智利竹鱼渔场海洋微生物的物种多样性及其生物活性筛选

利用东海水产研究所开展的2012年东南太平洋智利竹鱼(Trachurus murphyi)渔场生态环境调查,对该海域75个站点进行了海洋微生物样品的采集;通过菌株的16S rRNA测序及GenBank数据库比对,分析了从所采样品中分离获得的海洋微生物菌株的物种多样性;通过菌株发酵、代谢产物提取及其抗菌滤纸扩散分析,对其发酵代谢产物粗提物进行了抗菌活性筛选;通过类胡萝卜素合成通路中八氢番茄红素合成酶(crtB)及脱氢酶(crtI)基因分析,对筛选到的抗菌活性菌株进行了基因筛选。结果表明,累计分离获得可培养海洋微生物菌株628株,其隶属12个属,其中的优势菌属包括亚硫酸盐杆菌属(Sulfitobacter)、弧菌属(Vibrios)及气单胞菌属(Aeromonas)。其中16株菌株的发酵代谢产物具有明显的抗菌活性,而其中12株活性菌株具有合成类胡萝卜素的crtB、crtI分子遗传基础。     

两株海洋微生物抗肿瘤代谢产物的筛选及其菌种鉴定

对5株海洋微生物进行发酵培养,发酵液离心后,收集上清液,采用饱和正丁醇萃取制备海洋微生物的代谢产物,采用MTT活性跟踪法,以BEL-7402、RKO、A549、U251和MCF-7等细胞系为模型,对代谢产物进行抗肿瘤活性的筛选。结果显示,菌株S-1和N16代谢产物的活性组分具有较好的抗肿瘤活性:菌株S-1对MCF-7、U251和BEL-7402三种肿瘤细胞的IC50值分别为44、102和82μg/ml;菌株N16对MCF-7和BEL-7402两种肿瘤细胞的IC50值分别为84、133μg/ml。对菌株S-1和N16进行了16S r RNA序列分析、生理生化特征鉴定,结果显示,菌株S-1为短芽孢杆菌属(Brevibacillus sp.),N16为芽孢杆菌属(Bacillus sp.)。通过显微镜观察了菌株Brevibacillus sp.S-1和Bacillus sp.N16代谢产物的活性组分对BEL-7402细胞形态的影响,表明两株菌的活性组分可以改变肝癌细胞BEL-7402的细胞形态,抑制细胞增殖。该研究为新型抗肿瘤先导化合物的发现提供了新的海洋微生物种质资源。     

一株海洋源细菌素产生菌筛选及细菌素性质研究

化学食品防腐剂潜在的副作用使其使用受到越来越严峻的挑战,微生物产天然食品防腐细菌素成为关注的热点。本研究以L.monocytogenes、E.coli、S.aureus、B.subtilis、V.parahemolyticus、A.niger为指示菌,以牛津杯法评价抗菌活性,以南海海洋动物肠道作为分离材料,通过抗菌作用的排酸实验、过氧化氢排除实验以及蛋白酶敏感实验进行细菌素产生菌的筛选,并进行了细菌素物质的热稳定性与酸稳定性的研究。筛选出1株具有抗菌活性强,抗菌谱广的细菌素产生菌A0705,其发酵液在-20~100℃处理30 min后,再在p H2.5~11.5的范围内37℃温浴2 h,仍保持较稳定的抗菌活性,经16S r DNA序列同源性比对及系统发育树分析,鉴定菌株A0705为芽孢杆菌(Bacillus sp),该菌是1株值得进一步研究与开发的海洋源细菌素产生菌。     

具抑菌活性海洋微生物的筛选

从采集于上海郊区海域的海水和海泥样品中分离出 193株海洋细菌，从中筛选抑菌活性菌株。 分别利用琼脂块法和管碟法对分离出的菌株进行初筛和复筛，结果显示，有 29株海洋细菌具有抑菌活性， 占总分离菌株的 15%，其中抑菌能力较强的 G4，抑菌谱较广，能同时抑制革兰氏阳性和阴性指示菌。     

4株南极真菌的形态学和分子鉴定及其中3株的蛋白磷酸酶、脂肪酸成分分析

由于极地独特的地理、气候及环境特点,使得极地微生物及其代谢产物具有新颖性与多样性。采用3种选择性培养基的稀释平板涂布法,从南极普里兹湾海洋沉积物中分离得到4株真菌菌株,经过形态学、18s r DNA-ITS序列分析,分别鉴定为隐球酵母Cryptococcus sp.NJF1、曲霉Aspergillus sp.NJF3、枝孢霉Cladosporium sp.NJF4和Cladosporium sp.NJF6;对其中的曲霉NJF3、枝孢霉NJF4和NJF6等3株真菌开展了蛋白磷酸酶含量和大田软海绵酸OA对该酶的抑制作用测试,并分析了菌株提取物中的脂肪酸成分。结果显示:NJF3、NJF4和NJF6有很好的蛋白磷酸酶活性,以蛋白磷酸酶-PP为外标,测定酶含量分别为0.732 2 U·g-1、0.678 U·g-1和1.229 2 U·g-1,且100 n M OA对NJF3和NJF4蛋白磷酸酶有一定抑制作用,而NJF6需要200 n M OA才可以轻微抑制其蛋白磷酸酶活性;3株真菌的脂肪酸成分以油酸为主,相对含量分别达到32.4%、37.2%、40.1%,而NJF4和NJF6还含有较高含量的亚油酸,相对含量分别达到27.3%、29.8%。本研究可为下一步开展菌株的酶、天然代谢产物研究奠定基础。     

短小芽孢杆菌X93及胞外产物抑菌活性的研究

从健康养殖大黄鱼肠道中筛选出对病原弧菌有拮抗作用的潜在益生菌株X93,利用细菌快速鉴定系统和16S rDNA方法进行细菌种类鉴定;通过点种法对其抗菌谱进行测定;采用平板扩散法,研究了培养时间、温度、盐度、蛋白酶K和Fe3+等因素对其胞外产物抑菌活性的影响。结果显示:该菌株为短小芽孢杆菌(GenBank accession No.HM137033);对20株病原指示菌中的13株均产生不同程度的抑制作用,抗菌谱较广;菌株培养48 h后的胞外产物抑菌作用最强;在pH为7时,抑菌活性最大;达到最大抑菌活性的温度和盐度分别为28 ℃、5;蛋白酶K和不同浓度的FeCl3均会降低其抑菌活性。该菌对病原弧菌、迟缓爱德华菌和嗜水气单胞菌等水产上常见主要致病菌有良好的拮抗效果,且具有抑菌作用的胞外产物对温度、盐度和Fe3+有一定的适应范围,适宜作为益生菌株,有望在实际养殖环境中得到应用。     

泥鳅体内凡隆气单胞菌拮抗细菌CMA 1的分离筛选及其抑菌活性

本研究从泥鳅养殖水体、底泥、健康泥鳅和患病泥鳅肠道等环境中采集样品,从中分离纯化出对凡隆气单胞菌具有拮抗作用的细菌57株.在TSB平板上采用牛津杯法进行体外拮抗实验,筛选出一株对凡隆气单胞菌具有明显拮抗作用的菌株CMA-1,经形态观察、生理生化鉴定和16SrDNA序列系统发育分析,将该菌鉴定并命名为Bacillus methylotrophicus CMA-1.菌株CMA-1生长24 h后进入稳定期,36 h时发酵上清液抑菌活性达到最大.对菌株CMA-1的发酵上清液采用硫酸铵分级沉淀,测得75％硫酸铵饱和度下的沉淀粗提物抑菌活性最高.     

植物精油对冷藏大黄鱼优势腐败菌的抑制作用及其机制

为探讨天然植物精油对冷藏大黄鱼贮藏过程中优势腐败菌的抑制作用及其机制,实验首先对冷藏大黄鱼的优势腐败菌进行分离纯化,并通过16S r DNA序列分析及生理生化实验进行菌株鉴定;采用纸片扩散法和微量肉汤稀释法检测14种植物精油或其组分对腐败菌株的抑菌活性;在阐明植物精油抗菌活性的基础上,再对植物精油处理后的大黄鱼气味进行感官可接受度检验,以筛选出具有高效抗菌活性和良好感官接受性的精油保鲜剂;实验通过细菌形态扫描电镜观察、胞内乳酸脱氢酶及核酸释放研究山苍子精油对不同腐败菌株的膜损伤作用,以初步阐明其抗菌机制;最后,通过检测微生物数量、产硫菌数量、挥发性盐基氮(TVB-N)及组胺含量变化来考察山苍子精油对冷藏大黄鱼潜在的保鲜效果;从冷藏大黄鱼中分离得到6株优势腐败菌,分别为腐败希瓦氏菌4株,热杀索丝菌1株,温和气单胞菌1株;14种植物精油对腐败菌株具有不同抑制活性,其中肉桂醛抑菌活性最强,最低抑菌浓度为0.125μL/mL,其次为丁香酚、百里香酚、柠檬醛、柠檬草及山苍子精油,最低抑菌浓度均≤0.5μL/mL;丁香酚、百里香酚、柠檬醛、柠檬草及山苍子精油使用后未导致大黄鱼气味的感官劣化,且可矫正大黄鱼肉的腥味;山苍子精油可破坏腐败菌株的细胞膜,并导致胞内蛋白酶和核酸外泄。经4μL/mL山苍子精油处理后,冷藏过程中大黄鱼片的菌落总数、产硫菌数、TVB-N及组胺含量显著降低。研究表明,肉桂醛、丁香酚、百里香酚、柠檬醛、柠檬草及山苍子精油对大黄鱼特定腐败菌具有显著抑制作用,芳香酚和富含柠檬醛的精油使用后不影响大黄鱼的感官接受性,有望作为天然保鲜剂用于冷藏海产品的保鲜。     

罗非鱼肠道益生菌的筛选、鉴定及其抑菌性能研究

采用“先筛选后分离”方式对活体罗非鱼肠道菌进行分离,选取革兰氏阳性接触酶阴性的细菌,反复冻融法制备模板DNA,PCR扩增并测定了菌株的16S rDNA序列,得到1株乳酸乳球菌、1株德氏乳杆菌.采用牛津杯法测定其对病原菌的抑菌效果,结果表明,这2株乳酸菌对大肠杆菌、金黄色葡萄球菌有不同程度的抑制作用,为生态养鱼提供了发酵菌种.     

健康养殖南美白对虾肠道细菌的抗菌活性

用双层琼脂扩散法检测从健康养殖南美白对虾肠道内分离出的111株菌对8株病原菌的抗菌活性。结果表明:111株菌对白假丝酵母菌,非01群霍乱弧菌,嗜水气单胞菌指示菌无抗菌活性,48.6%的菌株对余下的5株指示菌有抗菌活性,其中气单胞菌属和发光杆菌属对荧光假单胞菌与金黄色葡萄球菌的抑制性最强。各菌属中以气单胞菌属,发光杆菌属的抗菌谱最广,弧菌属、肠杆菌科、黄单胞菌属、芽孢杆菌属、小球菌属次之。土壤杆菌属、棒状杆菌属、黄杆菌属、产碱杆菌属、假单胞菌属和色杆菌属则没有抗菌活性。     

褐藻多酚的生物活性及提取技术研究进展

本文对不同褐藻中的褐藻多酚存在的形式进行了分析,综述了褐藻多酚抗菌、抗氧化、抗凝血、抗肿瘤以及化学防御等生物活性的研究进展,并比较了有机溶剂浸提、超声波辅助提取、微波辅助提取以及有机溶剂萃取纯化等方法的提取效果,旨在为褐藻多酚的进一步研究和利用提供参考依据.     

鼠尾藻多酚分级组分的抑菌活性研究

采用平板生长抑制法对鼠尾藻Sargassum thunbergiikuntze多酚化合物各分级组分进行抑菌活性研究。结果表明,在一定浓度范围内各分级组分对副溶血弧菌、哈维氏弧菌、沙蚕弧菌、金黄色葡萄球菌、大肠杆菌、鳗弧菌、溶藻弧菌和四联微球菌等受试菌株均有抑菌活性。其中,分级组分I(Mr5.0×103)的抑菌效果优于其他组分,对副溶血弧菌、哈维氏弧菌和沙蚕弧菌的最低抑菌浓度(MIC)值为900μg/ml,对金黄色葡萄球菌、大肠杆菌、鳗弧菌和溶藻弧菌的MIC值为1 800μg/ml,对四联微球菌的MIC值为3 600μg/ml。另外,组分I(Mr5.0×103)显示出较好的热稳定性,在pH 3~4时对受试菌的抑菌活性最强。     

Study of Chemical Composition and Efficacy of Different Extracts of Iranian Propolis on the Microbiological and Sensory Parameters of Minced Cyprinus carpio Meat at 4°C Storage

This study has evaluated the effects of different concentrations of the water and ethanolic extracts of Iranian propolis (WEP and EEP) on the microbiological and sensory parameters of minced common carp meat that was stored at 4°C for 9 days. The chemical compounds of the extracts were identified by gas chromatography–mass spectroscopy (GC-MS) analysis. The results revealed that WEP and EEP extracts were efficient against the spoilage microorganisms, including total plate counts (TPCs), psychrotrophic populations (PTCs), lactic acid bacteria (LAB), and even Staphylococcus aureus. There was a significant difference in the bacteria growth between samples treated with WEP or EEP and the control sample during storage. Inhibitory effects were, respectively, 7% EEP > 5% EEP > 3% EEP > 7% WEP > 5% WEP > 3% WEP. In all treatments, PTC counts were higher than 7 log CFU/g after 6 days as well as TPC, LAB, and S. aureus populations after 9 days. Overall, the results show that EEP and WEP have good antimicrobial activity due to high phenolic and flavonoid compounds, including caffeic acid, tectochrysin, aceacetin, chrysin, pinostrobin, pinobanksin-3-acetate, galangin-3-methyl ester, and pinocembrin chalcone. Sensory evaluation shows very slight improvement in sensory properties (color, odor, texture, and overall acceptability) of the treated samples with the different extracts of Iranian propolis.     

氨基葡萄糖盐酸盐的防腐抗菌作用

实验表明：GAH对所测试的21种常见食品腐败菌均有抗菌作用，最小抑菌质量浓度对细菌为0．5g/L，对霉菌为2g/L，对酵母菌为1g/L，将GAH作为防腐剂应用于马蹄原汁保藏时显示了很好的防腐效果。     

Antimicrobial Activity of Selected Essential Oils on Pseudomonas Species Associated with Spoilage of Fish with Emphasis on Cinnamon Essential Oil

ABSTRACT

Eight plant essential oils (cardamom, cinnamon, clove, eucalyptus, lemongrass, lime, nutmeg and rosemary) were evaluated for their antimicrobial activity against three Pseudomonas species associated with microbial spoilage of refrigerated tilapia. In vitro test of the essential oils, using disc-diffusion method, showed various degrees of antimicrobial activity against the Pseudomonas species. Cinnamon essential oil (CEO) had the highest antimicrobial activity, followed by clove essential oil. The remaining essential oils showed consistently weak activity. The antimicrobial efficiency of CEO against the Pseudomonas species was confirmed by potential minimum inhibitory concentration and minimum bactericidal concentration values, ranging between 0.125 and 0.375 µL/mL and 0.250–0.500 µL/mL, respectively. The anti-Pseudomonas activity of CEO was also tested in fish extract model at refrigeration temperature. The essential oil reduced the Pseudomonas viable count in fish extract but to a lesser extent than when it was applied at the same concentrations in culture medium. The chemical composition of CEO showed that the oil contained 79.1% cinnamaldehyde, which was suggested to be responsible for its antimicrobial activity. These results revealed the potential of CEO as a promising natural antimicrobial agent that could efficiently contribute to the control of spoilage bacteria and retard microbial spoilage of fresh fish.     

海水人工湿地脱氮效果与系统内基质酶、微生物分析

利用人工湿地系统探讨了海水养殖外排水中不同形态氮的去除效果,分析了系统内部微生物空间分布、基质酶活性对氮去除效率的影响,深入研究了系统内部各微生物间相互作用、基质酶活性与微生物空间分布关系。选择互花米草为人工湿地植物,种植密度为64株/m2;基质填料选择细纱、高炉矿渣和珊瑚石。在3种不同工况条件下,研究人工湿地系统中氮的去除效果、微生物数量和基质酶活性。结果显示,系统对 TN、NH4-N 平均去除率分别为(25.02±12.69)%、(82.91±17.51)%;系统中不同基质层次的微生物数量和脲酶、脱氢酶活性不同,基质中、上层好氧微生物数量和脲酶、脱氢酶活性显著大于下层,系统下行池中、上层硝化细菌、亚硝化细菌显著高于下层,中、上层反硝化细菌数量明显低于下层;细菌总数与 TN(R2=0.50)和 NH4-N(R2=0.61)去除率存在一定正相关性,基质脲酶与 TN 去除率存在显著正相关性(R2=0.86)。研究结果将有助于理解海水人工湿地处理系统中氮的迁移转化机制。     

Characterization of Spray-Dried Hydrolyzed Proteins from Pink Perch Meat Added with Maltodextrin and Gum Arabic

In the present study, spray-dried pink perch meat protein hydrolysate (PPH) and hydrolysate with maltodextrin and gum arabic (PPHMG) were characterized in relation to their physical, rheological, functional, antioxidant, thermal, and sensory properties. The Herschel–Bulkley model was suitable to explain the flow behavior of PPH and PPHMG emulsions. Both the samples exhibited functional and antioxidant properties. Antioxidant activities were significantly higher for PPH compared with PPHMG (p<0.05). PPH and PPHMG alone did not exhibit antimicrobial activity but enhanced the activity of chitosan against pathogens. Sensory acceptability of the samples in soup revealed that PPHMG of up to 4% was highly scored without any traces of bitterness, whereas for PPH, a concentration of up to 3% was acceptable.     

Antimicrobial Susceptibility of Coagulase-Positive and Coagulase-Negative Staphylococci in Ready-to-Eat Sashimi

The aim of this study was to investigate the antimicrobial susceptibility pattern of Staphylococcus spp. isolated from sashimi meals collected in Japanese restaurants from northern Portugal. One hundred and thirty-six isolates of Staphylococcus spp. were recovered from sashimi samples. The antimicrobial susceptibility test showed high levels of resistance to β-lactams, macrolides, and lincosamides. All Staphylococcus isolates were sensitive to trimethoprim-sulfamethoxazole, ciprofloxacin, and chloramphenicol. Multidrug-resistant staphylococci (25%) were found in sashimi samples. It is of the utmost need to improve the hygienic practices in handling in order to reduce the occurrence of Staphylococcus species, potentially resistant to antimicrobial agents.     

Putative virulence factors of atypical Aeromonas salmonicida isolated from Arctic charr,Salvelinus alpinus (L.), and European grayling,Thymallus thymallus (L.)

Atypical Aeromonas salmonicida (AAS) causes generalized lethal infections in farmed Arctic charr, Salvelinus alpinus (L.), and European grayling, Thymallus thymallus (L.), and is thus a serious threat for culture of these fish species. Virulence factors were studied among isolates of AAS from Arctic charr (n = 20), European grayling (n = 19) and other fish species (n = 20), of which 48 were of Finnish and 11 of Swedish origin. All isolates produced an A-layer. Extracellular products (ECP) of the AAS isolates did not produce detectable gelatinase and caseinase activity in test assays. Analysis of the same ECP preparations with substrate sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed weak proteolytic activity, indicating the different sensitivity of the detection methods used. The ECP from AAS isolates showed low cytotoxic activity against cultured cells. However, the ECP did not induce mortality in challenged Arctic charr. The results suggest that toxic components, like ECP, secreted by the bacterium may not be the major virulence factor in AAS-infection in Arctic charr and European grayling, and hence the pathogenesis also differs from the pathogenesis of AAS-infection in Atlantic salmon, Salmo salar L.     

中国对虾中抗菌短肽的分离纯化与功能分析

以水产病害的病原菌为目标菌，从中国对虾（Penaeuschinesis）体液中分离筛选抗菌肽，通过Sephadex G-50、RP-HPLC等分离纯化技术，得到一种抑制水产病原菌的抗菌肽PC-V-2。经初步鉴定，该抗菌肽具有广谱抗性，不仅抗鲁克氏耶尔森氏菌（Yersinia ruckeri），而且对金黄色葡萄球菌（Staphyloccus auleus）、枯草杆菌（Bacillus subttilis）等革兰氏阳性菌，大肠杆菌（Escherichia coli）等革兰氏阴性菌和白色念珠菌（Candida albicans）等真菌也有较强的抗性。另外，该抗菌肽还有一定的抑制凝血的活性，但没有检测到溶血和丝氨酸蛋白酶抑制活性。经MALDI-TOF质谱分析，其分子量为1848D，但其序列及其他生物特性还有待进一步研究。