Isolation and Identification of a Bacterium from Marine Shrimp Digestive Tract：A New Degrader o

Asian Case Reports in Veterinary Medicine 亚洲兽医病例研究, 2016, 5(1), 5-10Published Online January 2016 in Hans. /journal/acrpvm/10.12677/acrpvm.2016.51002Identification and Isolation of a Wild Strain of Infectious Bursal Disease VirusMengjiao Fu, Yongqiang Wang, Xiaoqi Li, Hong Cao, Fuyong Chen, Shijun Zheng*College of Veterinary Medicine, China Agricultural University, BeijingReceived: Jan. 5th, 2016; accepted: Jan. 26th, 2016; published: Jan. 29th, 2016Copyright © 2016 by authors and Hans Publishers Inc.This work is licensed under the Creative Commons Attribution International License (CC BY)./licenses/by/4.0/AbstractThe kidney, spleen and bursa of fabricius samples were collected from diseased chickens on a farm in Beijing suburban areas. A wild strain of infectious bursal disease virus (IBDV), temporarily called BY-2015-1, was isolated by embryonic inoculation and identified via RT-PCR assay and se-quence analysis. Gene sequence analysis of VP2 showed that the homology between BY-2015-1 and OKYM strains was 97.7%. The amino acid sequence analysis of VP2 high variable region indi-cated that BY-2015-1 isolate had similar characteristics of very virulent strains, suggesting that the isolate might be a circulating virulent strain in the flocks on the farm. The isolation and identi-fication of IBDV BY-2015-1 strain would help to prevent and control IBDV in the local areas.KeywordsInfectious Bursal Disease Virus, VP2, Virulence一株传染性法氏囊病毒野毒株的分离与鉴定付梦姣，王永强，李晓齐，曹红，陈福勇，郑世军*中国农业大学动物医学院，北京收稿日期：2016年1月5日；录用日期：2016年1月26日；发布日期：2016年1月29日*通讯作者。

山西农业科学 2023，51（6）：690-695Journal of Shanxi Agricultural Sciences黄芪根腐病病原菌的分离鉴定及对其抑制作用分析牛景萍 1，燕翔 1，石志勇 1，宋诗娟 1，刘娇娇 1，杜杰 1，原艳欣 1，史将 1，梁建萍1，2（1.山西农业大学 生命科学学院，山西 太谷030801；2.山西农业大学 中兽医药现代化山西省重点试验室，山西 太谷030801）摘要：黄芪（Astragalus membranaceus ）是膜荚黄芪和蒙古黄芪的干燥根，根部受病原菌侵染会导致根腐病发生，根腐病会严重影响黄芪的药用价值及其产业的发展。

黄酮和皂苷类物质属于黄芪的次级代谢物，也是黄芪的主要药用成分。

为了明确黄芪根腐病致病菌类型以及黄芪黄酮和皂苷对病原菌的抑制作用，为防治该致病菌引起的根腐病以及研究黄芪次级代谢物参与黄芪抗病奠定基础，从山西省浑源县取回黄芪病株进行病原菌的分离鉴定，并研究黄芪根部总黄酮和总皂苷对病原菌生长的抑制作用；通过形态学观察、ITS 序列分析和致病性鉴定，明确黄芪根腐病病原菌为腐皮镰刀菌HYFS -1。

结果表明，30%的无水乙醇对腐皮镰刀菌菌落生长无影响，可作为总黄酮和总皂苷的稀释液；当总黄酮质量浓度为0.3 mg/mL 时，对菌落生长具有显著抑制作用，平均抑制率为16.42%；当总皂苷质量浓度为0.5 mg/mL 时，对菌落生长具有显著抑制作用，平均抑制率高达85.42%。

关键词：黄芪根腐病；腐皮镰刀菌；总黄酮；总皂苷；抑制作用中图分类号：S435.67 文献标识码：A 文章编号：1002‒2481（2023）06‒0690‒06Isolation and Identification of Pathogens Causing Astragalus membranaceusRoot Rot and Analysis of Its Inhibition EffectsNIU Jingping 1，YAN Xiang 1，SHI Zhiyong 1，SONG Shijuan 1，LIU Jiaojiao 1，DU Jie 1，YUAN Yanxin 1，SHI Jiang 1，LIANG Jianping 1，2（1.College of Life Sciences ，Shanxi Agricultural University ，Taigu 030801，China ；2.Shanxi Key Lab. for Modernization of TCVM ，Shanxi Agricultural University ，Taigu 030801，China ）Abstract ：Astragalus membranaceus is the dried root of Astragalus membranaceus (Fisch.) Bge. and Astragalus membranaceus Bunge var. Mongholicus (Bge.) Hsiao. Infection of Astragalus membranaceus roots by pathogen can lead to the occurrence of root rot which seriously affects the medicinal value of Astragalus membranaceus and the development of its industry. Flavonoids and saponins belong to the secondary metabolites and are also the main medicinal components of Astragalus membranaceus . In order to clarify the types of the pathogen of root rot and the inhibition effects of flavonoids and saponins on the pathogen, and lay a foundation for prevention and control of root rot caused by the pathogen and study on the involvement of secondary metabolites in Astragalus membranaceus disease resistance, in this study, the pathogenic bacteria were isolated and identified from the diseased plant from Hunyuan, Shanxi province, and the inhibition effects of total flavonoids and astragalosides on the growth of pathogenic bacteria were studied. Through morphological observation, ITS sequence analysis, and pathogenicity identification, the pathogen was identified as Fusarium solani HYFS -1. The results showed that 30% of anhydrous ethanol had no effect on the growth of Fusarium solani and could be used as diluent of total flavonoids and astragalosides. When the mass concentration of total flavonoids was 0.3 mg/mL, the colony growth was significantly inhibited and the average inhibition rate was 16.42%. When the mass concentration of astragalosides was 0.5 mg/mL, the colony growth was significantly inhibited and the average inhibition rate was up to 85.42%.Key words ：Astragalus membranaceus root rot; Fusarium solani ; total flavonoids; total astragalosides; inhibition effects黄芪是豆科多年生草本药用植物，黄芪根腐病的发生会严重影响其药用价值。

动物医学进展,2021,2(1):50-55Progress in Veterinary Medicine应用CRISPR/Cas9系统构建大肠埃希氏菌irp2基因缺失株富国文，单春兰，敖平星，赵汝，高丽波，刘超英，高洪*（云南农业大学动物医学院，云南昆明650201）摘要：CRISPR/Cas9是一个新兴的基因编辑技术，利用该技术对临床分离的撒坝猪致病性大肠埃希氏菌中的irp2基因进行编辑，为进一步研究该基因在致病中的作用提供基础。

首先构建靶向irp2基因的sgRNA,PCR扩增上、下游同源臂，利用重叠PCR技术连接sgRNA与上、下游同源臂，再通过酶切与酶连构建出靶向irp2基因的pTargetF重组质粒。

将pCas与pTargetF重组质粒转化临床分离的撒坝猪致病性大肠埃希氏菌中，成功构建了该菌株的irp2基因缺失菌株，为进一步研究其致病机制奠定基础。

关键词：CRISPR/Cas9；大肠埃希氏菌；irp2基因缺失菌株中图分类号:S85.2.612；Q789文献标识码:A文章编号：1007-5038（2021）01-0050-06耶尔森氏菌强毒力岛（Yersinia high-pathogeni-簇［1］。

研究表明，耶尔森氏菌HPI不仅可以通过耶ticity island,HPI）主要含有与摄铁有关的毒力基因尔森氏菌素（Yersiniabactin,Ybt）的铁载体夺取宿收稿日期：2020-0327基金项目：国家自然科学基金项目（31660704,31960692）作者简介：富国文（1980—），男（满族），辽宁西丰人，博士研究生，主要从事特种经济动物饲养研究。

*通讯作者and antimicrobial susceptibllty of Mannheimia haemolytica,PasLeurelta mulbocida,and IUsLophilus somni isolated fromthelowerrespiratorytractofhealthyfeedlotca t leandthosediagnosed wth bovine respiratory disease[J].Vet Microbiol,2017，208:118-125[14]MICHAEL G B,BOSS?JANINE T,STEFAN S.Antimicro-bialresistanceinPasteure l aceaeofveterinaryorigin[J]Mi-crobiolSpectrum，2018，6(3):331-363[15]KLIMA CASSIDY L，HOLMAN DEVIN B，RALSTONBRENDAJ，etalLowerrespiratorytractmicrobiomeandre-sistomeofnovinerespiratorydiseasemortalities[J]Microbia-lecology，2019，78(2):446-456Isolation?Identification and Drug Sensitivity Tfest of Pasteurella multocida in Cattle MAO Chang-si^DANG Qiao2,WEI Xing4,KONG Ling-cong2，(1.College of Veterinary Mediciee, China Agricultural UniversiLy, Beijing,100093,China；2.College of Animal Science and Technology,JUn Agricutural UniversiLy,Changchun,J i l i n,30118,China；3.Key Laboratory of Anima l Production and Product Qua liLy and Safety^Ministry of Educabion,Changchun,J i l i n,30118,China；4.Animal Disease PrevenLinn and Control CenLer of Liaoyuan,Liaoyuan,Jilin,136220,China)Abstract：The aim of this study was to investigate the pathogen of bovine respiratory diseases in a cattle farm Biochemicalandmolecularbiologicalidentificationmethodswereusedtoisolateandidentifythepath-ogenic bacteria from nasal swabs of sick ca t le and lungs of dead ca t le，and LD50wasperformed Further，themicrobrothdilutionmethodandplatedoubledilutionmethodwereusedtodetecttheantimicrobialsen-sitivityofthepathogenicbacteria Fina l y，pulsed-fieldgelelectrophoresiswasusedfortyping Theresults showed that three strains of bovine capsular type A PasteurelLa multocida were isolated from a healthy bo-vinenasalswabandtwobovinelungsthatdiedofbovinerespiratorydiseases Thethreestrainshadthe samegenotypeand werea l resistanttociprofloxacin，enrofloxacin，sulfamethoxazole，sulfamethoxazole，clindamycin and tilmicosin It is only sensitive to tetracycline and florfenicol In a word，the pathogen cau­sing respiratory diseases in this cattle farm is type A bovine capsular PasteurelLa multocida,and the isola-tedbacteriahaveshowndi f erentdegreesofdrugresistancetocommonlyusedclinicaldrugsKey words：cattle；Pasieurelta multocida；isolation and identification；drug resistance富国文等:应用CRISPR/Cas9系统构建大肠埃希氏菌irp2基因缺失株51主中的铁元素进而加重机体的感染，而且可在耶尔森氏菌和大肠埃希氏菌（E.oi）之间水平传播，与致病性E.cl的毒力进化有着密切的关系⑵。

Isolation of Bacillus sp.strains capable of decomposing alkali lignin and their application in combination with lactic acid bacteria for enhancing cellulaseperformanceYoung-Cheol Chang a ,⇑,1,DuBok Choi b ,⇑,1,Kazuhiro Takamizawa c ,Shintaro Kikuchi aaDivision of Applied Sciences,College of Environmental Technology,Graduate School of Engineering,Muroran Institute of Technology,27-1Mizumoto,Muroran 050-8585,Hokkaido,Japan bDepartment of Pharmacy,College of Pharmacy,Chungbuk National University,Cheongju 361-763,Republic of Korea cDepartment of Applied Life Science,Faculty of Applied Biological Sciences,Gifu University,Gifu 501-1193,Japanh i g h l i g h t sTwo alkali lignin-degrading bacteria (CS-1and CS-2)were isolated from forest soils in Japan. CS-1and CS-2displayed alkali lignin degradation capability. High laccase activities were observed in crude enzyme extracts. Improving surface area accessible to cellulose is an important.A two-step procedure is effective at accelerating cellulase performance.a r t i c l e i n f o Article history:Received 23September 2013Received in revised form 11November 2013Accepted 13November 2013Available online 21November 2013Keywords:Lignin-degradation Bacillus sp.Lactic acid bacteria Cellulase performance Laccasea b s t r a c tEffective biological pretreatment method for enhancing cellulase performance was investigated.Two alkali lignin-degrading bacteria were isolated from forest soils in Japan and named CS-1and CS-2.16S rDNA sequence analysis indicated that CS-1and CS-2were Bacillus sp.Strains CS-1and CS-2displayed alkali lignin degradation capability.With initial concentrations of 0.05–2.0g L À1,at least 61%alkali lignin could be degraded within 48h.High laccase activities were observed in crude enzyme extracts from the isolated strains.This result indicated that alkali lignin degradation was correlated with laccase activities.Judging from the net yields of sugars after enzymatic hydrolysis,the most effective pretreatment method for enhancing cellulase performance was a two-step processing procedure (pretreatment using Bacillus sp.CS-1followed by lactic acid bacteria)at 68.6%.These results suggest that the two-step pretreatment procedure is effective at accelerating cellulase performance.Ó2013Elsevier Ltd.All rights reserved.1.IntroductionIn contrast to fungal lignin degradation,the enzymology of bac-terial lignin breakdown is currently not well understood,but extra-cellular peroxidase and laccase enzymes appear to be involved (Bugg et al.,2011).The advantage of taking a lignin-degrading en-zyme from bacteria rather than fungi is that bacteria are much more amenable to genetic modification.This means allows scien-tists to transfer genes that codes for enzymes into different species of bacteria,such as the industrial workhorse Escherichia coli ,andalso modify the metabolic pathways to enhance the enzyme’s lig-nin-degrading activity (Bugg et al.,2011).In addition,harnessing the biosynthetic ability of microorganisms is becoming an increas-ingly important platform for producing value-added chemical products (Du et al.,2011).To date,extensive research and developmental studies on the effective utilization of lignocellulosic materials has been con-ducted.However,the largest obstacle to the economic production of cellulosic biofuels is cost-effectively releasing sugars from recal-citrant lignocellulose (Zhang,2008).One of the key problems hin-dering the effective utilization of this renewable resource as a raw material for chemical reactions and feeds is the low susceptibility of lignocellulose to hydrolysis,which is attributable to the crystal-line structure of cellulose fibrils surrounded by hemicellulose and the presence of the lignin seal which prevents penetration by degrading enzymes (Gong et al.,1999).0960-8524/$-see front matter Ó2013Elsevier Ltd.All rights reserved./10.1016/j.biortech.2013.11.032⇑Corresponding authors.Tel./fax:+81143465757(Y.-C.Chang),tel./fax:+82634662984(D.Choi).E-mail addresses:ychang@mmm.muroran-it.ac.jp (Y.-C.Chang),choidubok@ ,choidubok@ (D.Choi).1These authors contributed equally to this work.Therefore,an ideal pretreatment is needed to reduce the lignin content and crystallinity of cellulose,and increase the surface area of these materials(Wang et al.,1998).Removal of lignin from bio-mass before biological processing improves cellulose digestibility, reduces downstream agitation power requirements,provides less sites for nonproductive cellulase adsorption,reduces dissolved lig-nin compounds that are toxic to fermentations,facilitates cell and enzyme recovery and recycling,and simplifies the distillation steps (Wyman et al.,2004).Dilute acid treatment is one of the most effective pretreatment methods for lignocellulosic biomass.A common pretreatment uses dilute sulfuric acid(50–300mM)at100–200°C.During hot acid pretreatment,some polysaccharides are hydrolyzed,mostly hemi-cellulose(Zhu et al.,2009;Lloyd and Wyman,2005;Mosier et al., 2005).The resulting free sugars can degrade to furfural(from pen-toses)or to5-hydroxymethylfurfural(HMF;from hexoses)(Agbor et al.,2011).These compounds inhibit yeast cells and lead to de-creased specific growth rates,specific ethanol production rates and ethanol yields.To resolve this inhibitor problem,organic acids (maleic acid and fumaric acid)have been suggested as alternatives to sulfuric acid during pretreatment.Both organic acids promote the hydrolysis of polysaccharides but,unlike sulfuric acid,neither promotes the degradation of free sugars to furfural and HMF (Kootstra et al.,2009).Recently,Rollin et al.(2011)reported that improving the surface area accessible to cellulose is a more impor-tant factor for achieving a high sugar yield rather than attempting to improve the enzymatic digestibility of biomass by removing anic acids that do not result in inhibition,such as furfural and HMF,may increase porosity and improves enzymatic digest-ibility,resulting in hemicellulose removal(Kootstra et al.,2009). However,this sophisticated method requires a heating process of 130–170°C.Thus,although hemicellulose can be eventually re-moved from substrates,the high energy requirements will remain problematic.In the current study,numerous forest soil samples from throughout Japan(from Hokkaido to Okinawa)were collected to better understand the diversity of lignin-decomposing bacteria. After the isolation process,two isolated strains(Bacillus sp.strains) were further studied to evaluate their alkali lignin-degrading abil-ity.In addition,their application in lignin degradation was exam-ined using rice straw.A biological pretreatment method was also optimized,which focused on the development of an environmen-tally-friendly and low energy method for the removal of lignin and to enhance cellulase performance.Two lactic acid bacteria (Lactobacillus bulgaricus and Streptococcus thermophilus)were also examined in an attempt to increase the surface area accessible to cellulose resulting in hemicellulose elimination.Application of Bacillus sp.strains in combination with lactic acid bacteria for lig-nin degradation and enhancing cellulase performance were also studied.2.Methods2.1.Soil samples and isolation procedureSoil samples were taken randomly from different forests(Mt. Asahi,Mt.Fuji,and Mt.Yonahadake)located in Hokkaido,Shi-zuoka,and Okinawa Island(Supplementary data1).Mt.Fuji is the highest mountain in Japan at3776m and an active stratovol-cano.In addition,recently it was registrated as a World Heritage site.Mt.Asahi is also an active stratovolcano and the tallest peak in Hokkaido(2290m).Mt.Yonahadake is the highest mountain on Okinawa Island at503m.The temperature of sampling sites was9°C(Mt.Asahi),13°C(Mt.Fuji),25°C(Mt.Yonahadake), respectively.Soil samples were taken at0–15cm depth.Sixty eight soil samples(36samples(Mt.Asahi);20samples (Mt.Fuji);12samples(Mt.Yonahadake))from the above men-tioned sites were used as the source of inoculum.As a rapid screen-ing method for detection of ligninolytic ability decolorization of Remazol Brilliant Blue R(RBBR)has been used.RBBR decoloriza-tion experiments were set up in20mL test tube containing 10mL of a basal salt medium.The basal salt medium used in this study contained0.05g of K2HPO4,0.05g of KH2PO4,0.1g of NaCl, 0.3g of MgSO4Á7H2O,0.2g of CaCl2Á2H2O,0.6mg of H3BO3, 0.169mg of CoCl2Á6H2O,0.085mg of CuCl2Á2H2O,0.099mg of MnCl2Á4H2O,and0.22mg of ZnCl2,and was supplemented with 0.01%(w/v)RBBR,1.0%(w/v)glucose,0.018%(w/v)yeast extract, and0.5%(w/v)peptone(BSGYP)in1000mL of deionized water (pH6.0).Cultures were performed under aerobic conditions by inoculating1g of each soil sample.Isolation procedures were performed using cultures from the forest soil samples which represented RBBR-decolorizing activity. The cultures in which RBBR decolorization was observed were sub-sequently transferred to fresh medium.To isolate colonies,10-fold dilution of log-phase cells of cultures were spread on petri plates containing BSGYP medium with1.5%agar.Plates were then incu-bated under aerobic conditions at30°C.The ability of RBBR-decol-orization was determined by inoculating colonies into liquid BSGYP medium supplemented with0.01%(w/v)RBBR and decolor-ization of RBBR was monitored using a UV–Vis spectrophotometer (UV1800;Shimadzu,Japan)at592nm ing this isola-tion procedure,some representative RBBR-decolorizing bacteria were successfully isolated.The purity of isolated cultures were confirmed using an inverted microscope(Diaphot TMD300;Nikon, Tokyo,Japan).2.2.16S rDNA sequence determination and physiological characteristicsFor phylogenetic identification of two representative isolates (strains of Bacillus sp.),the16S rRNA gene fragment was amplified by polymerase chain reaction with a pair of universal primers,27f and1392r,and DNA sequencing was determined as described by Chang et al.(2011).Phylogenetic analysis was determined as de-scribed by Okeke and Lu(2011).Physiological characteristics of the isolates were also determined using commercially available identification systems(API20E,API10S,API50CHE,API20NE; API Staph,API Coryne,API20AÒ,API20C AUX,APIÒ50CH,APIÒ50CHB,rapid ID32A API,API Coryne;bioMérieux,Kobe,Japan).2.3.Biodegradation of alkali ligninFor biodegradation of alkali lignin,two most effective RBBR-decolorizing strains(Bacillus sp.CS-1and CS-2)were selected.Bio-degradation experiments were carried out in BSGYP(as mentioned above)containing0.05g LÀ1of alkali lignin.Two isolates were pre-grown on BSGYP medium for24h.Erlenmeyerflasks(250mL)con-taining100mL of autoclaved(20min,121°C)BSGYP(pH6.0)were inoculated with2mL of pregrown pure culture(0.65mg pro-tein mLÀ1)in log phase.The uninoculated(control)and bacterial inoculatedflasks were incubated at30°C on a rotary shaker (120rpm)in dark conditions for3days.The time course of lignin degradation was followed while shaking theflasks for3days.Dis-appearance of alkali was monitored by aseptically removing1mL samples for measurement of ultraviolet absorption spectra at 280nm after centrifugation at6000Âg for5min.All assays were performed at least in duplicate with their corresponding controls. Both non-inoculated media(blanks)and inoculated autoclaved samples(controls)were used.430Y.-C.Chang et al./Bioresource Technology152(2014)429–4362.4.Effects of temperature and pH on alkali lignin degradationOne hundred ml of sterile production medium for bacteria was prepared in different conicalflasks at pH8.0and inoculated with 2%inoculum.Eachflask was incubated at different temperatures (15,25,30,37,and40°C)for48h.Total protein concentrations were determined using a Bio-Rad protein assay kit,which con-tained a bovine c-globulin standard and a bovine serum albumin standard.Eachflask was adjusted to a different pH(4,5,6,8, and10)using0.1N NaOH and0.1N HCl.After sterilization,flasks were inoculated with2%inoculum.Flasks were then incubated at 37°C for48h.2.5.Enzyme activityLignin peroxidase(LiP,EC1.11.1.14)and manganese peroxidase (EC1.11.1.1)activity were determined as described by Yang et al. (2011).Laccase activity was determined in both culturefluid and using a crude intracellular ccase activity was measured in 1mL reaction measurements containing75mM catechol as the substrate in50mM sodium phosphate buffer,pH5and200l L of culturefluid.The progress of the reaction was monitored at 440nm for10min.One unit of laccase activity was defined as a change in A440of1mL in1min(Ramesh et al.,2008).To determine intracellular laccase activity,bacterial cultures were centrifuged(6,000Âg)for20min at4°C to precipitate cellu-lar debris and obtain clear supernatants.Bacterial pellets were then washed with Tris–HCl buffer(0.1M;pH7.5)containing 10mM of phenylmethylsulfonylfluoride to inhibit protease activ-ity in the supernatant before sonication.Cell extract was obtained by centrifugation(14,000Âg)at4°C for20min and used as a crude intracellular enzyme.All spectrophotometric measurements were carried out using a UV–Vis spectrophotometer(UV1800;Shima-dzu,Japan).All assays were carried out in triplicate.2.6.Biodegradation of lignin in rice straw by Bacillus sp.CS-1Strain CS-1was pregrown on BSGYP medium for24h.Erlen-meyerflasks containing100mL of growth medium(pH8)and 3g of milled rice straw was inoculated with2mL of pregrown pure culture(0.65mg protein mLÀ1).Uninoculated(control)and bacte-rial inoculatedflasks were incubated at37°C on a rotary shaker (120rpm)in dark conditions for3days.Rice straw was provided by a farmhouse which cultivates Kor-ean rice in Icheon,Gyeonggi,Korea.Air dried raw material without classification were cut into3–5cm lengths and stored in sealed plastic bags at room temperature for pretreatment.Prior to compo-sition analysis,biomass was ground using a Wiley mill and,parti-cles between the sizes of40and80mesh were led rice straws were washed with water to remove dust and dried in an air forced oven at60°C for48h.As a control,Thermobifida fusca(NBRC14071T),obtained from NITE Biological Resource Center in Japan was used for the removal of lignin in rice straw.Cultivation was performed with nutrient broth medium(pH6.0)and incubated at45°C on a rotary shaker (170rpm)in dark conditions for10days.2.7.Determination of components of rice strawThe components(cellulose,Klason lignin,acid soluble lignin, and ash)of native or pretreated rice straw were determined as de-scribed previously(Zhu et al.,2009;Taniguchi et al.,1982).Holo-cellulose content was determined after solubilization with72% sulfuric acid according to the phenol–sulfuric acid method using glucose as the standard(Zhu et al.,2009;Taniguchi et al.,1982).Hemicellulose content was calculated by subtracting the weight of cellulose from that of holocellulose.2.8.Biological pretreatment methods for enhancing cellulase performanceExperiments were planned and divided into four different pro-cessing procedures as follows:(1)single pretreatment of rice straw by Bacillus sp.CS-1;(2)single pretreatment by two lactic acid bac-teria(Lactobacillus bulgaricus(NBRC13953)and Streptococcus ther-mophiles(NBRC13957));(3)two-step processing procedure (sequential pretreatment using lactic acid bacteria followed by Bacillus sp.CS-1;(4)two-step processing procedure(sequential pretreatment using Bacillus sp.CS-1followed by lactic acid bacte-ria).After each pretreatment processing procedure,the content of holocellulose(hemicellulose and cellulose),cellulose,and lignin in rice straw were determined(Zhu et al.,2009;Taniguchi et al., 1982).For the two-step processing procedure using lactic acid bacteria and Bacillus sp.CS-1,the following experiment was conducted.To obtain enough cell volume,pre-cultivation using lactic acid bacte-ria was performed using each optimum medium as documented by Chang et al.(2012b).Lignin and hemicellose degradation experi-ments used300mL glass-stoppered Erlenmeyerflasks containing 100mL of growth medium and3g of milled rice straw.Growth medium had the following composition in1000mL of deionization water:3g peptone;15g malt extracts;and40g glucose.Each pre-cultivated culture(0.25mg protein mLÀ1)of the two lactic acid bacteria were inoculated in100mL of growth medium with milled rice straw and incubated for3days at120rpm on a rotary shaker at30°C for thefirst processing procedure.Autoclaved growth medium served as the control,with the pH of the growth medium adjusted to7with NaOH(1.0M)before led rice straws were then separated by centrifugation(2180Âg for 15min),rinsed with deionized water twice and reacted for a fur-ther3days in a BSGYP medium also containing Bacillus sp.CS-1 (the second processing procedure).2.9.Enzymatic hydrolysisAfter removing microorganisms growing on the rice straw as completely as possible,pretreated rice straw was enzymatically solubilized in a50mM sodium citrate buffer(pH5.0).Hydrolysis experiments were conducted in a shaking water bath at120rpm and40°C for48h.Cellulase(Cellulase Y-NC;Yakult Pharmaceuti-cal Industry,Tokyo,Japan)was used at a protein concentration of 100mg LÀ1.The specific activity of cellulase was30,000U gÀ1, according to the manufacturers’data.Carboxymethyl cellulose was used as a substrate to measure cellulase activity.The concen-tration of pretreated rice straw was10g LÀ1.After an appropriate incubation time,the reaction mixture was centrifuged(8000Âg for5min),and the supernatant wasfiltrated with a glassfilter (G-100;Advantec Toyo,Tokyo,Japan)to remove the residual sub-strate.Total soluble sugar and glucose in the resultingfiltrate were determined using the phenol–sulfuric acid method(Masai et al., 2007)and high performance liquid chromatography using a Bio-Rad HPX-87H column,respectively.The net yields of total soluble sugar(TS)and glucose(G)were determined on the basis of the amounts of holocellulose(H:cellu-lose and hemicellulose)and cellulose(C)in untreated rice straw, respectively,as follows:Net yield of TS(%)={(amount of TS produced from residual straw after pretreatment)/(amount of H in untreated straw)}Â162/180Â100.Y.-C.Chang et al./Bioresource Technology152(2014)429–436431Net yield of G(%)={(amount of G produced from residual straw after pretreatment)/(amount of C in untreated straw)}Â162/ 180Â100.2.10.Data analysisAll results are shown as mean values with standard deviations (±95%confidence interval)from triplicate experiments,except for the pH and temperature effects on alkali lignin degradation exper-iments.Statistically significant differences were determined using Student’s t tests with a p value of<0.05.2.11.Nucleotide sequence accession numberThe16S rRNA gene sequences of the isolates(strain CS-1and strain CS-2)determined in this study have been deposited at the DNA Data Bank of Japan under Accession No.AB795826and AB795827,respectively.3.Results and discussion3.1.Isolation of bacterial strainsOf the sixty eight soil samples,only four samples(Mt.Asahi in Hokkaido(one sample),Mt.Fuji in Shizuoka(one sample),and Mt. Yonahadake on Okinawa Island(two samples))showed RBBR-decolorizing ability.Of these,the Mt.Asahi soil sample showed the highest decolorizing activity.The soil sample from Mt.Fuji showed relatively low RBBR-decolorizing activity(data not shown).Although RBBR-decolorizing activity varied,several RBBR-decolorizing bacteria were isolated from soil throughout Japan (from Hokkaido to Okinawa),clarifying the wide distribution of RBBR-decolorizing bacteria in Japanese forest soil(Table.1).The RBBR-decolorizing rate of each isolate was determined.Identifica-tion of isolates was conducted using commercially available API identification systems(Table1).As a results,several strains were isolated from Mt.Asahi and Mt.Yonahadake.When soil samples from Mt.Fuji was used in the isolation process,only a few genera of strains could be isolated.Among the isolates,two strains of Bacillus sp.from Mt.Asahi and Mt.Yonahadake showed the highest RBBR-decolorizing rates(Table1).Until a few years ago,strains of Bacillus have not been well known as lignin-degrading -tely,several lignin-degrading strains of Bacillus sp.have been iso-lated from around the world and their lignin-degrading and/or decolorizing abilities have been investigated(Bandounas et al., 2011).In current study,Bacillus sp.strains were found in all RBBR-decolorizing soil samples.These results indicate that Bacillus sp.may be an important microorganism and play a key role in deg-radation of lignin.On the other hand,bacteria of several genera,including Pseudo-monas,Alcaligenes,Arthrobacter,Nocardia and Streptomyces,can readily degrade the single-ring aromatic compounds that build up the lignin macromolecule(Bugg et al.,2011).There are also a number of literature reports on bacteria(Streptomyces viridosporus T7A,Nocardia,and Rhodococcus)that are able to break down lignin (Yang et al.,2012).Strains of Pseudomonas putida mt-2and Rhodo-coccus jostii RHA1were found to show lignin-degrading activity that is comparable with S.viridosporus T7A(Yang et al.,2012). There was also a report of a lignin degradation bacterial consor-tium named LDC that was screened from the sludge of a reed pond. It could break down60.9%of the lignin in the reeds at30°C under static culture conditions(Wang et al.,2013).Interestingly,strains isolated which are capable of decolorizing RBBR from the soil of Okinawa origin involved several of the same genus that have al-ready been reported as lignin-and aromatic compound-degrading bacteria,such as Pseudomonas,Arthrobacter,Nocardia,and Rhodo-coccus(Bugg et al.,2011;Li et al.,2009).These results indicate that more various lignin-degrading bacteria could be distributed in Mt. Yonahadake compared with those of Mt.Asahi and Mt.Fuji.Two RBBR-decolorizing bacteria(strains of Bacillus sp.)were isolated and named CS-1and CS-2.16S rDNA sequence(1455bp) analysis indicated that CS-1from Mt.Asahi and CS-2from Mt. Yonahadake were Bacillus sp.(100%identity with Bacillus subtilis (1514bp);GenBank Accession No.HQ727971.1and100%identity with Bacillus sp.B37(1508bp);GenBank Accession No. JN656409.1),respectively(data not shown).The isolated strains were Gram-positive and rod-shaped bacte-rium.CS-1and CS-2were able to produce catalase,oxidase,and urease but not indole(Supplementary data2).Utilization of citrate was positive but not propionate.The strains were positive for ni-trate reduction and hydrolysis of casein tests but not H2S produc-tion.CS-1and CS-2could grow using the following carbon sources: D-glucose,fructose,sucrose,glycerol,D-xylose,maltose,lactose,and D-mannitol.These data indicated that CS-1and CS-2resem-bled a member of the Bacillus genus.3.2.Biodegradation of alkali ligninAs a result of isolation and screening,two strains of Bacillus sp. (CS-1and CS-2)were selected for alkali lignin degradation experi-ments on the basis of their RBBR-decolorizing efficiency(Table1). With initial concentrations of0.05–2.0g LÀ1,at least61%of alkali lignin could be degraded within48h(data not shown).There was a significant biodegradation of alkali lignin(0.5g LÀ1)in the culture after24h;the removal ratio of lignin was up to40%,reach-ing80%on the second day of incubation(Fig.1).The maximum lig-nin degradation rate of CS-1was estimated to be99.5%at a concentration of0.05g LÀ1(data not shown).This result is much better than that previously reported(Tuomela et al.,2000).The re-moval percentages of lignin by CS-1were higher than several novel actinomycete strains,including Streptomyces spp.strains F-6and F-7(Yang et al.,2012).Cell growth was in accordance with the lignin degradation ratio (Fig.1).On the other hand,CS-1and CS-2were not able to use lig-nin as the sole carbon source(data not shown).Environmental parameters showed great influence on the growth of organisms and the degradation of lignin.The optimum temperature for the best degradation of bacterial isolates wasTable1Decolorization of Remazol Brilliant Blue R(RBBR)by various soil samples.Source Species Percentage depletion after3days aMt.Fuji,Shizuoka Bacillus sp.27Actinomyces sp.20Pseudomonas sp.18Mt.Asahi,Hokkaido Bacillus sp.100Burkholderia sp.38Ralstonia pickettii37Mt.Yonahadake,Okinawa Island Bacillus sp.95 Pseudomonasfluorescens38 Arthrobacter spp.59 Nocardia spp.49 Sphingomonas sp.48 Rhodococcus sp.25The initial concentration of RBBR was0.01%(v/w).Identification was only per-formed using isolates capable of decolorizing RBBR more than15%.a Results are expressed as a percentage of the decolorized RBBR after3days ofincubation.The initial percentage before incubation was considered to be0%.Eachvalue represents an average of two analyses(differences of data obtained from twoanalyses was within3.8%).432Y.-C.Chang et al./Bioresource Technology152(2014)429–436determined using various temperatures (Supplementary data 3).Experiments indicated that this was 37°C.Temperature influence on the growth of CS-1and the degradation of lignin was in accor-dance with cell growth (Supplementary data 3).Results also indi-cated that the optimum pH for the degradation of lignin was around 8.Like temperature,degradation activity relied on cell growth of CS-1.A number of bacteria capable of degrading lignin have been re-ported (Bugg et al.,2011;Tuomela et al.,2000).Of those bacteria,lately,several Bacillus sp.strains have been reported as lignin-degrading bacteria.Bacillus sp.(CSA105)was isolated from sedi-ment core from the pulp and paper mill industries and purified lig-ninolytic enzyme from the cell extract.In addition,several kraft lignin-degrading Bacillus sp.have been isolated (Bandounas et al.,2011;Chandra et al.,2007;Raj et al.,2007).These results indicate that Bacillus sp.may be an important microorganism and may play a key role in lignin biodegradation.3.3.Enzyme activityThe activities of three enzymes were determined.Both Bacillus sp.strain CS-1and CS-2showed very low manganese peroxidase activity (data not shown).Laccase activity of both CS-1and CS-2were at high levels (Fig.2).LiP activity was not observed (data not shown).Laccase activity was associated with growth in CS-1and CS-2(Fig.2).Intracellular laccase activity was 3.4times higher than the extracellular laccase activity (data not shown).This result suggests that more local activity was cell-associated.Alkali lignin degradation rate of CS-1was slightly higher than that of CS-2(data not shown).This result seems to be resulted in the difference of laccase activity (Fig.2).The effect of temperature on laccase activity using a crude intracellular enzyme was investi-gated.The optimum temperature for ABTS oxidation was deter-mined to be between 55–75°C.For ABTS oxidation,a steady increase of activity up to 70°C was monitored,demonstrating the high temperature tolerance of laccase (data not shown).Due to this unusual property,laccase from Bacillus sp.CS-1and CS-2may be of significant importance in industrial applications.Gener-ally,the rate of biological pretreatment is too slow for industrial purposes.However,the two isolated Bacillus sp.strains,CS-1and CS-2,possessed high lignolytic enzyme activities (laccase activi-ties),and lignin-degrading time is very fast.Laccase and laccase-producing microorganisms play an impor-tant role in bioremediation of aromatic compounds from contam-inated soils,industrial pollutants and ccases are generally found in plants and fungi,but they have also been re-ported in a few bacteria,including Azospirillum lipoferum ,Bacillus sphaericus ,Marinomonas mediterranea ,Streptomyces griseus ,and Serratia marcescens (Sheikhi et al.,2012).Bacterial laccases are more amenable to genetic manipulation than fungal laccases.Therefore,research and study of bacterial laccases is very interesting.Bacterial laccase of Bacillus genus was first reported by Claus and Filip (1997).Since,then,more bacterial laccases have been found.B.subtilis WPI showed laccase-like activity towards the oxi-dizing substrates ABTS and guaiacol (Sheikhi et al.,2012).How-ever,Bacillus megaterium and Bacillus sp.(CSA105)strain showed no correlation with laccase activity on bioalteration of kraft lignin (Kharayat and Thakur,2012).These results indicate that the type of ligninolytic enzymes involved in lignin degradation might be differ from the biochemical characteristics,even if the strain is of the same genus.3.4.Biodegradation of lignin in rice straw by Bacillus sp.CS-1Native straw was composed of cellulose (38%),hemicellulose (25%),Klason lignin (21%),acid soluble lignin (4.8%)and other materials,mainly ash (11.2%).The growth of CS-1resulted in weight loss of dry rice straw (data not shown).All the main com-ponents (cellulose,hemicellulose,and Klason lignin)were partially degraded (Table 2).In bacterial pretreatment,3.2%cellulose and 20%Klason lignin were degraded with CS-1.The ratio of hemicel-lulose removed was only 19.2%(Table 2).Thermobifida fusca (NBRC 14071T ),is considered as one of the most effective fungi for the selective removal of lignin on rice straw (McCarthy and Broda,1984).In this study,T.fusca was able to remove 18%of Klason lignin in rice straw during incubation (Fig.3).This degradation activity was lower than that of other fun-gi,for example,P.ostreatus (30%Klason lignin),but comparable to that of P.simplicissimum (15.1%)(Yang et al.,2012).CS-1tested in this study might be promising because the re-moval of Klason lignin on rice straw was comparable with that of fungi (T.fusca and P.ostreatus ).Otherwise,the lignin-degrading activity was lower than that of other fungi,for example,Fusarium moniliforme (34.7%)and Penicillium sp.strain apw-tt2(66.3%),Y.-C.Chang et al./Bioresource Technology 152(2014)429–436433。

Bacterial Isolate Sample Bacterial isolation is a crucial technique in microbiology that involves separating a single strain of bacteria from a mixed culture. This process is essential for studying the characteristics and behavior of specific bacterial species. When it comes to bacterial isolate samples, there are several key considerations and challenges that researchers and microbiologists encounter. In this discussion, we will delve into the requirements, techniques, and significance of bacterial isolate samples, as well as the potential problems and solutions associated with this process. First and foremost, the process of bacterial isolation requires meticulous attention to detail and adherence to strict aseptic techniques. Contamination can easily occur during the isolation process, leading to inaccurate results and potentially compromising the integrity of the bacterial sample. It is imperative to ensure that the equipment, media, and environment used for isolation are sterile and free from any potential sources of contamination. Additionally, proper identification and labeling of the isolated bacterial sample are essential to avoid mix-ups and misinterpretation of results. Moreover, the selection of an appropriate growth medium is critical for the successful isolation of bacterial strains. Different bacterial species have specific nutritional requirements, and the choice of growth medium can significantly impact the growth and viability of the isolated bacteria. Factors such as pH, temperature, and the presence of specific nutrients must be carefully considered to create an environment conducive to the growth of the target bacterial strain. Furthermore, the incubation conditions, including temperature and duration, play a crucial role in promoting the growth of the isolated bacteria while inhibiting the growth of contaminants. In addition to the technical aspects of bacterial isolation,ethical considerations also come into play when working with bacterial isolate samples. It is essential to adhere to ethical guidelines and regulations governing the use of bacterial samples, especially those obtained from clinical or environmental sources. Proper consent and ethical approval must be obtained when working with human-derived bacterial isolates, and researchers must handle and dispose of bacterial samples in a manner that minimizes potential risks to human health and the environment. Furthermore, the significance of bacterial isolatesamples in various fields, such as medicine, agriculture, and environmental science, cannot be overstated. These samples serve as the foundation for understanding the pathogenicity of bacteria, developing antimicrobial agents, and elucidating the role of bacteria in ecological processes. Bacterial isolates are invaluable for studying antibiotic resistance, identifying novel bacterial species, and exploring the potential applications of beneficial bacteria in diverse industries. Despite the importance of bacterial isolate samples, several challenges and potential problems can arise during the isolation process. One common issue is the presence of contaminants or non-target bacteria in theisolated sample, which can confound the results of subsequent analyses. Contamination can occur at any stage of the isolation process, from the initial sampling to the plating and incubation of bacterial cultures. Rigorous quality control measures and the use of selective media can help mitigate the risk of contamination and ensure the purity of the isolated bacterial strain. Another challenge in bacterial isolation is the difficulty of obtaining pure cultures of certain bacterial species, particularly those with fastidious growth requirements or those that are outcompeted by other microorganisms in their natural environment. In such cases, innovative techniques such as selective enrichment, co-culturing with companion organisms, or the use of specialized growth conditions may be necessary to successfully isolate the target bacteria. Additionally, the emergence of antibiotic-resistant strains and the presence of biofilms in environmental samples can pose significant hurdles to the isolation of pure bacterial cultures. In conclusion, the process of bacterial isolation is a multifaceted endeavor that requires careful attention to technical, ethical, and scientific considerations. From ensuring aseptic conditions and selecting appropriate growth media to navigating ethical guidelines and addressing potential challenges, the isolationof bacterial samples demands a comprehensive and meticulous approach. Despite the inherent complexities and potential pitfalls associated with bacterial isolation, the rewards are substantial, as these samples form the basis for groundbreaking research, medical advancements, and a deeper understanding of the microbial world. As researchers and microbiologists continue to refine and innovate bacterialisolation techniques, the impact of bacterial isolate samples on human health, agriculture, and the environment will undoubtedly continue to expand and evolve.。

第 43 卷第 1 期2024年 1 月Vol.43 No.1Jan. 2024中南民族大学学报（自然科学版）Journal of South-Central Minzu University（Natural Science Edition）一株内切葡聚糖酶产生菌株的分离鉴定及其产酶条件优化赵思卡1，许倍滔1，冯喆1，王力2，张建国2，李晓华1*（1 中南民族大学a. 生命科学学院；b. 微生物资源与利用湖北省工程技术研究中心，武汉430074；2 江苏神力生态农业科技有限公司，江苏宜兴214211）摘要从湖南省长沙县养殖场牛胃秸秆发酵物中分离获得了一株高产内切葡聚糖酶的菌株，命名为SCUEC7. 通过菌落形态、生理生化特性及16S rDNA序列分析，鉴定SCUEC7菌株为枯草芽胞杆菌（Bacillus subtilis）. 当培养时间为12 h、pH值为6.0、培养温度为37 °C、碳源为20.0 g·L-1麦芽糖、氮源为15.0 g·L-1胰蛋白胨、金属离子为1.5 g·L-1的Mn2+的条件下，枯草芽胞杆菌SCUEC7菌株菌体生长量和内切葡聚糖酶活性均较高. 在单因素实验的基础上，响应面法分析显示：培养11.9 h，初始pH值为5.9，麦芽糖含量为19.8 g·L-1时，预测最高酶活力为409.0 U·mL-1，内切葡聚糖酶的酶活力实测值与预测值之间有较高的拟合度. SCUEC7菌株产生内切葡聚糖酶活性较高，为其秸秆饲料中的应用奠定基础.关键词枯草芽胞杆菌SCUEC7菌株；内切葡聚糖酶；产酶条件优化；响应面法优化中图分类号Q93 文献标志码 A 文章编号1672-4321（2024）01-0024-08doi：10.20056/ki.ZNMDZK.20240104Isolation and identification of an endoglucanase producing strain and optimization of its enzymatic production conditionsZHAO Sika1，XU Beitao1，FENG Zhe1，WANG Li2，ZHANG Jianguo2，LI Xiaohua1*（1 South-Central Minzu University a. College of Life Sciences； b. Hubei Provincial Engineering and Technology Research Center for Resources and Utilization of Microbiology， Wuhan 430074，China； 2 Jiangsu Shenli Ecological AgriculturalScience and Technology Co Ltd， Yixing 214211， Jiangsu China）Abstract A strain with high production of endoglucanase was selected from the straw fermentation of cattle stomach in Changsha County， Hunan Province， and was named SCUEC7. Based on the morphological observation， physiological and biochemical characteristics， and 16S rDNA sequence comparison analysis， SCUEC7 strain was identified as Bacillus subtilis. The culture conditions for the better growth of SCUEC7 strain were as follows： 12 h of culture period， pH 6.0， 37 °C of culture temperature， 20.0 g·L-1 maltose as carbon source， 15.0 g·L-1 tryptone as nitrogen source， 1.5 g·L-1 Mn2+ as metal ion. Response surface methodology analysis showed that the highest predicted enzyme activity was 409.0 U·mL-1 when the initial pH value was 5.9， maltose content was 19.8 g·L-1， and culture period was 11.8 h. There was a high degree of fit between the measured and predicted values of endoglucanase activity. SCUEC7 strain has a strong ability to produce endoglucanase， which can provide a basis for the development of animal feed.Keywords Bacillus subtilis SCUEC7 strain； endoglucanase； optimization of enzymatic production conditions； response surface optimization收稿日期2021-09-24* 通信作者李晓华（1968-），男，教授，博士，研究方向：微生物资源与应用， E-mail：*********************基金项目国家自然科学基金资助项目（31070087）；中央高校基本科研业务费专项资金资助项目（CZY19018）；中南民族大学企业委托资助项目（HZY20042，HZY21015）第 1 期赵思卡，等：一株内切葡聚糖酶产生菌株的分离鉴定及其产酶条件优化纤维素酶是作用于纤维素中β-1，4-葡萄糖苷键的一组酶［1］，能够使纤维素变为葡萄糖和纤维二糖［2-3］，根据酶功能不同主要分为3种：外切葡聚糖酶、内切葡聚糖酶和葡萄糖苷酶［4］，纤维素酶在各行业应用广泛，在全球市场占据了酶产业份额的15%［5］. 目前筛选到具有产纤维素酶能力的真菌主要来自木霉属（Trichoderma）、青霉属（Penicillium）、曲霉属（Aspergillus）和脉孢菌属（Neurospora）等；细菌主要来自芽胞杆菌属（Bacillus）、高温单胞菌属（Thermomonospora）、瘤胃球菌属（Ruminococcus）等；放线菌主要有链霉菌属（Streptomyces）、小单胞菌属（Micromonospora）、分枝杆菌属（Mycobacterium）等［6］. 秸秆主要成分为木质纤维素［7］，具有致密结构，在自然条件下难以被动物体降解和吸收［8］，纤维素酶在秸秆饲料中的应用可以起到增加饲料适口性和动物体吸收率等作用［9］.本研究从健康的牛胃秸秆发酵物中分离内切葡聚糖酶产生菌株，并通过菌落特征、生理生化性质、16S rDNA 序列对比分析对菌株进行鉴定，进一步对菌株产酶条件进行研究，为内切葡聚糖酶在秸秆饲料中应用奠定基础.1　材料与方法1.1　实验材料实验样品采自于湖南省长沙县养殖场健康牛胃中的秸秆发酵物.1.2　培养基（1）基础发酵培养基：葡萄糖5.0 g·L-1，酵母浸粉10.0 g·L-1，pH值自然.（2）牛肉膏蛋白胨培养基：NaCl 5.0 g·L-1，蛋白胨10.0 g·L-1，牛肉膏3.0 g·L-1，琼脂18.0 g·L-1，pH 值7.0.（3）CMC-Na培养基：羧甲基纤维素钠10.0 g·L-1，酵母浸粉0.5 g·L-1，KH2PO4 1.5 g·L-1，蛋白胨0.5 g·L-1，MgSO4 0.3 g·L-1，NaCl 5.0 g·L-1，琼脂15.0 g·L-1，pH值自然.1.3　实验方法1.3.1　产内切葡聚糖酶菌株的筛选将牛胃秸秆发酵物在CMC-Na液体培养基中37 ℃，180 r·min-1，富集培养24 h，取富集培养液1 mL，稀释涂布于CMC-Na固体培养基中，37 ℃培养24 h，多次分离纯化挑取单菌落.用牛津杯法［10-11］测定降解圈大小，实验设置3次重复.1.3.2　菌株特征、生理生化特性及16S rDNA序列的扩增分析将筛选得到的菌株划线接种后，37 ℃培养12 h，革兰氏染色［12］，镜检观察.根据《常见细菌鉴定手册》对菌株进行生理生化鉴定［13］. 提取菌株基因组DNA，使用16S rDNA通用引物［14］扩增. PCR产物由武汉擎科生物公司测序，利用BLAST对16S rDNA 序列比对分析，使用MEGA7.0构建系统进化树. 1.3.3　菌株产酶条件单因素优化以培养时间、pH值、温度、碳源、氮源、无机盐作为影响因素，依次改变其中一个因素，研究各因子对SCUEC7菌株产内切葡聚糖酶的影响.以1%接种量将SCUEC7菌株接种到牛肉膏蛋白胨培养基中，37 °C，180 r·min-1培养48 h，每隔4 h 测定一次菌体量和酶活性；分别将pH值设置为2.0、3.0、4.0、5.0、6.0、7.0、8.0、9.0、10.0、11.0，37 °C、180 r·min-1培养12 h测定菌体量和酶活性；分别将培养温度设置为25、30、37、42、50、55 °C，180 r·min-1培养12 h，每个处理设置3次重复，测定菌体量和酶活性.以发酵培养基为基础，分别将唯一碳源设置为浓度分别为10 g·L-1的蔗糖、麦芽糖、果糖、淀粉、魔芋粉；唯一氮源设置为浓度分别为10 g·L-1的胰蛋白胨、牛肉膏、酪蛋白、尿素、硫酸铵；分别向培养基中添加浓度为1 g·L-1的K+、Cu2+、Na+、Mn2+、Mg2+，37 °C、180 r·min-1培养12 h，每个处理设置3次重复，测定菌体量和酶活性.按国际单位规定：在37 °C、pH值7.0条件下，每分钟催化CMC-Na水解产生1 μg葡萄糖所需要的酶量定义为1个内切葡聚糖酶酶活性单位（1 U）. 1.3.4　菌株产酶条件响应面法优化在单因素实验的基础上，采用Box-Behnken响应面设计，分析各因素的交互作用，构建二次响应面回归模型，计算方差，得出最佳酶活性条件并验证. 响应面试验各因素与水平如表1所示.表1　响应面试验因素与水平Tab.1　Analytical factors and levels for response surface methodology 水平-11因素培养时间/h91215pH值567麦芽糖含量/（g·L-1）15202525第 43 卷中南民族大学学报（自然科学版）2　结果与讨论2.1　产内切葡聚糖酶菌株筛选将牛胃中秸秆发酵物在CMC-Na液体培养基中富集培养，用生理盐水进行梯度稀释，涂布于CMC-Na固体培养基上，利用牛津杯法测定，结果如表2所示，11株菌株中纤维素降解圈在11.38～22.83 mm之间，G2菌株直径最小，为11.38 mm，G7菌株直径最大，为22.83 mm，将G7菌株命名为SCUEC7菌株.2.2　内切葡聚糖酶产生菌SCUEC7菌株的鉴定将SCUEC7菌株接种到牛肉膏蛋白胨固体培养基上，菌落扁平，呈圆形，边缘不规则，无光泽，呈现不透明黄白色，易挑起，无色素. SCUEC7菌株生理生化特性测定结果：革兰氏染色试验、酪素利用试验、甲基红试验、伏-普试验、柠檬酸盐利用试验、淀粉利用试验、丙酸盐利用试验和硝酸盐还原试验结果呈阳性，硫化氢试验、枸橼酸盐利用试验和吲哚试验结果呈阴性.以SCUEC7菌株基因组DNA为模板，PCR法扩增菌株的16S rDNA序列，利用BLAST进行同源性序列比对，使用MEGA7.0软件N-J法构建系统进化树，结果如图1所示，SCUEC7菌株与Bacillus subtilisstrain ATCC 6015相似度达到99%，结合SCUEC7菌株形态特征、生理生化特性，初步鉴定SCUEC7菌株为枯草芽胞杆菌.2.3　单因素实验优化产酶条件2.3.1　培养时间以1%接种量将SCUEC7菌株接种到牛肉膏蛋白胨培养基中，结果表明（图2），培养时间在0～12 h 范围内，菌株生长量和酶活性随时间的增加而增加；培养时间在12～48 h范围内，菌株生长量和酶活性随时间的增加而下降.2.3.2　pH值以1%接种量将SCUEC7菌株接种到不同初始pH值的牛肉膏蛋白胨培养基中，培养12 h后，pH值在2.0～6.0范围内，菌株的生长量和内切葡聚糖酶活性随pH值的增加而增加（图3）； pH值在6.0～11.0范围内，菌株生长量和酶活性随pH值的增加而逐渐降低. pH值为6.0时，SCUEC7菌株产酶活性显著高于其他实验组（P <0.05）. 结果表明， pH值为6.0时，枯草芽胞杆菌SCUEC7菌株生长量和酶活性较高.2.3.3　温度培养温度在25～37 °C范围内，菌株生长量和表2　11株菌株降解圈大小Tab.2　Size of CMC-Na degradation circle of 11 strains菌株编号G1G2G3G4降解圈直径/mm15.40±2.2511.38±1.5314.57±1.5312.13±1.70菌株编号G5G6G7G8降解圈直径/mm18.77±1.1520.52±0.9222.83±1.5617.40±0.70菌株编号G9G10G11降解圈直径/mm21.23±1.7915.77±0.4213.67±3.36图1 基于16S rDNA 的SCUEC7菌株系统进化树Fig.1　Phylogenetic tree of SCUEC7 strain based on 16S rDNATime/hOD6酶活性/(U·mL-1)图2 培养时间对SCUEC7菌株生长和产酶活性的影响Fig.2　Effects of culture time on the growth and enzyme productionof SCUEC7 strain26第 1 期赵思卡，等：一株内切葡聚糖酶产生菌株的分离鉴定及其产酶条件优化酶活性随培养温度的升高而增加（图4）；培养温度在37～55 °C 范围内，菌株生长量和酶活性随温度的升高而下降.温度为37 °C 时，SCUEC7菌株产酶活性显著高于其他实验组（P <0.05）. 因此，培养温度为37 °C 时，枯草芽胞杆菌SCUEC7菌株生长量和酶活性较高.2.3.4　碳源当以麦芽糖作为碳源时（图5），枯草芽胞杆菌SCUEC7菌株的内切葡聚糖酶活性较高，除与葡萄糖、果糖实验组差异不显著外，显著高于其他三个实验组（P <0.05）. 结果表明，麦芽糖为碳源时，枯草芽胞杆菌SCUEC7菌株的内切葡聚糖酶活性较高.将培养基中的麦芽糖浓度分别设为5.0、10.0、15.0、20.0、25.0、30.0 g ·L -1的梯度，培养12 h 后发现（图6），麦芽糖浓度在5.0～20.0 g ·L -1范围内，酶活性从203.8 U ·mL -1增加到242.4 U ·mL -1，麦芽糖浓度在20.0～30.0 g ·L -1范围内，酶活性从242.4 U ·mL -1下降到198.0 U ·mL -1 . 麦芽糖浓度为20.0 g ·L -1时，SCUEC7菌株产酶活性显著高于其他实验组（P <0.05）.所以麦芽糖浓度为20.0 g ·L -1时，枯草芽胞杆菌SCUEC7菌株的内切葡聚糖酶活性较高.2.3.5　氮源当胰蛋白胨作为唯一氮源时，SCUEC7菌株的内切葡聚糖酶活性显著高于其他五个实验组（P <0.05）. 结果表明：胰蛋白胨为氮源时，枯草芽胞杆菌SCUEC7菌株的内切葡聚糖酶活性较高（图7）.将胰蛋白胨浓度分别设置成5.0、10.0、15.0、20.0、25.0、30.0 g ·L -1的浓度梯度，培养12 h 后，测定内切葡聚糖酶活性，结果如图8所示：胰蛋白胨的浓度在5.0~15.0 g ·L -1范围内，酶活性由198.0 U ·mL -10.01.02.03.04.0pH50100150200O D 600酶活性/(U ·m L -1)a 、b 、c 、d 、e 、f 、g 和h 代表不同pH 值对SCUEC7菌株产酶活性影响的显著性差异分析（P <0.05）.图3 pH 值对SCUEC7菌株生长和产酶活性的影响Fig.3　Effects of pH value on the growth and enzyme production ofSCUEC7 strain-01234Temperature/℃O D 600酶活性/(U ·m L -1)a 、b 、c 、d 、e 和f 代表不同温度对SCUEC7菌株产酶活性影响的显著性差异分析（P <0.05）.图4 培养温度对SCUEC7菌株生长和产酶活性的影响Fig.4　Effects of culture temperature on the growth and enzymeproduction of SCUEC7 strain葡萄糖蔗糖麦芽糖果糖淀粉魔芋粉50100150200250碳源aaabcd酶活性/(U ·m L -1)a 、b 、c 和d 代表不同碳源对SCUEC7菌株产酶活性影响的显著性差异分析（P <0.05）.图5 不同碳源对SCUEC7菌株生长和产酶活性的影响Fig.5　Effects of different carbon sources on the growth and enzymeproduction of SCUEC7 strain150175200225250麦芽糖浓度/(g·L -1)酶活性/(U ·m L -1)a 、b 、c 和d 代表不同浓度麦芽糖对SCUEC7菌株产酶活性影响的显著性差异分析（P <0.05）.图6 不同浓度麦芽糖对SCUEC7菌株产酶活性的影响Fig.6　Effects of different concentration maltose on enzyme productionof SCUEC7 strain27第 43 卷中南民族大学学报（自然科学版）增加到222.0 U ·mL -1，胰蛋白胨的浓度在15.0~30.0 g ·L -1范围内，酶活性由222.0 U ·mL -1下降到185.1 U ·mL -1. 胰蛋白胨浓度为15.0 g ·L -1时，SCUEC7菌株产酶活性显著高于其他实验组（P <0.05）.结果表明，胰蛋白胨浓度为15.0 g ·L -1时，枯草芽胞杆菌SCUEC72.3.6　金属离子以1%接种量将SCUEC7菌株分别接种到添加1.0 g ·L -1的Na +、K +、Mn 2+、Cu 2+和Mg 2+离子的基础发酵培养基中，培养12.0 h 后（图9），添加Mn 2+的实验组，SCUEC7菌株的内切葡聚糖酶活性显著高于其他四个实验组（P <0.05）. 因此，添加Mn 2+，枯草芽胞杆菌SCUEC7菌株的内切葡聚糖酶活性较高.将Mn 2+浓度分别设置成0.5、1.0、1.5、2.0、2.5、3.0 g ·L -1的梯度，培养12.0 h 后，测定内切葡聚糖酶活性，结果如图10所示，Mn 2+浓度在0.5～1.5 g ·L -1范围内，酶活性由311.6 U ·mL -1增长到354.3 U ·mL -1，Mn 2+浓度在1.5-3.0 g ·L -1范围内，酶活性由354.3 U ·mL -1下降到310.6 U ·mL -1 . Mn 2+浓度为1.5 g ·L -1时，SCUEC7菌株产酶活性显著高于其他实验组（P <0.05）.2.4　响应面试验优化结果2.4.1　响应面设计与结果选取培养时间、pH 值、麦芽糖含量3个独立变量为考察因素，设计响应面试验，测定每组实验菌株酶活性，作为响应面法分析的依据，表3为响应面试验设计及结果.2.4.2　响应曲面模型构建及回归方程结果显著性分析使用Design -Expert 10软件分析表4实验数据，建立多元回归模型.拟合培养时间（A ）、pH 值（B ）、麦芽糖含量（C ）之间的二次多项回归方程如下：酶酵母浸粉胰蛋白胨牛肉膏酪蛋白尿素硫酸铵60120180240氮源abcdee酶活性/(U ·m L -1)a 、b 、c 、d 和e 代表不同氮源对SCUEC7菌株产酶活性影响的显著性差异分析（P <0.05）.图7 不同氮源对SCUEC7菌株产酶活性的影响Fig.7　Effects of different nitrogen sources on enzyme production ofSCUEC7 strain酶活性/(U ·m L -1)胰蛋白胨含量/(g·L -1)a 、b 、c 、d 和e 代表不同浓度胰蛋白胨对SCUEC7菌株产酶活性影响的显著性差异分析（P <0.05）.图8 不同浓度胰蛋白胨对SCUEC7菌株产酶活性的影响Fig.8　Effects of different concentration tryptone on enzyme productionof SCUEC7 strainNa +K +Mn 2+Cu 2+Mg 2+100150200250300350金属离子acdeb酶活性/(U ·m L -1)a 、b 、c 、d 和e 代表不同金属离子对SCUEC7菌株产酶活性影响的显著性差异分析（P <0.05）.图9 不同金属离子对SCUEC7菌株产酶活性的影响Fig.9　Effects of different metal ions on the growth of SCUEC7 strainMn 2+浓度/(g·L -1)酶活性/(U ·m L -1)a 、b 和c 代表Mn 2+浓度对SCUEC7菌株产酶活性影响的显著性差异分析（P <0.05）.图10 不同浓度Mn 2+对SCUEC7菌株产酶活性的影响Fig.10　Effects of different concentration Mn 2+ on the growth ofSCUEC7 strain28第 1 期赵思卡，等：一株内切葡聚糖酶产生菌株的分离鉴定及其产酶条件优化活Y=408.78-3.38A-3.22B-1.67C-2.40AB-15.97AC+ 0.042BC-22.73A2-23.89B2-10.11C2.如表4所示，模型的F=11.7，模型P=0.0019< 0.01，表明模型极为显著. R2=0.9377，表明该方程与对应的响应值吻合程度达93.77%. 由F检验可知影响菌株SCUEC7产酶活性的主次因素为培养时间>pH值>麦芽糖含量. 通过Design-Expert 10软件计算得出：当预测的响应值最大时，3个因素所对应的最佳值是11.9 h，初始pH值5.9，麦芽糖含量19.8 g·L-1.在此条件下所预测的最大酶活为 409.0 U·mL-1 .2.4.3　响应曲面交互作用及结果分析培养时间、pH、麦芽糖含量3个因素对SCUEC7菌株产酶活性影响的响应面图和等高线图见图11. 由图可知，pH值、培养时间之间存在较弱的交互作用；pH值、麦芽糖浓度之间和培养时间、麦芽糖浓度之间存在很强的交互作用.2.4.4　模型检验试验结果将SCUEC7菌株最佳产酶活条件调整为培养时间11.9 h，初始pH值6.0，麦芽糖含量19.8 g·L-1，此条件下进行3组平行实验，所得平均酶活力为409.2 U·mL-1，与预测值409.0 U·mL-1差距不大，表明该结果符合客观实际. 所以采用响应面法优化SCUEC7产内切葡聚糖酶条件可行.3　结语本研究从湖南省长沙县养殖场健康牛胃秸秆发酵物中分离得到枯草芽胞杆菌（Bacillus subtilis）SCUEC7菌株，通过响应面分析，预测最优产酶条件下培养时间为11.9 h，初始pH值为5.9，麦芽糖含量为19.8 g·L-1.在动物饲料加工过程中，内切葡聚糖酶可高效降解秸秆饲料中的纤维素［15］，增加动物体营养物质利用率［16-18］.陈龙等研究Bacillus velezensis 157发现，以碱处理玉米秸秆和豆粕的质量比为1.0∶1.0，底物与水分质量比为1.0∶0.5，于37 °C培养温度下发酵24 h后，其内切葡聚糖酶活性为56.83 U·mL-1［19］；何楠等从玉米秸秆中分离出一株枯草芽胞杆菌BS-DX4，在最适生长温度 40 °C，最适pH值7.0 条件下，该菌株胞外分泌液内切葡聚糖酶最高活性为358.3 U·mL-1 ［20］，其最高酶活低于SCUEC7菌株，最适pH值与最适温度高于SCUEC7菌株；马振刚等从自然界分离出一株蜡样芽胞杆菌Bacillus cereus strain CQNUX 3-1，在70 °C、pH值8.0条件下该菌株胞外分泌液内切葡聚糖酶最高活性为107.7 U·mL-1 ［21］，最高酶活低于SCUEC7菌株，最适pH值与最适温度高于SCUEC7. 虽然研究者已从芽胞杆菌中分离出多株产内切葡聚糖酶菌株，但远不能满足工业生产对产酶菌株需求和酶性质的要求，产内切葡聚糖酶菌种资源筛选备受关注. 本研究为内切葡聚糖酶的表3　响应面各因素水平与产酶活性Tab.3　Factor level of response surface methodology and enzyme activity实验序号1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17A培养时间/h11-11-1-1-11B pH值-11-11-1-111C麦芽糖含量/（g·L-1）-1-11-111-11酶活力/（U·mL-1）377.0388.8372.6368.5362.2347.6369.4396.8371.9408.1360.6413.4416.4395.0380.1409.2357.7表4　回归模型方差分析Tab.4　Regression model and analysis of variance来源ModelABCABACBCA2B2C2残差失拟项净误差总离差平方和6754.3291.2382.9122.3823.011020.140.0007082175.412402.55430.01449.09225.47223.617203.41df911111111173416均方750.4891.2382.9122.3823.011020.140.0007082175.412402.55430.0164.1675.1655.90F值11.701.421.290.350.3615.900.00001433.9137.456.701.34P值0.00190.27190.29300.57330.56810.00530.99190.00060.00050.03600.3785显著性*******注：“**”，差异极显著（P<0.01）；“*”，差异显著（P<0.05）.29第 43 卷中南民族大学学报（自然科学版）工业化生产提供了微生物资源.参 考 文 献［1］ HU Y W ， KANG G B ， WANG L ， et al. 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乔虹，黎松松，周南希，等. 海洋来源金属离子螯合肽研究进展[J]. 食品工业科技，2024，45（1）：368−377. doi: 10.13386/j.issn1002-0306.2023030124QIAO Hong, LI Songsong, ZHOU Nanxi, et al. Research Progress in Metal Ion Chelated Peptides of Marine Sources[J]. Science and Technology of Food Industry, 2024, 45(1): 368−377. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2023030124· 专题综述 ·海洋来源金属离子螯合肽研究进展乔　虹1，黎松松1，周南希1，徐同成2，宗爱珍2，费晓伟3，魏代磊3，孙　杰1,*（1.青岛大学生命科学学院，山东青岛 266071；2.山东省农业科学院，山东济南 250100；3.山东金胜粮油食品有限公司，山东临沂 276600）摘　要：海洋多肽是生物多肽的重要来源之一，海洋来源多肽种类多并且易于被金属离子修饰，因此海洋来源的肽制备金属离子螯合肽有着天然优势，有望成为未来金属螯合肽研究热点。

本文综述了海洋来源金属螯合肽的螯合机理，对单齿螯合模式、双齿螯合模式以及 α 螯合模式三种螯合模式进行了说明。

同时归纳总结了肽的大小、肽的氨基酸种类和位置以及一些特殊的基团和残基等因素对螯合效果的影响。

列举了海洋来源金属螯合肽的制备及纯化方法，分析了不同方法的优缺点。

此外，总结了金属螯合肽促进金属离子吸收、抑菌和抗氧化等生物活性，可为金属离子螯合肽制备、功能活性分析及螯合机理研究提供参考。

关键词：海洋来源，金属离子螯合肽，螯合机理，制备，生物活性本文网刊:中图分类号：TS201.2 文献标识码：A 文章编号：1002−0306（2024）01−0368−10DOI: 10.13386/j.issn1002-0306.2023030124Research Progress in Metal Ion Chelated Peptides of Marine SourcesQIAO Hong 1，LI Songsong 1，ZHOU Nanxi 1，XU Tongcheng 2，ZONG Aizhen 2，FEI Xiaowei 3，WEI Dailei 3，SUN Jie 1, *（1.College of Life Sciences, Qingdao University, Qingdao 266071, China ；2.Shandong Academy of Agricultural Sciences, Jinan 250100, China ；3.Shandong Jinsheng Cereals & Oils Foods Group Co., Ltd., Linyi 276600, China ）Abstract ：Marine peptides are one of the important sources of biological peptides. Marine peptides have many types and are easy to be modified by metal ions. Marine peptides have natural advantages in the preparation of metal ion chelated peptides, and are expected to become a research hotspot of metal ion chelated peptides in the future. In this paper, the chelating mechanism of metal ion chelated peptides from ocean is reviewed. Single dentate chelating mode, double dentate chelating mode and α chelating mode, three chelating modes are described. At the same time, the effects of peptide size,amino acid type and position, some special residues on the chelation effect are summarized. The preparation and purification methods of metal chelate peptides from ocean are listed. The advantages and disadvantages of different methods are analyzed. In addition, the biological activities of metal ion chelated peptides such as promoting metal ion absorption, bacteriostatic and antioxidant are summarized. This paper can provide technical support for the preparation,functional activity analysis and chelating mechanism study of metal ion chelated peptides in the future.Key words ：marine sources ；metal ion chelated peptide ；chelation mechanism ；preparation ；biological activity肽是以食用蛋白为原料，经过酶解、分离、纯化等制成的新型蛋白水解产品，担负着信息传递、营养收稿日期：2023−03−13基金项目：山东省重点研发计划（2021TZXD010）；2020 年烟台市“双百人才”项目；青岛市科技惠民示范引导专项（20-3-4-33-nsh ；23-2-8-xdny-6-nsh ；23-3-8-xdny-1-nsh ）；2021 年山东省重点扶持区域引进急需紧缺人才项目；2019 年山东省人才引进成果示范推广项目；山东省 2018 年度农业重大应用技术创新项目。

环境昆虫学报2021, 43 (1)： 158-169Joornat cf Exvimnsentol Extosologyhttp ： 〃hjkcxb. alljournals. netdoi ： 1003969 /ai s n01674 -085802021001016徐天梅，黄锤，肖关丽，陈斌.白背飞虱雌雄成虫肠道可培养细菌种类组成及差异分析[J ]-环境昆虫学报，2021, 43 (1)： 158 -169-白虫 养细菌种类组成及差异分析徐天梅S 黄 锂2，肖关丽2",陈 斌"(1-云南农业大学植物保护学院,云南生物资源保护与利用国家重点实验室，云南昆明650201；2-云南农业大学农学与生物技术学院,云南昆明650201)摘要：为弄清白背飞虱成虫肠道细菌组成及雌雄成虫之间的组成差异，本研究采用传统培养法，对云南省元江县 水稻田白背飞虱雌雄成虫肠道细菌种类组 分离 ， 用 及16S RNA 序列比对方法进行细菌的种属鉴定。

结 表明：从白 飞 道内分离鉴定出细菌3门8科12属20种，优势菌门均菌门Firmicides ( 50% ),雌成虫肠道优势菌属为葡萄球菌属Stnppylococcs ( 25% ),优势菌种为松鼠葡萄球菌Stnppymcoccvc rpoirret ( 25% )；雄成虫肠道优势菌属为芽抱杆菌属BachOs ( 22% )和微杆菌属MOsbader(22% ),优势菌种为产左聚糖微杆菌Microbacteriom lodanObrsan (22% )(其中，雌性成虫肠道内共分离获得细菌3门8科8属12种，包括芽抱杆菌属Bndf m 3种；葡萄球菌属Stnphylococcs S 种；微小杆菌属ExifoobncdWom1种；不动杆菌属ASnedbader 1种；泛菌属Pnxtoen 1种；假单抱菌属Psodfnoxas 2种；苍白杆菌属OcymbncSom 1种；微杆菌属Microbacteriom 2种。

10福建畜牧兽医第42卷第5期2020年乳鸽源绿脓杆菌的分离与鉴定李冰心1程柏丛2王俊3杨秀环2孙梦许3(1.北京市大兴区动物疾病控制中心北京102600；2.北京市畜牧兽医总站北京100107；3.中国农业大学动物医学院北京100193)摘要2019年6月，河北省石家庄市某养鸽场的乳鸽发病，病死率超30%遥经病理剖检和实验室病原分离、鉴定，确诊为绿脓杆菌感染，并对分离菌株进行了药敏试验遥现将诊断情况报道如下袁为预防和控制该病的流行提供参考依据遥关键词乳鸽绿脓杆菌分离鉴定药敏试验文献标识码:A文章编号：1003-4331(2020)05-0010-03Isolation and identification of Pseudomonas aeruginosa of pigeonLi Bingxin1Cheng Bocong2Wang Jun3Yang Xiuhuan2Sun Mengxu3(1.Beijing Daxing Center for Animal Disease Control,Beijing102600;2.Beijing Municipal Animal Husbandry and Veterinary General Station,Beijing100107;3.College of Veterinary Medicine,China Agricultural University,Beijing100193)Abstract In June2019,an outbreak occurred in newborn pigeon which were raised in a pigeon farm in Shijiazhuang country of Hebei province.The mortality rate was up to30%.Based on the necropsy examination and laboratory pathogen isolation and diagnosis,the pathogen was identified as Pseudomonas aeruginosa,and drug susceptibility testing was performed with the isolated strain.The diagnosis process for this case is reported below,which will provide reference for preventing and controlling the epidemic of the disease.Key words Newborn pigeon Pseudomonas aeruginosa Isolation and identification Drug susceptibility testing绿脓杆菌（Pseudomonas aeruginosa,PA）又称为铜绿假单胞菌,Gersard于1882年首次从伤口脓液中分离得到，该菌广泛存在于自然界的水和土壤中以及动物肠道和皮肤表面，是典型的人兽共患条件致病菌，通常引起人皮肤化脓性感染,常见于烧烫伤患者。