抑制馒头中腐败霉菌活性乳酸菌的筛选及其应用

采用稀释涂布平板法从发霉馒头中分离主要腐败霉菌,并通过形态观察及分子生物学技术对其进行鉴定。以分离得到的腐败霉菌为指示菌,从实验室保藏乳酸菌中筛选抑菌效果较好的乳酸菌,对其发酵上清液中抑菌物质的理化特性进行分析,并将其应用于馒头的防腐。结果表明,分离到两株主要腐败霉菌,经鉴定分别为扩展青霉（Penicillium expansum）和黄曲霉（Aspergillus flavus）。筛选到一株具有较强抑制霉菌活性的植物乳杆菌（Lactobacillus plantarum）LP-1,其发酵上清液对温度、过氧化氢酶、胰蛋白酶和蛋白酶K处理均不敏感,在pH为3～4时有较好的抑菌活性,初步推测L. plantarum LP-1代谢产生的有机酸为主要抑菌物质。利用L. plantarum LP-1发酵制备酸面团,酸面团添加量为40%时,防霉效果与0.5 g/kg脱氢乙酸钠相当,37 ℃防霉效期可达4 d。     

原桃胶中1株芽孢杆菌的分离鉴定及其主要抗菌物质

从天然原桃胶分离到1 株产抑菌活性物质的芽孢杆菌TJ12。其发酵产物对金黄色葡萄球菌（Staphylococcusaureus）、藤黄微球菌（Micrococcus luteus）、蜡样芽孢杆菌（Bacillus cereus）和串珠镰刀菌（Fusarium moniliforme）有抑菌活性。结合菌落形态、生理生化指标、16S rRNA序列和gyrB序列将TJ12菌株鉴定为地衣芽孢杆菌（Bacillus licheniformis）。通过硫铵沉淀、有机溶剂萃取、羟丙基葡聚糖凝胶、高效液相色谱分离纯化,得到其主要活性物质。该物质对pH值、温度和3 种蛋白酶（胰蛋白酶、蛋白酶K、胃蛋白酶）有较好的稳定性。经电喷雾电离质谱分析,初步鉴定该株地衣芽孢杆菌TJ12的主要抗菌物质为杆菌肽A。     

胡麻枯萎病生防芽孢杆菌筛选及抑菌效果研究

为筛选拮抗胡麻枯萎病菌的生防微生物,对采自我国不同省份胡麻根围土壤进行了细菌分离和拮抗菌筛选。根据土样中151株病原菌尖孢镰刀菌[Fusarium oxysporum f.sp.lini（FOL）]对峙培养获得21株拮抗芽孢杆菌,其中XJ2-20拮抗效果最好,盆栽实验的胡麻枯萎病防效可达56.3%,经鉴定为枯草芽孢杆菌。菌株XJ2-20对151株镰刀菌株的抑菌率最高可达64.5%,其发酵滤液可抑制尖孢镰刀菌生长及抑制分生孢子萌发。菌株XJ2-20的抑菌活性物质的最佳硫酸铵沉淀浓度为70%,活性物质对热和胰蛋白酶敏感,最适pH为7。其最佳发酵条件为LB液体培养基（初始pH 7.2）、每300mL三角瓶装液量50～100mL,29℃培养5d。     

浏阳豆豉发酵中高产酶活菌株的分离鉴定及酶活性分析

从浏阳豆豉发酵过程中分离产高酶活菌株,通过形态观察结合分子生物学技术进行鉴定,并对其产蛋白酶、脂肪酶及纤维素酶的活性进行分析。结果表明,分离得到3株菌（编号为000、5132、621）均被鉴定为溜曲霉菌（Aspergillus tamarri）。3株菌的蛋白酶、脂肪酶及纤维素酶的活性测定结果表明,菌株621蛋白酶活性最强,为（207.98±3.20）U/mL;菌株5132的纤维素酶活性最强,为（3.40±1.40）U/mL;菌株000的脂肪酶活性最高,为（90.7±0.64）U/mL。     

广谱拮抗菌株的筛选诱变及抗菌物质分离鉴定

分离和鉴定产生广谱抗菌活性物质的菌株,并对抗菌物质分离鉴定。通过形态学、生理生化特征和16S rDNA序列分析鉴定菌株。采用紫外线-硫酸二乙酯化学复合诱变的方法,获得抗菌能力强、遗传稳定的高产菌株。分别用甲醇提取、凝胶过滤色谱（Sephadex G-10）和半制备高效液相色谱分离纯化抗菌物质,基质辅助激光解吸电离飞行时间质谱（matrix-assisted laser desorption/ionization time of flight mass spectrometry,MALDI-TOF-MS）技术对抗菌物质鉴定。本研究从土壤中分离出67 株芽孢杆菌。其中8 株表现出较强的抗菌作用,菌株XF32表现出显著广谱抑制活性,并鉴定为地衣芽孢杆菌（Bacillus licheniformis）。菌株XF32对金黄色葡萄球菌、枯草芽孢杆菌、蜡样芽孢杆菌、大肠杆菌、沙门氏菌、志贺氏菌、铜绿假单胞菌7 种病原菌具有抗菌活性,对酿酒酵母、黑曲霉、深绿木霉和尖孢镰刀菌有抗真菌活性。经诱变筛选得到突变菌株XF32-22,对金黄色葡萄球菌的抑菌活性比原始菌株显著提高,分离纯化抗菌物质,得到单一的抗菌成分。MALDI-TOF-MS分析表明抗菌物质为Fengycin系脂肽化合物。采用复合诱变技术,可以有效地提高地衣芽孢杆菌脂肽的产量。成功分离得到对多种病原细菌具有较强抑制作用的Fengycin类脂肽,同时该菌株在拮抗植物病原真菌方面也有明显效果。XF32-22菌株在食品保鲜和农业生物防治方面具有很好的应用前景。     

烟草镰刀菌根腐病（Fusarium spp.）拮抗菌L210的鉴定及生防潜力评价

为筛选烟草镰刀菌根腐病（Fusarium spp.）的高效拮抗菌株,通过平板对峙、盆栽和大田复筛试验,从平顶山烟区烟株根际土壤中分离筛选拮抗菌;利用形态观察、生理生化测试和16S rDNA序列分析对拮抗菌进行鉴定,并对拮抗菌抗镰刀菌根腐病的机理进行研究。结果表明：从10份土壤样品中共分离土壤微生物菌株512株,其中以L210菌株对镰刀菌根腐病抑制效果最好,平板对峙抑菌直径71.4 mm,抑菌率为68.2%;盆栽和大田防病试验的发病率分别为0、6%,病情指数分别为0、1.5,防治效果分别为100%和77.9%。初步鉴定L210菌株为液化沙雷氏菌（Serratia liquefaciens）,其抑菌机理为溶解尖孢镰刀菌的菌丝,使其失去侵染能力,进而抑制病原菌的生长。菌株发酵液中含有几丁质酶和纤维素酶,可溶解病原菌的细胞壁,还可显著诱导提高烟苗中多酚氧化酶（PPO）、过氧化物酶（POD）和苯丙氨酸解氨酶（PAL）等防御酶的活性。因此,菌株L210在烟草镰刀菌根腐病的生物防治中具有较大的应用潜力。     

海洋真菌产酶能力的研究

从大连丽娇湾和金石滩采集获得海水、海藻等样品,采用PDA培养基分离海洋真菌,并对产酶真菌进行筛选及鉴定,及其产酶能力进行初步研究。结果显示,共筛选得到真菌32株,产酶菌株18株,其中有1株同时产4种酶,3株同时产3种酶,8株同时产2种酶。初步鉴定18株产酶菌分布于4个属,其中优势属为曲霉属（Aspergillus sp.）和青霉属（Penicillium sp.）,各6株,均占总筛选产酶菌株数的33.33%,其次为酵母属（Saccharomyces sp.）,共4株,占总筛选产酶菌株数的22.22%。11株产植酸酶、10株产纤维素酶、6株产淀粉酶、5株产蛋白酶、3株产脂肪酶。     

拮抗黄曲霉海洋放线菌的筛选及发酵条件优化

该研究从黄海海域沉积物中筛选一株能高效抑制黄曲霉（Aspergillus flavus）的海洋放线菌,通过形态观察、生理生化试验及 分子生物学技术对其进行鉴定,并以抑菌圈直径为响应值,通过Plackett-Burman试验、最陡爬坡试验及响应面试验对其产抑菌活性 物质的发酵条件进行优化。 结果表明,筛选获得一株对黄曲霉抑制活性较高的菌株B11,并鉴定其为锈赤蜡黄链霉菌（Streptomyces rubiginosohelvolus）;菌株B11产抑菌活性物质的主要影响因素为发酵温度、发酵时间及接种量,最优发酵条件为：发酵温度29 ℃、发 酵时间11 d、接种量5%。 在此最优发酵条件下,抑菌圈直径达到（2.9±0.15）cm,抑菌圈直径比优化前（1.4±0.15）cm增大51.72%。     

拮抗砀山梨炭疽菌的内生菌筛选及抑菌效果分析

从健康砀山梨中分离出15 株内生菌,筛选出1 株对砀山梨胶孢炭疽菌抑菌活性较高的菌株,命名为DSL-9,初步鉴定为枯草芽孢杆菌（Bacillus subtilis）。DSL-9菌株对炭疽菌的抑制主要是通过胞外分泌物作用的结果。采用硫酸铵沉淀法获得DSL-9的粗蛋白提取液,蛋白提取液在30～60?℃抑菌率无明显变化,大于60?℃抑菌率下降;在pH?7～10区间抑菌活性较高,且无明显变化;紫外线对抑菌物质无明显影响;蛋白提取液经蛋白酶处理后,抑菌活性下降。研究内生菌DSL-9对感染炭疽菌砀山梨防御酶活性的影响,发现内生菌DSL-9可提高砀山梨果实对病菌的抗性。从砀山梨中分离筛选的枯草芽孢杆菌DSL-9?对砀山梨炭疽病害的生物防治具有潜在的应用价值。     

抑制呕吐毒素生物合成的乳酸菌的筛选及鉴定

以禾谷镰刀菌ACCC36938为指示菌,测定不同乳酸菌对其抑制作用,筛选得到抑菌效果较强的1株乳酸菌A14-2。进一步研究不同温度、p H值和蛋白酶处理对乳酸菌A14-2抑菌活性的影响,结果表明p H值变化对其影响最大,同时也存在非蛋白类热敏感物质具有一定抑菌作用。为了探究乳酸菌A14-2对禾谷镰刀呕吐毒素(deoxynivalenol,DON)生物合成的影响,选用麦芽汁作为培养基,将乳酸菌和禾谷镰刀菌混合在麦芽汁培养基中共同培养,分析培养基中DON质量浓度变化,结果发现乳酸菌培养物及其上清液均能够在抑制禾谷镰刀菌生长的同时,也显著降低了DON的合成量,但乳酸菌细胞对DON无吸附作用。最后对乳酸菌A14-2进行理化及分子鉴定,显示其为植物乳杆菌(Lactobacillus plantarum)。     

1株产脂肽类抗生素芽孢杆菌的鉴定及脂肽类抗生素相关基因克隆

从小麦根际土壤中分离得到一株对禾谷镰刀菌有拮抗作用的菌株7F1。以菌株7F1基因组总DNA为模板,通过16S rDNA和gyrB基因序列分析,鉴定7F1为多粘类芽孢杆菌。本实验对该菌种产拮抗物的稳定性进行了研究,同时,应用特异性引物对7F1中脂肽类抗生素合成相关基因进行检测。结果表明：该菌产生的拮抗物在40～90 ℃保持较高的活性;在蛋白酶K、胃蛋白酶和胰蛋白酶处理后,抑菌活性显著下降;在pH 3.0～10.0具有抑菌活性;使用已报道的脂肽类抗生素基因片段设计的引物进行聚合酶链式反应扩增,从该菌的基因组DNA中扩增得到了3 种bamC、eriSa、ituC脂肽类抗生素合成酶基因。此研究结果对脂肽类抗生素进一步的分离提供了重要参考。     

海洋细菌BMF04菌株的抑菌和毒素去除作用

为了明确海洋细菌BMF04菌株的抑菌作用和毒素去除作用及其降解毒素机理。采用平板对峙法测定BMF04菌株及无菌发酵液对小麦赤霉病菌（Fusarium graminearum）、小麦雪腐镰刀菌（Fusarium nivale (Fr) Ces.）的抑制作用;采用含毒平板法和HPLC法测定该菌株、无菌发酵液、细胞壁悬浮液、胞内液对玉米赤霉烯酮（ZEN）毒素的去除作用和高温以及蛋白酶处理对BMF04菌株去除ZEN毒素作用的影响,明确该菌株去除毒素的作用机理。结果表明,BMF04菌株及无菌发酵液对小麦赤霉病菌和小麦雪腐镰刀菌具有较强的抑菌作用;BMF04菌株对ZEN毒素具有较强的去除作用,当培养液中ZEN毒素浓度为15 μg/mL时,菌株活菌、无菌发酵液、细胞壁悬浮液和胞内液均对ZEN毒素具有较强的去除作用,去除率分别98.92%±0.07%、98.70%±0.19%、97.85%±0.07%和98.54%±0.10%,菌液在121 ℃条件下高温处理30 min,灭活菌液和灭活无菌发酵液对ZEN毒素的去除率明显下降,仅为60.32%±0.21%、2.09%±1.15%,胰蛋白酶、胃蛋白酶和蛋白酶K处理后,去除率分别下降到3.52%±0.77%、0.50%±0.39% 和0.18%±0.12%,说明高温灭活和蛋白酶对BMF04菌株发酵液去除ZEN毒素作用具有显著影响;菌株去除ZEN毒素既有胞外蛋白质降解作用也有细胞壁的吸附作用。研究结果为微生物去除污染粮食、饲料中的ZEN毒素提供了新的菌种资源。     

酱油渣中具有抑菌活性的乳酸菌的筛选及其抑菌特性

本研究以实验室前期从酱油渣中分离的16株乳酸菌ZW1~ZW16为出发菌株,拟筛选对大肠杆菌、金黄色葡萄球菌抑制效果较强的菌株,并对菌株产生的抑菌物质、生长特性及其影响因素进行分析。结果显示,乳酸菌ZW2、ZW9、ZW14对大肠杆菌、金黄色葡萄球菌均有较高的抑菌活性,其中ZW9抑菌效果最好,排除有机酸和H₂O₂影响后,胰蛋白酶、胃蛋白酶、蛋白酶K、α-蛋白酶、碱性蛋白酶、中性蛋白酶处理后抑菌活性均显著性降低（P<0.05）,初步推断抑菌物质是一种具有蛋白质属性的细菌素。抑菌动力学曲线显示,菌株培养至20 h抑菌活性趋于稳定。抑菌稳定性显示,抑菌物质在40~100 ℃具有较好的热稳定性,在紫外线照射、表面活性剂处理下均保持较高的抑菌活性;在pH2.0~6.0抑菌活性无明显变化,pH10.0抑菌能力完全丧失。酱油渣中筛选的3株乳酸菌具有抑菌功能且产生的抑菌物质稳定,对开发成新型生物抑菌剂具有重要参考价值。     

Acetylcholinesterase‐inhibitor hydrolysates obtained from ‘in vitro’ enzymatic hydrolysis of mannoproteins extracted from different strains of yeasts

In vitro inhibitory activity against acetylcholinesterase (AChE) of peptides obtained by enzymatic hydrolysis of mannoproteins extracted from strains of yeasts was investigated. Yeast mannoproteins were extracted from strains belonging to the genera Brettanomyces, Candida, Pichia and Saccharomyces isolated from dairy products. They were obtained by heat treatment in citrate buffer and purified by affinity chromatography with concanavalin A. Each purified extract was subsequently hydrolysed with proteolytic enzymes (trypsin, pepsin, chymotrypsin and proteinase K) applied individually or in combination, thus generating smaller peptides. Inhibitory activity of the latter against AChE was determined. The molecular weight of mannoproteins, determined by SDS‐PAGE, was between 6.5 and 30 kDa. As regards AChE inhibition, a preliminary screening of all hydrolysed extracts was performed, yielding variable results with 59% maximum inhibition. Subsequently, when inhibitory concentration 50 (IC50) was determined, the extracts showed higher inhibitory activity (between 6.75 and 12.3 mg mL^?1). Results showed that the mannoproteins separated from yeast strains of food origin generated bioactive peptides by enzymatic hydrolysis, which can be of interest to the manufacturing of food with potential functional properties.     

产宽谱pH 细菌素乳酸菌的筛选鉴定、毒力检测及细菌素特性研究

采用琼脂扩散法从分离自川西高原奶渣的34株疑似乳酸球菌中筛选细菌素产生菌,初筛检测发酵上清液抑菌活性,复筛排除有机酸和H2O2干扰,检测蛋白酶敏感性,测定抑菌物质盐析液和粗提液抑菌活性,确定菌株SAU-2为细菌素产生菌。根据形态、生理生化指标和16S rDNA序列系统发育分析,将其鉴定为Enterococcus。肠球菌SAU-2溶血素表型阴性,agg、gelE、cylM、cylB、cylA、esp、efaAfm、cpd、cob、ccf、cyILL、cyILS、fsrB和hyLEfm等毒力因子基因型阴性,表明肠球菌SAU-2是安全的。所产细菌素可耐受121℃条件20min;在pH2.0～12.0有抑菌活性;对胰蛋白酶和蛋白酶K敏感,对木瓜蛋白酶和胃蛋白酶不敏感;主要对近缘乳酸菌和G+细菌有抑菌活性,除对1株铜绿假单胞菌和1株红酵母有抑菌活性外,对其余G－细菌和真菌无抑制作用。     

产抗李斯特菌细菌素乳酸菌的筛选与鉴定

从51株肠球菌中筛选出2株具有抑制李斯特菌活性的肠球菌菌株:肠球菌KLDS6.0610和肠球菌KLDS6.0611。在排除有机酸和过氧化氢的干扰后,2株菌的无细胞发酵上清液对指示菌还有强烈的抑制作用。经蛋白酶K处理后,抑菌活性丧失,证明无细胞发酵上清液中含有蛋白性质的抑菌物质,即细菌素。经菌体形态和16SrDNA序列同源性分析鉴定2株肠球菌均为粪肠球菌。     

The effect of fungal competition on colonization of maize grain by Fusarium moniliforme, F. proliferatum and F. graminearum and on fumonisin B1 and zearalenone formation

The effect of water activity (0.98, 0.95, 0.93) and temperature (15, 25 degrees C) on fungal growth and toxin production from interactions between isolates of Fusarium moniliforme and F. proliferatum producing fumonisin, and an isolate of F. graminearum producing zearalenone, incubated at the same time on irradiated maize grains were determined in vitro. Populations (CFUs) of F. moniliforme and F. proliferatum were reduced to a greater or lesser extent by the presence of F. graminearum under all conditions tested, while that the presence of F. moniliforme or F. proliferatum had a minor inhibitory effect on fungal populations of F. graminearum. Fumonisin B, production by F. proliferatum was inhibited under all conditions tested, while fumonisin B1 production by F. moniliforme was inhibited at 15 degrees C and enhanced at 25 degrees C in the presence of F. graminearum. The level of zearalenone was not significantly modified in the presence of F. moniliforme and F. proliferatum under the conditions tested.     

Promising antifungal effect of some Euro-Asiatic plants against dangerous pathogenic and toxinogenic fungi

BACKGROUND: Increasing evidence of fungicide‐resistant toxinogenic and pathogenic fungal species is obvious. Looking for new possibilities of antifungal treatment or sources of antifungal substances is a major problem. Some medicinal plants exert strong antifungal properties and could be conveniently used as a promising alternative source for presently problematic antifungal treatment in many areas with respect to their natural origin. Methanol extracts of 46 medicinal plants from the Eurasian area were used in a screening assay for antifungal activity in this study. The growth inhibitory effect was tested against six significant pathogenic and toxinogenic fungal species: Fusarium oxysporum, F. verticillioides, Penicillium expansum, P. brevicompactum, Aspergillus flavus and A. fumigatus. RESULTS: For 14 plant species, the possibility of using them as natural fungicides was indicated. The extract from Grindelia camporum showed significant activity against all target fungal species. The most sensitive target fungus was the toxinogenic and human pathogenic species A. fumigatus. CONCLUSION: This study has identified 14 extracts of medicinal plants with a potential use as an antifungal treatment in various areas. One of them showed promising efficiency against all selected significant pathogenic and toxinogenic fungal species. Copyright © 2010 Society of Chemical Industry     

Antimicrobial activity and antibiotic resistance of lactic acid bacteria isolated from Armada cheese (a Spanish goats’ milk cheese)

Thirty-one lactic acid bacteria isolated from Armada cheese and previously selected in accordance with their technological properties, were screened for antimicrobial activity one against another. Four strains showed inhibitory activity against 14 strains when tested by well diffusion assay after the effects of organic acid and hydrogen peroxide were eliminated. Extracts of the strains did not show inhibitory activity after treatment with proteinase K, trypsin, α-chymotrypsin or pepsin. None of the strains showed antimicrobial activity against several pathogenic and spoilage reference strains. Eighteen representative strains were tested for their antibiotic resistance. None of the strains were totally susceptible to all antibiotics tested and multiple resistance was observed. Most of the tested strains were resistant to cefotoxin, oxacillin, vancomycin, teicoplanin, nitrofurantoin and trimethoprim.