesequences,namedtheeventualpla ntGFPsubcellularlocalizationvectorboxpCEG.ByAgrobacteri um-mediatedtransformationmethod,pCEGinstantaneousexp ressedintobaccoleaves,andGFPproteinsubcellularlocalization wasobservedbylaserconfocalmicroscope.Theresultdisplayedt hattheGFPproteinsexpressedinbothcytoplasmandnucleus,thi smeansthepCEGboxcanbeusedconvenientlyforplantgenessub cellularlocalizationanalysisandprovidealotofusablerestrictio nenzymecuttingsiteforplantgenesfusionedwithGFP,andthepC EGboxisconvenientforplantgenesfunctionanalysisandhasgre atusevalue.Keywords:constructionofvectorbox;subcellularlocalization ;GFP;tobacco在基因的功能研究过程中，外源重组基因表达产物的功能与其在宿主细胞中的定位有重要的关系，特别是对于真核细胞，蛋白质位于不同的细胞部位所行使的功能也不同，所以研究其在宿主细胞收稿日期：2011-01-12基金项目：国家高科技发展计划（863计划）（2008AA10Z153）；国家自然科学基金资助项目（30570990）；黑龙江省科技厅重大攻关项目（GA06B10）；黑龙江省教育厅科技项目（11521024）；黑龙江省教育厅科技项目（11521021）作者简介：朱丹（1986-），女，硕士研究生，研究方向为植物基因工程与分子生物学。
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Characterization of a novel bacteriocin produced by Lactobacillus sakei LSJ618isolated from traditional Chinese fermented radishJie Jiang,Bo Shi,Deqiang Zhu,Qingxia Cai,Yiran Chen,Jinnian Li,Kezong Qi,Ming Zhang *College of Life Sciences,Anhui Agricultural University,130#ChangJiang Road,Hefei 230036,PR Chinaa r t i c l e i n f oArticle history:Received 1March 2011Received in revised form 20July 2011Accepted 26July 2011Keywords:BacteriocinLactobacillus sakei SakacinBiopreservativea b s t r a c tA novel bacteriocin,sakacin LSJ618,produced by the strain Lactobacillus sakei LSJ618isolated from traditional Chinese fermented radish,was studied.L .sakei LSJ618was identi fied by both phenotypical and physiological tests combined with 16S rDNA sequence analysis.Sakacin LSJ618is sensitive to hydrolytic enzymes including lipase,is stable between pH 2e 8,and is heat resistant (30min at 121 C).Sakacin LSJ618exhibits inhibitory activity against food-spoiling bacteria and food-borne pathogens,including the Gram-positive Listeria monocytogenes ,Staphylococcus aureus ,Sarcina sp.,Micrococcus luteus ,and the Gram-negative Proteus sp.and Escherichia coli ,but not against most of the lactic acid bacteria tested.Maximal production of bacteriocin was reached in the late stationary phase,and inhibitory activity declined within 26h.The mode of action of sakacin LSJ618was determined to be bactericidal,as evidenced by its action upon Micrococcus tetragenus .After partial puri fication by ammonium sulfate precipitation and Sephadex G-25chromatography,the molecular weight of sakacin LSJ618was determined to be 5.2kDa by Tricine-SDS-PAGE.The identi fied properties of sakacin LSJ618indicate that it is a novel bacteriocin with potential application as a biopreservative in the food industry.Ó2011Elsevier Ltd.All rights reserved.1.IntroductionFood spoilage and food-borne pathogenic bacteria such as Lis-teria monocytogenes ,Escherichia coli ,Salmonella sp.,Staphylococcus aureus ,Bacillus cereus and Clostridium botulinum not only endanger human health,but also lead to severe losses of foodstuff.Although chemical additives can,to some extent,effectively inhibit the growth of bacteria,some of them have been proven harmful to human health as well as the environment.Fortunately,many lactic acid bacteria (LAB)can produce inhibitory compounds such as organic acids,hydrogen peroxide,diacetyl,enzymes and bacterio-cins (Deegan,Cotter,Hilla,&Ross,2006)that are effective against spoilage and pathogenic bacteria.Among these antibacterial compounds,bacteriocins represent a potentially safe class of bio-preservatives for use in the food industry because of their sensi-tivity to proteolytic enzymes.For example,Nisin,a bacteriocin produced by Lactococcus lactis ,has been used in many countries to extend the shelf-life of food (Deegan et al.,2006).Lactobacillus sakei is widely observed in diverse habitats such as meat,vegetables,dairy and bakery products;they are widely used asstarter cultures in dry sausage production,and contribute to their characteristic color,texture and flavor.Since the L .sakei Lb 706product sakacin A (Schillinger &Lücke,1989)was identi fied,more than ten other bacteriocins have been well studied,including sakacin P,sakacin K,sakacin 23K,sakacin Q,sakacin 2a,sakacin C2,bacteriocin AMA-K and bacteriocin ST8KF (Gao,Jia,Gao,&Tan,2010;Hugas,Garriga,Aymerich,&Monfort,1995;Kjos,Snipen,Salehian,Nes,&Diep,2010;Mathiesen,Huehne,Kroeckel,Axelsson,&Eijsink,2005;Powell,Witthuhn,Todorov,&Dicks,2007;Tichaczek,Vogel,&Hammes,1994;Todorov,Nyati,Meincken,&Dicks,2007).The most important characteristic of bacteriocins produced by L.sakei is their activity against Listeria sp.(Schillinger &Lücke,1989;Todorov et al.,2010;Trinetta,Floros,&Cutter,2010).Some antilisterial bacteriocins and their producing strains can control a number of viable L.monocytogenes cells in different meat productions for 3e 4weeks (Katla et al.,2002;Schillinger,Kaya,&Lücke,1991).In addition,the individual 50%inhibitory concentra-tions of sakacin P and sakacin A are less than that of nisin,and thus are considered to be safe (Dortu,Huch,Holzapfel,Franz,&Thonart,2008;Jones,Zagorec,Brightwell,&Tagg,2009;Katla,Naterstad,Vancanneyt,Swings,&Axelsson,2003;Trinetta et al.,2010).The aforementioned studies have revealed that these bacteriocins are very promising biological food preservatives,especially in meat products.In fact,most bacteriocin-producing strains of L .sakei have been isolated from meat products;only two strains,L.sakei C2(Gao*Corresponding author.Room 315,College of Life Sciences,Anhui Agricultural University,130#ChangJiang Road,Hefei 230036,PR China.Tel.:þ865515786319;fax:þ865515785833.E-mail address:zhangming@ (M.Zhang).Contents lists available at ScienceDirectFood Controljournal homep age:/locate/foodcont0956-7135/$e see front matter Ó2011Elsevier Ltd.All rights reserved.doi:10.1016/j.foodcont.2011.07.027Food Control 23(2012)338e 344et al.,2010)and L.sakei5(Vaughan,Eijsink,O’Sullivan,O’Hanlon,& Van Sinderen,2001)were isolated from vegetable sources(tradi-tional Chinese fermented cabbage and malted barley,respectively).In an effort tofind a bacteriocin that can be used as a food preservative,a strain producing a novel bacteriocin,termed sakacin LSJ618,was isolated from the traditional Chinese fermented radish. The strain was identified as L.sakei strain LSJ618.Furthermore,this study reports the characteristics and mode of action of sakacin LSJ618.2.Materials and methods2.1.Strains,media and culture conditionsThe24types of traditional Chinese fermented foods used in this study were purchased from Hubei Province,China.Bacterial strains used in this study are shown in Table1.The bacteriocin-producing strain L.sakei LSJ618,isolated from traditional Chinese fermented radish,as well as the indicator strains of LAB,were cultured in MRS medium(Generay Biotech Co.Ltd., Shanghai,China(Sangon));other indicator strains were cultured in LB medium(Sangon).The strains were stored atÀ70 C in MRS or LB broth supplemented with DMSO(7.5%,v/v)(Sigma e Aldrich, St.Louis,MO),and were propagated twice in MRS or LB medium before use.Seed cultures of L.sakei LSJ618were prepared by culti-vating the cells in MRS broth with shaking(120r/min),and main-tained at37 C for12h.For fermentation,4%of the seed culture was inoculated into1000ml of MRS broth and maintained at37 C for24h.2.2.Isolation and identification of bacteriocin-producing strainsThe traditional Chinese fermented radish was added to a bottle containing50ml of sterilized water and was mixed by shaking.The suspension was diluted,and a0.1ml sample was plated on MRS agar with0.01%(w/v)bromocresol purple(Chemapol Asia Pte Ltd). After the plates were incubated at37 C for48h,colonies that changed the color of the medium from purple to yellow were restreaked onto MRS plates.The bacteriocin activities of the strains were determined by the agar well diffusion test(Ennahar, Sashihara,Sonomoto,&Ishizaki,2000).Strains that inhibited the indicator strains were selected for further studies.Strain identification was based initially on phenotypical and physiological tests(Kandler&Weiss,1986).Further identification was carried out by sequence analysis of16S rDNA.Bacterial DNA was isolated with a UNIQ-10Column Bacterial Genomic DNA Isolation Kit(Sangon);16S rDNA was amplified by PCR(94 C, 5min,for predegeneration,then94 C for45s,55 C for45s and 72 C for90s,30cycles)with primers P1(50-AGAGTTT-GATCCTGGCTCAG-30),and P2(50-TACGGTTACCTTGTTACGACTT-30) using a PCR amplification kit(Sangon).The PCR products were sequenced and sequence homologies were examined by comparing DNA databases(/BLAST).A multiple sequence alignment was carried out using CLUSTAL X,and a boot-strap consensus tree(1000copies)was constructed by the Neighbor-Joining(NJ)method using Mega software,version4.1 (Kumar,Tamura,&Nei,2004;Thompson,Higgins,&Gibson,1994).2.3.Preparation of the crude bacteriocinThe cell-free supernatant of the L.sakei LSJ618culture was prepared by centrifugation(9000g for15min)andfilter-sterilization(0.45m m pore-size membrane).The cell-free super-natant was then dialyzed(MW cutoff500Da)against double-distilled(dd)H2O(pH6.0)for6h at4 C to remove organic acids,H2O2and other small antibacterial materials;the solution was then lyophilized.The resulting supernatant powder(SP)was used as the sample for further analysis of the characteristics of the bacteriocin.2.4.Assay of bacteriocin activity and antibacterial spectrumBacteriocin activity was measured by the agar well diffusion test (Ennahar et al.,2000)using Micrococcus tetragenus as the indicator strain.The bacteriocin titer was determined by measuring the diameter of the inhibition zone around the wells,and expressed as arbitrary units(AU)per ml;one AU was defined as the reciprocal of the highest serial two-fold dilution producing distinct inhibition of the indicator lawn(Todorov&Dicks,2005).All of the indicators for the antibacterial spectrum are listed in Table1.2.5.Preliminary characterization of sakacin LSJ618To avoid artifacts caused by the presence of hydrogen peroxide, organic acids and other small molecular antibacterial compounds in the cell-free supernatant,SP was used in the agar well diffusion test(Ennahar et al.,2000).All experiments were conducted in triplicate.To test thermostability,SP samples were heated to60 C,80 C, 100 C and121 C for30min,and then placed in an ice bath for 10min.Residual bacteriocin activity was then determined; unheated SP served as the control.To determine pH stability,SPTable1Inhibition of various bacteria by the supernatant powder(SP)of Lactobacillus sakeiLSJ618as determined by the well diffusion agar test.Indicator strains Source a Inhibitory activity bEscherichia coli ECX1This Lab eEscherichia coli ECX2This Lab eEscherichia coli ECX3This Lab eEscherichia coli ECX4This LabþProteus sp.This LabþþþPseudomonas aeruginosa This Lab eBacillus subtilis This Lab eBacillus thuringiensis This Lab eSalmonella pullorum CIVDC eListeria monocytogenes GIMþþþListeria innocua GIM1.230GIMþMicrococcus tetragenus This LabþStaphyloccocus aureus This LabþSarcina sp.This LabþþMicrococcus luteus GIM1.226GIMþþþPediococcus acidilactici This Lab eStreptococcus thermophilus This Lab eEnterococcus faecalis This Lab eLactobacillus sp.LSX801This Lab eLactobacillus plantarum This Lab eLactobacillus lactis cremoris This Lab eLactobacillus brevis This LabþLactobacillus pentosaceus This Lab eLactobacillus fructivorans This Lab eLactobacillus helveticus This Lab eLactobacillus casei This Lab eLactobacillus fermentum This Lab eLactobacillus bulgaricus This Lab eLactobacillus sakei LSJ618This Lab eLactobacillus sakei K14-1This Lab eLactobacillus sakei L5-1This Lab eLactococcus lactis K303This Lab ea CIVDC(China Institute of Veterinary Drug Control,Beijin,PR China);GIM(Guangdong Institute of Microbiology,Kuanzhou,PR China).b Wells(8mm in diameter)werefilled with200m l SP,Mean counts ofthree duplications.þ,Diameter of inhibition zone:8.00e11.00mm;þþ,11.00e15.00mm;þþþ,more than15.00mm;À,no inhibition zone.J.Jiang et al./Food Control23(2012)338e344339samples were adjusted to pH2,4,6,8,10and12with sterile1.0M NaOH or1.0M HCl;SP maintained at pH6.0served as the control. Following6h of incubation at37 C,samples were readjusted to pH 6.0with sterile1.0M NaOH or1.0M HCl,adjusted to the same volume,andfinally tested for residual bacteriocin activity(Todorov et al.,2010).The sensitivity of the antibacterial compound to hydrolytic enzymes,including a-chymotrypsin(Bio BASIC Inc.),protease K (Sigma),trypsin(Ameresco),lipase(Sigma),papainase(Sangon) and pepsin(Bio BASIC Inc.),was examined by incubating the SP for 6h in the presence of6.0mg/ml of each enzyme at37 C at optimal pH.The samples were then heated for10min at100 C to inactivate the enzyme.SP without enzyme and6.0mg/ml enzyme solution without SP served as controls.All samples were readjusted to pH 6.0with sterile1.0M HCl or1.0M NaOH,and the residual bacte-riocin activity was tested(Minamikawa,Kawai,Inoue,&Yamazaki, 2005).The SP was then incubated for4h in the presence of a1% chemical reagent such as Tween20(Bio BASIC Inc.),Tween80(Bio BASIC Inc.),urea(Bio BASIC Inc.),sodium dodecyl sulfate(SDS)(Bio BASIC Inc.)or EDTA(Bio BASIC Inc.)(v/v or w/v)at37 C,and the residual bacteriocin activity was examined;SP without1%reagent and1%reagent solutions without SP served as controls(Todorov et al.,2007).2.6.Mode of action of sakacin LSJ618Seed cultures of M.tetragenus were prepared by cultivating the cells in LB broth with shaking(120r/min)at37 C for14h (OD600¼2.73).To assess the mode of action of sakacin LSJ618,5% (v/v)of the seed culture was inoculated into200ml of LB broth in a250mlflask and cultured with shaking(120r/min)at37 C for 16h.When the growth of M.tetragenus reached the late-logarithmic phase,320AU/mL of sakacin LSJ618was added to the culture.The OD600and viable cell count of the culture were determined once every hour(Gao,Li,Sheng,&Liu,2011;Smaoui et al.,2010).2.7.Cell growth,pHfluctuation and production of sakacin LSJ618Seed culture of strain LSJ618was inoculated into500ml of MRS broth at4%(V/V)and maintained at37 C.Samples were taken every2h to determine the OD600,the pH and the antibacterial activity against M.tetragenus.2.8.Partial purification of sakacin LSJ618and molecular mass determinationCell-free supernatant(2000ml)was precipitated with80% ammonium sulfate saturation at4 C over2h with vigorous stir-ring.After overnight precipitation at4 C,the proteins were iso-lated by centrifugation(9000g for45min at4 C)and resuspended in80ml of dd H2O.The resuspended solution was dialyzed(MW cutoff500Da)for6h at4 C to remove the sulfate,then lyophilized and stored atÀ20 C.The lyophilized samples were dissolved in dd H2O,and5ml was applied to a2.6cmÂ100cm Sephadex G-25 column equilibrated withfiltered dd H2O.The proteins were eluted with dd H2O(0.5ml/min);1ml fractions were monitored for protein content by measuring the absorbance at280nm and then assayed for bacteriocin activity.Fractions with bacteriocin activity were collected and lyophilized.Active fractions eluted from the Sephadex G-25column were analyzed by Tricine-SDS-PAGE.Electrophoresis was performed using an18%polyacrylamide gel at a constant voltage of120V for 3.5h(Schägger,2006).The gel wasfixed in40%(v/v)methanol/l0% glacial acetic acid for2h.Half of the gel was then stained with Coomassie brilliant blue R-250(Bio BASIC Inc.)for molecular weight determination;the other half was used for the antimicrobial activity assay.The antimicrobial assay was performed by washing the gel in sterile water for4h,overlaying with nutrient soft agar seeded with1.5%(v/v)M.tetragenus,and culturing at37 C for14h to determine the inhibitory effect(Gao et al.,2010;Todorov et al., 2010).3.Results and discussion3.1.Isolation and identification of the bacteriocin-producing strainStrain LSJ618was isolated from a particular kind of Chinese fermented radish,whose cell-free supernatant demonstrated activity against Gram-positive and Gram-negative bacteria(data not shown).In order to confirm that the active compound was a protein,the antibacterial activity of the sample was tested, following precipitation,using solid ammonium sulfate to80% saturation.The results of the antibacterial assay revealed that the sample demonstrated activity against a number of Gram-positive and Gram-negative bacteria(Table1),and was also sensitive to proteolytic enzymes(data not shown).These results indicated that the active compound was a protein,and was named sakacin LSJ618.Strain LSJ618was a rod-shaped,Gram-positive,catalase-negative bacterium which produced white circular colonies on MRS plates. The organism grew in MRS broth from4 C to41 C,but not at 45 C;this strain also grew in7.5%NaCl,but not10%NaCl.To further characterize the strain,fermentable carbohydrates were examined and16S rDNA sequences were analyzed.Strain LSJ618did not grow on mannitol,monohydrate,xylose,glycerol,maltose, melezitose,raffinose,sorbose,sorbitol,erythritol,inositol,rham-nose or soluble starch,but grew well on glucose,fructose,galactose, arabinose,mannose,trehalose,sucrose,lactose,cellobiose,meli-biose,ribose,esculin hydrate,sodium D-gluconate and D-(þ)salicin. The sequence of the partial16S rDNA of strain LSJ618(GenBank access number is HQ992696)showed99%similarity to that of L. sakei BMG168(Fig.1).All of the above results indicated that LSJ618 was a L.sakei strain.Most reported bacteriocin-producing L.sakei strains have been isolated from cheese or meat products(Samelis,Roller,& Metaxopoulos,1994;Zdolec et al.,2008);only the sakacin C2 producing strain,L.sakei C2,was isolated from a vegetable product. Strain LSJ618,examined in this study,is the second strain to be isolated from a vegetable product.This suggests that bacteriocins produced by L.sakei strains may be important in these vegetable products.3.2.Effects of different treatments on the antibacterial activity of sakacin LSJ618The antibacterial activity of sakacin LSJ618was affected,to varying degrees,by hydrolytic enzymes,chemical reagents, temperature and pH(Table2).Sakacin LSJ618was completely inactivated by pepsin,papain,trypsin and lipase,and was partially inactivated by proteinase K and a-chymotrypsin.It remained stable for30min in solutions of1%Tween20,Tween80or acetonitrile, but was completely inactived by1%urea.These results demon-strated that the active compound was proteinaceous in nature.All chemical reagents tested had no effect on the growth of M.tetragenus by themselves(data not shown);the effects of1% EDTA and SDS on the inhibitory activity of sakacin LSJ618could not be ascertained from these experiments because these reagents themselves have been shown to inhibit the indicator strain.In the thermostability experiments,sakacin LSJ618retained more than84%and80%of its activity at100 C and121 C,J.Jiang et al./Food Control23(2012)338e344 340respectively for 30min.Assessments of pH stability revealed that the antibacterial activity decreased somewhat between pH 4and 6,and completely disappeared at pH 10.The highest residual activity was greater than 81%when the solution pH was between 2and 6.Thus,sakacin LSJ618was identi fied as being less pH-stable than sakacin 2a (produced by L .sakei 2a),which was reported to be stable from pH 1.5to 10(de Carvalho et al.,2010).The enzyme sensitivity tests demonstrated that this strain was a novel sakacin,based on its sensitivity to lipase;other known bacteriocins produced by L .sakei were not sensitive to lipase,although the bacteriocin-like inhibitory substances from patho-genic Vibrio harveyi VIB 571and Enterococcus faecium 62-6were sensitive to lipase (Kelly,Mequio,&Pybus,2003;Prasad,Morris,Hansen,Meaden,&Austin,2005).These results suggest that sakacin LSJ618may be a lipoprotein whose lipidic moiety is an important factor for its antibacterial activity.3.3.Antibacterial spectrum of sakacin LSJ618The antibacterial spectrum of the SP is shown in Table 1.Sakacin LSJ618inhibited the Gram-positive bacteria S .aureus ,Sarcina sp.,Micrococcus luteus ,L.monocytogenes and Listeria innocua ,but had no effect on most of the LAB tested,except L .brevisi .Sakacin LSJ618was also signi ficantly active against the Gram-negative bacteria Proteus sp.and E .coli ECX4.These results demonstrate the broad antibacterial activity of sakacin LSJ618.Until now,most bacteriocins produced by L .sakei strains have demonstrated inhibitory activity against Gram-positive bacteria but not against Gram-negative bacteria;sakacin C2is an exception,as it was shown to inhibit L actobacillus delbrueckii ,L actobacillus acidophilus ,L actobacillus plantarum and Sarcina flava ,L.innocua ,Streptococcus thermophilus ,S .aureus ,E .coli ,Salmonella typhimu-rium ,Shigella flexneri and B .cereus .Although sakacin LSJ618was active against Gram-positive and Gram-negative bacteria,its anti-bacterial spectrum was different from that of sakacin C2.Interest-ingly,sakacin LSJ618showed inhibitory activity against all of the food spoilage and pathogenic bacteria tested,including L.mono-cytogenes ,S .aureus ,M .tetragenus ,E .coli and Sarcina sp.However,sakacin LSJ618was ineffective against LAB strains including E .faecalis ,L .plantarum ,L .pentosaceus ,L .fructivorans ,L .helveticus ,L .casei ,L .fermentum ,L .bulgaricus ,L .sakei ,Lactococcus lactis lactis ,L .lactis cremoris and Pediococcus acidilactici ;interestingly,this strain did exhibit activity against L .brevis .By de finition,bacteriocins can inhibit bacteria closely related to the producer strain,and most bacteriocins have been shown to have this characteristic (Simon,Fremaux,Cenatiempo,&Berjeaud,2002;Vaughan et al.,2001).A notable exception is Warnericin RB4,produced by S .warneri RB4,which demonstrated antibacterial activity against two genus of bacteria,Alicyclobacillus and Micrococcus ,including A .acid-oterrestris ,A .acidocaldarius and M .luteus ;however,this strain was not effective against genus Staphylococcus bacteria,the same genus as the Warnericin RB4-producting strain itself (Minamikawa et al.,2005).Furthermore,Warnericin RK,like sakacin LSJ618,demon-strated no activity against the same species,S .warneri 447(Héchard,Férraz,Bruneteau,Steinert,&Berjeaud,2005;Verdon,Berjeaud,Lacombe,&Héchard,2008).Thus,Warnericin RB4,Table 2Effect of enzymes,chemical reagents,pH and temperature on the antibacterial activity of sakacin LSJ618.TreatmentResidual activity (ÆSD)%a Untreated bacteriocin 100EnzymesProteinase K 81.25Æ0Chymotrypsin 72.40Æ3.68Pepsin 0Lipase 0Papain 0TrypsinChemical reagents Tween 2096.30Æ5.24Tween 8094.44Æ3.30Acetonitrile 96.30Æ5.24Urea0Temperature 4 C,30min 98.08Æ0.7437 C,30min 99.04Æ1.0060 C,30min 90.38Æ1.3980 C,30min 85.58Æ0.03100 C,30min 84.62Æ3.00121 C,30min 80.77Æ1.65pH 2.070.83Æ2.034.081.25Æ06.0100Æ08.072.92Æ3.1110.0012.0aResults are presented as the mean value of triplicate trials Æstandard deviation(SD).Fig.1.Phylogenetic tree derived from the 16S rDNA sequence of the L.sakei strain LSJ618.All of the sequences used here were from LAB-type strains.Numbers at the nodes indicate the bootstrap values on neighbor-joining analyses of 1000replicates data sets.Bar 0.02represents sequence divergence.J.Jiang et al./Food Control 23(2012)338e 344341Warnericin RK and sakacin LSJ618are similar,in that they harbor inhibitory activity against some Gram-positive and Gram-negative bacteria,but not against closely related bacteria.This property strongly suggests that the activity spectrum of sakacin LSJ618, Warnericin RB4and Warnericin RK are unlike those of most other bacteriocins.There are many different genus of LAB with different roles in naturally fermented food;sakacin LSJ618only inhibited certain food spoilage and pathogenic bacteria,leaving most LAB unaf-fected.This property might make sakacin LSJ618a good bio-preservative candidate because it inhibits food spoilage and pathogenic bacteria but not LAB,which are generally used as fermentation starters.3.4.Partial purification and molecular weight of sakacin LSJ618Sakacin LSJ618was partially purified from MRS medium using a two-step procedure comprised of ammonium sulfate precipita-tion and Sephadex G-25chromatography.Only one of the four eluted peaks showed antibacterial activity.The activity,yield and the purification fold of sakacin LSJ618,along with the purification procedure,are summarized in Table3.The purification fold was2.8,with a3.0%recovery of activity.An active fraction from Sephadex G-25chromatography was analyzed by Tricine-SDS-PAGE,which yielded a single band at w5.2kDa on the stained gel.Moreover, antibacterial activity against M.tetragenus strain was also demon-strated(Fig.2).In recent years,a large number of new bacteriocins produced by L.sakei have been identified and characterized.Aside from sakacin C2(5.5kDa)which is produced by L.sakei C2,the molecular masses of all the bacteriocins produced by other L.sakei species are in the range of3.8e4.6kDa(Gao et al.,2010).However, to our knowledge,there is no bacteriocin produced by any L.sakei strain with a molecular mass of5.2kDa with similar characteristics to sakacin LSJ618.Thus,it is possible that sakacin LSJ618(5.2kDa) may be a novel bacteriocin produced by L.sakei.In future studies, further mass spectrometry assessments will be performed for more accurate size determination(Zhang et al.,2009).3.5.Production and mode of action of sakacin LSJ618Cell growth,pH variation and bacteriocin production of sakacin LSJ618were examined at37 C.Increased bacteriocin production accompanied cell growth during both the logarithmic and stationary phase;production of bacteriocin started at the early exponential phase and reached its maximum at the late stationary phase(Fig.3).This result suggests that sakacin LSJ618was produced in a growth-associated manner.The pH of the culture declined from 6.8to 4.8in the logarithmic phase,and then remained constant over the course of bacteriocin production.No significant decrease in bacteriocin activity was observed in the culture broth until26h,compared to15h for the bacteriocin GM005(Onda,Yanagida,Tsuji,Shinohara,&Yokotsuka,2003).This result suggested that L.sakei LSJ618either secreted extracellular proteases in the later-stages of culture,or secreted a lower quantity of bacteriocin.To understand the mode of action of sakacin LSJ618against M. tetragenus,320AU/ml of sakacin LSJ618was added to a stationary-Table3Purification and activity of sakacin LSJ618.Purification Step Volume(ml)Total activity(AU)a Total protein(mg)Specific activity(AU/mg)PurificationfoldYield%Culture supernatant200 5.40Â104176230.661100 Ammonium sulfate precipitate12 1.71Â104330.4851.82 1.6931.7 Gelfiltration9 1.62Â10318.8685.90 2.80 3.0a Antibacterial activity(in arbitrary units[AU])was assayed by agar well diffusion test using M.tetragenus as an indicatorstrain.Fig.2.Tricine-SDS-PAGE and antibacterial analyses of sakacin ne M:low molecular weight markers;Lane1:sakacin LSJ618;Lane2:gel overlaid with nutrient broth soft agar surface seeded with1.5%(v/v)M.tetragenus.246810 OD6nmTime (h)pHFig.3.Production of sakacin LSJ618during the growth of L.sakei LSJ618in MRS broth at37 C.Activity of bacteriocin(1),optical density at600nm(-)and changes in pH(:).J.Jiang et al./Food Control23(2012)338e344 342phase culture of M.tetragenus .The log 10of the colony forming units (CFU)quickly dropped from 11.08to 8.75.After w 2h,the CFU/ml demonstrated a downward tendency,but the optical density readings of this indicator microorganism (OD 600)remained constant (Fig.4).These results indicated that sakacin LSJ618had a bactericidal function,with no clear evidence for lysis as a mode of action.However,further experimentation is needed to elucidate the mode of action of sakacin LSJ618,including its effects on leakage of certain intracellular substances and on cell morphology.In conclusion,in the present study,a new bacteriocin-producing strain,L .sakei LSJ618,was isolated from the traditional Chinese fermented radish.Sakacin LSJ618,a w 5.2kDa lipoprotein produced by L .sakei LSJ618,inhibited the growth of food-spoiling bacteria and food-borne pathogens,including the Gram-positive bacteria S .aureus ,Sarcina sp.,M .luteus ,L.monocytogenes and the Gram-negative bacteria Proteus sp.and E .coli .The identi fied strain did not,however,inhibit most of the LAB tested.Sakacin LSJ618was stable to changes in environmental factors,and demonstrated less degradation over the course of production.Furthermore,its mode of action was determined to be bactericidal.These properties indicate that sakacin LSJ618has potential utility as a bio-preservative in the food industry.AcknowledgmentsThis work was funded by the China National 863Program (2008AA10Z319and 2006AA10Z320).The authors would like to thank Dr.Jianhong Li and Dr.Leni Sun for their critical reading of the manuscript.Referencesde Carvalho,K.G.,Bambirra,F.H.S.,Kruger,M.F.,Barbosa,M.S.,Oliveira,J.S.,Santos,A.M.C.,et al.(2010).Antimicrobial compounds produced by Lactoba-cillus sakei subsp.sakei 2a,a bacteriocinogenic strain isolated from a Brazilian meat product.Journal of Industrial Microbiology and Biotechnology,37(4),381e 390.Deegan,L.H.,Cotter,P.D.,Hilla,C.,&Ross,P.(2006).Bacteriocins:Biological toolsfor bio-preservation and shelf-life extension.International Dairy Journal,16,1058e 1071.Dortu,C.,Huch,M.,Holzapfel,W.H.,Franz,C.M.A.P.,&Thonart,P.(2008).Anti-listerial activity of bacteriocin-producing Lactobacillus curvatus CWBI-B28and Lactobacillus sakei CWBI-B1365on raw beef and poultry meat.Letters in Applied Microbiology,47(6),581e 586.Ennahar,S.,Sashihara,T.,Sonomoto,K.,&Ishizaki,A.(2000).Class IIa bacteriocins:biosynthesis,structure and activity.FEMS Microbiology Reviews,24,85e 106.Gao,Y.,Jia,S.,Gao,Q.,&Tan,Z.(2010).A novel bacteriocin with a broad inhibitoryspectrum produced by Lactobacillus sake C2,isolated from traditional Chinese fermented cabbage.Food Control,21(1),76e 81.Gao,Y.,Li,D.,Sheng,Y.,&Liu,X.(2011).Mode of action of sakacin C2againstEscherichia coli .Food Control,22,657e 661.Héchard,Y.,Férraz,S.,Bruneteau,E.,Steinert,M.,&Berjeaud,J.M.(2005).Isolationand characterization of a Staphylococcus warneri strain producing an anti-Legionella peptide.FEMS Microbiol Lett,252,19e 23.Hugas,M.,Garriga,M.,Aymerich,M.T.,&Monfort,J.M.(1995).Inhibition of Listeriain dry fermented sausages by the bacteriocinogenic Lactobacillus sake CTC494.Journal of Applied Microbiology,79(3),322e 330.Jones,R.,Zagorec,M.,Brightwell,G.,&Tagg,J.(2009).Inhibition by Lactobacillussakei of other species in the flora of vacuum packaged raw meats during pro-longed storage.Food Microbiology,26(8),876e 881.Kandler,O.,&Weiss,N.(1986).Genus Lactobacillus Beijerinck 1901.InP.H.A.Sneath,N.S.Mair,M.E.Sharpe,&J.G.Holt (Eds.),Bergey ’s manual of systematic bacteriology,Vol.2(pp.1209e 1234).Baltimore:Williams &Wilkins.Katla,T.,Møretrø,T.,Sveen,I.,Aasen,I.M.,Axelsson,L.,Rørvik,L.M.,et al.(2002).Inhibition of Listeria monocytogenes in chicken cold cuts by addition of sakacin P and sakacin P-producing Lactobacillus sakei .Journal of applied microbiology,93(2),191e 196.Katla,T.,Naterstad,K.,Vancanneyt,M.,Swings,J.,&Axelsson,L.(2003).Differ-ences in susceptibility of Listeria monocytogenes strains to sakacin P,sakacin A,pediocin PA-1,and nisin.Applied and Environmental Microbiology,69(8),4431e 4437.Kelly,M.C.,Mequio,M.J.,&Pybus,V.(2003).Inhibition of vaginal lactobacilli bya bacteriocin-like inhibitor produced by Enterococcus faecium 62-6:potential signi ficance for bacterial vaginosis.Infectious Diseases in Obstetrics Gynecology,11(3),147e 156.Kjos,M.,Snipen,L.,Salehian,Z.,Nes,I.F.,&Diep,D.B.(2010).The Abi proteins andtheir involvement in bacteriocin self-immunity.Journal of Bacteriology,192(8),2068e 2076.Kumar,S.,Tamura,K.,&Nei,M.(2004).MEGA3:integrated software for molecularevolutionary genetics analysis and sequence alignment.Brie fings in Bio-informatics,5(2),150e 163.Mathiesen,G.,Huehne,K.,Kroeckel,L.,Axelsson,L.,&Eijsink,V.G.H.(2005).Characterization of a new bacteriocin operon in sakacin P-producing Lacto-bacillus sakei ,showing strong translational coupling between the bacteriocin and immunity genes.Applied and Environmental Microbiology,71(7),3565e 3574.Minamikawa,M.,Kawai,Y.,Inoue,N.,&Yamazaki,K.(2005).Puri fication andcharacterization of warnericin RB4,anti-Alicyclobacillus bacteriocin,produced by Staphylococcus warneri RB4.Current Microbiology,51(1),22e 26.Onda,T.,Yanagida,F.,Tsuji,M.,Shinohara,T.,&Yokotsuka,K.(2003).Production andpuri fication of a bacteriocin peptide produced by Lactococcus sp strain GM005,isolated from Miso-paste.International Journal of Food Microbiology,87(1-2),153e 159.Powell,J.E.,Witthuhn,R.C.,Todorov,S.D.,&Dicks,L.M.T.(2007).Characterizationof bacteriocin ST8KF produced by a ke fir isolate Lactobacillus plantarum ST8KF.International Dairy Journal,17,190e 198.Prasad,S.,Morris,P.C.,Hansen,R.,Meaden,P.G.,&Austin,B.(2005).A novelbacteriocin-like substance (BLIS)from a pathogenic strain of Vibrio harveyi.Microbiology-Sgm,151,3051e 3058.Samelis,J.,Roller,S.,&Metaxopoulos,J.(1994).Sakacin B,a bacteriocin produced byLactobacillus sake isolated from Greek dry fermented sausages.Journal of applied microbiology,76(5),475e 486.Schägger,H.(2006).Tricine-SDS-PAGE.Nature Protocols,1(1),16e 22.Schillinger,U.,Kaya,M.,&Lücke,F.K.(1991).Behaviour of Listeria monocytogenes inmeat and its control by a bacteriocin-producing strain of Lactobacillus sake .Journal of applied microbiology,70(6),473e 478.Schillinger,U.,&Lücke,F.K.(1989).Antibacterial activity of Lactobacillus sakeisolated from meat.Applied and Environmental Microbiology,55(8),1901e 1906.Simon,L.,Fremaux,C.,Cenatiempo,Y.,&Berjeaud,J.M.(2002).Sakacin G,a newtype of antilisterial bacteriocin.Applied and Environmental Microbiology,68(12),6416e 6420.Smaoui,S.,Elleuch,L.,Bejar,W.,Karray-Rebai,I.,Ayadi,I.,Jaouadi,B.,et al.(2010).Inhibition of fungi and gram-negative bacteria by bacteriocin BacTN635produced by Lactobacillus plantarum sp.TN635.Applied Biochemistry and Biotechnology,162(4),1132e 1146.Thompson,J.D.,Higgins,D.G.,&Gibson,T.J.(1994).CLUSTAL W:improving thesensitivity of progressive multiple sequence alignment through sequence weighting,position-speci fic gap penalties and weight matrix choice.Nucleic Acids Research,22(22),4673e 4680.Tichaczek,P.S.,Vogel,R.F.,&Hammes,W.P.(1994).Cloning and sequencing of sakPencoding sakacin P,the bacteriocin produced by Lactobacillus sake LTH 673.Microbiology,140(2),361e 367.Todorov,S. D.,&Dicks,L.M.T.(2005).Lactobacillus plantarum isolated frommolasses produces bacteriocins active against Gram-negative bacteria.Enzyme and Microbial Technology,36(2-3),318e 326.Todorov,S.D.,Nyati,H.,Meincken,M.,&Dicks,L.M.T.(2007).Partial character-ization of bacteriocin AMA-K,produced by Lactobacillus plantarum AMA-K isolated from naturally fermented milk from Zimbabwe.Food Control,18,656e 664.Todorov,S.D.,Rachman,C.,Fourrier,A.,Dicks,L.M.T.,van Reenen,C.A.,Prévost,H.,et al.(2010).Characterization of a bacteriocin produced by Lactobacillus sakei R1333isolated from smoked salmon.Anaerobe,17(1),23e 31.L o g 10(C F U m l -1)Time (h)2468101214O D 600 n mFig.4.The effect of sakacin LSJ618on the growth of M .tetragenus .Data shown are optical density at 600nm without (:)and with (Δ)sakacin LSJ618and viable cell counts (CFU/ml)without (C )and with (B )sakacin LSJ618.J.Jiang et al./Food Control 23(2012)338e 344343。