海洋源蛋白酶产生菌筛选及酶学特性的初步研究

从海洋源鱿鱼中筛选高产蛋白酶菌株。通过检测蛋白酶产生水解圈结合蛋白酶活性测定的方法筛选高产蛋白酶菌株,采用PCR技术对筛选菌株进行16S rRNA鉴定,并构建目的菌株的系统发育树,同时研究粗蛋白酶的酶学特性。结果表明:筛选得到的10株产蛋白酶活力较高的菌株,经鉴定分别属于芽孢杆菌属(Bacillus sp.)、普罗威登斯菌属(Providencia sp.)和假单胞菌属(Pseudomonas sp.)。其中SW5菌株产酶活性最高达257.67±2.44 U/mL,为甲基营养型芽孢杆菌(Bacillus methylotrophicus)。该菌株所产粗蛋白酶的酶学特性研究发现,其最适pH为8.0,最适温度为40℃,终离子浓度为1 mmol/L时Mn2+、Ba2+和Ca2+对该蛋白酶活性有较高的激活作用,而Fe~(2+)和Zn~(2+)能明显抑制该蛋白酶活性。     

天山一号冰川沉积层低温脂肪酶产生菌的筛选及初步鉴定

从新疆天山一号冰川西支尾部的底部沉积层中分离筛选出高产低温脂肪酶菌株,进行初步鉴定及产酶探讨。使用罗丹明-B、吐温80、维多利亚蓝选择培养基以及三丁酸甘油酯酶活检测板,结合棕榈酸对硝基苯酯(p-NPP)比色法,筛选出产低温脂肪酶酶活较高的菌株,研究其生长特性及常规生理生化实验,并根据16S rRNA基因序列初步确定其所属菌属。共分离筛选到5株产低温脂肪酶酶活较高的菌株,经初步鉴定其中4株为假单胞菌属(Pseudomonas sp.),1株为芽孢杆菌属(Bacillus sp.)。该芽孢杆菌属产脂肪酶的能力略高于其余菌株。分离得到的5株菌均属于耐冷菌,有较好的耐盐性,能在低温条件下发酵产酶,其中4株在36h左右达到产酶高峰,为开发冰川环境下丰富的低温酶资源奠定了基础。     

三株产几丁质酶菌株的筛选、产酶条件优化及水解虾壳的研究

目的:筛选鉴定产几丁质酶的菌株并优化其发酵条件,最终应用于虾壳降解研究。方法:以盐城市滩涂海泥为样品,利用平板筛选水解几丁质的菌株,运用生物信息学方法鉴定菌株,通过单因素优化其发酵条件,并将筛选得到的菌株和优化后的发酵条件用于虾壳发酵。结果:鉴定得到三株显著降解胶体几丁质的菌,分别是发光杆菌（Photobacterium sp. LYM-1）、需钠弧菌（Vibrio sp. WM-1）和希瓦氏菌（Shewanella sp. ZXY-1）;优化发酵条件:发光杆菌的碳源为几丁质10 g/L,氮源为NH₄Cl 2.0 g/L,接种量为3%,发酵液pH为6.5,温度为32 ℃,发酵1 d酶活最高为15.37±0.55 U/mL,是优化前的4.37倍;需钠弧菌的碳源为几丁质10 g/L,氮源为NH₄Cl 2.0 g/L,接种量为3%,发酵液pH为7.5,温度为22 ℃,发酵2 d酶活最高为40.82±6.03 U/mL,是优化前的1.60倍;希瓦氏菌的碳源为几丁质10 g/L,氮源为(NH₄)₂SO₄ 2.0 g/L,接种量为3%,发酵液pH为6.5,温度为22 ℃,发酵1 d酶活最高为25.64±3.29 U/mL,是优化前的2.47倍;三株菌均能利用虾壳产几丁质酶,但利用效率均低于几丁质,酶活力分别为10.25±0.95、32.16±2.25和21.81±4.27 U/mL。结论:本研究从盐碱地筛选得到三株产几丁质酶的菌株,优化后酶活力均得到提高,且均能利用虾壳产几丁质酶,为发酵虾壳制备几丁质酶提供新的菌株来源。     

植物源益生乳酸菌的筛选及其特性

为筛选具有益生特性的植物源乳酸菌,以传统发酵蔬菜中分离的1 000 株乳酸菌为出发菌株,进行了耐酸性、耐胆盐能力、抑菌性、体外抗氧化能力、药敏性、溶血性和氨基酸脱羧酶活性等特性研究,并对筛选菌株进行了16S rDNA 鉴定。经pH 3.0 MRS培养得乳酸菌82 株,再经pH 2.5 MRS培养得乳酸菌49 株;49 株菌经0.3%胆盐测试,均具有耐胆盐能力;根据镜检形态结合发酵植物源的不同从中挑选19 株乳酸菌进行药敏性、溶血性、抑菌性、氨基酸脱羧酶活性和1,1-二苯基-2-三硝基苯肼自由基清除实验。结果表明,19 株菌对所选20 种抗生素多数表现敏感,其中4 株菌对20 种抗生素都较敏感;19 株菌对供试致病菌都有不同程度抑制能力且都无溶血性;经氨基酸脱羧酶活性试剂盒结合聚合酶链式反应扩增检测表明,19 株菌无产生物胺的潜在威胁;有5 株菌体外抗氧化能力高于40%。可见19 株菌均具有益生菌的基本特性。经16S rDNA鉴定,7 株为发酵乳杆菌、6 株为植物乳杆菌,嫩江杆菌、戊糖片球菌、利莫西杆菌、戊糖乳杆菌、屎肠球菌、短乳杆菌分别各1 株。     

黄酒中不产生物胺乳酸菌的筛选及应用

为筛选不产生物胺的乳酸菌菌株,对黄酒发酵醪液及米浆水中的微生物进行培养及与分离纯化,发现3株菌不具有氨基酸脱羧酶活性,但具有一定的生物胺降解能力。使用黄酒发酵液培养该3株菌,发现在2.50%Vol的黄酒培养液中,3株菌能正常生长;在8.80%Vol黄酒培养液中,菌株5-4和8-3生长能力明显弱于14-2-1;在15.70%Vol黄酒培养液中,3株菌均受到严重抑制。菌株14-2-1生长速率最快,繁殖最旺,经鉴定为植物乳杆菌,同时菌株产酸能力较强,产酸速率也较快,表现出一定的优越性,应用于黄酒酿造中,能有效降低黄酒中生物胺的含量。     

一种海洋几丁质酶产生菌的筛选及酶学性质研究

海洋是自然界几丁质主要来源地，滋养出种类繁多可降解几丁质的微生物，因此从海洋中筛选产高活性几丁质酶的细菌，是获得高产几丁质酶微生物的有效途径。该文以胶体几丁质为唯一碳源，从渤海滩涂筛选到一株具有降解几丁质特性的细菌Y-8,对其进行鉴定并研究其几丁质酶酶学性质。Y-8菌株经分子鉴定为副溶血性弧菌(Vibrio parahaemolyticus),发酵上清液经SDS-PAGE及蛋白质谱分析，发现其存在2种几丁质酶，分别为Chi1几丁质外切酶，氨基酸残基数为848,理论分子质量为87.6 kDa; Chi2几丁质内切酶，氨基酸残基数为1 054,理论分子质量为112.9 kDa。Y-8所产生的几丁质酶能够高效降解胶体几丁质，获得单一产物N-乙酰氨基葡萄糖，其最适反应温度为55℃,45℃保温1 h,仍能保持70%以上活性；最适pH为6.0,在pH 4.0～9.0、37℃保温1 h后相对酶活性保留55%以上，具有较好的稳定性。10 mmol/L的Mn²⁺能使几丁质酶活性提高362%,而EDTA以及SDS、吐温-20、吐温-80对酶活力具有抑制作用。     

一株几丁质酶产生菌的筛选及产酶条件优化

从浙江省台州市剑门港海区采集海底淤泥样本,以几丁质作为惟一营养因子,筛选出5株几丁质酶产生菌,对其中一株产酶活性较高的菌株从形态学、生理生化、脂肪酸组成及含量、G+C含量、醌型及分子生物学特征进行了鉴定(gene Bank:HM136777)。结果表明,该菌属于慢生根瘤菌科(Bradyrhizobiaceae),芽生杆菌属(Blastobacter)中的一种,命名为Bradyrhizobiaceae blastobacter SYBC-H2。P-B实验结果显示,几丁质、葡萄糖及玉米浆粉是该菌株产几丁质酶的关键营养因子,利用中心组合设计实验(Contral Composite Design)对该菌株的产酶培养基进行了成分优化,结果表明,当培养基主要成分几丁质、葡萄糖和玉米浆粉质量浓度分别为2.7、0.85、2.64 g/L时,几丁质酶活性最高,达到5.70 U/m L。     

降解玉米秸秆产纤维素酶和木质素酶菌种的筛选

筛选能降解玉米秸秆产生纤维素酶和木质素酶的菌种,并测定纤维素酶系和木质素酶系的相关酶活.经过筛选得到10株菌株,经鉴定为:Penicillium sp、Altemaria sp、Aspergillus fumigatus、Aspergillus sp、Pestalotiopsis sp、Tricherderma sp、Cephalsporium sp、Pleurotus sp.酶活分析显示,筛选得到的10株菌都有CMC酶、滤纸酶、微晶纤维素酶、Mn过氧化氢酶、漆酶等酶活性;筛选得到的茵株都具有较全面的降解玉米秸秆所需要的酶系,具有较好的玉米秸秆生物降解的研究价值.     

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。     

TUV26菌株的几丁质酶动力学研究

TUV26是以Pseudomonas sp.为出发菌株,经诱变得到的一株几丁质酶高产菌。本实验以胶体几丁质为底物,对TUV26产几丁质酶的动力学进行研究,实验结果表明:该酶的最佳反应温度为48.5℃,最佳反应pH6.8;在0～60min反应速度为常数,反应初速度为54.199μg/ml·h;米氏常数Km=24.04μg/ml,最大反应速度Vmax=218.68μg/ml·h,酶转化数Kcat=0.08025h-1。这些研究结果为酶法生产几丁寡糖提供必要的工艺参数。     

产纤溶酶乳酸菌筛选及安全性评估

以从传统发酵食品中分离出的2株产纤溶酶的粪肠球菌HYD09与HYN06为对象,测定其发酵上清液酶活,对菌株进行安全性评估。选较安全的菌株进行胃肠道生存能力测试;同时对发酵上清液进行蛋白酶处理试验;从发酵液中获得粗酶液,进行活性电泳。结果表明:HYD09和HYN06发酵上清液酶活分别为115.3 U/mL和107.2 U/mL;HYD09和HYN06吲哚试验、硝酸盐还原酶试验、氨基脱羧酶试验、溶血试验均为阴性,对14种抗生素无多重耐药性;HYD09无毒力基因检出,HYN06检出esp毒力基因。证明HYD09菌株是安全的。进一步研究发现,菌株HYD09有一定的耐酸耐胆盐能力;HYD09发酵上清液经胃蛋白酶和胰蛋白酶处理仍具有纤溶活性;且菌株HYD09发酵上清液的粗酶中有2种主要的组分,但具有纤溶活性的组分只有1种。     

Characterization and technological properties of Staphylococcus xylosus strains isolated from a Tunisian traditional salted meat

The technological properties of strains of Staphylococcus xylosus were studied to select the most suitable for use as starter cultures for the production of dried fermented meat products. Strains of S. xylosus were isolated from traditional salted Tunisian meat and were identified by biochemical and molecular methods. Thirty strains of S. xylosus were studied to evaluate their catalase, nitrate reductase, lipolytic, proteolytic and antibacterial activities as well as growth ability at different temperatures, pH's and NaCl concentrations. All strains of S. xylosus had catalase activity and were able to reduce nitrates to nitrites. The nitrate reductase activity increased when the strains were kept under anaerobic conditions. Proteolytic activity on milk and on gelatin agar was demonstrated for 100% and 83.3% of the S. xylosus isolates, respectively. However extracellular proteolytic activity as assessed by the azocasein method was poor in all the strains. Lipolytic activity as assessed by the agar method showed that 76.6% of strains of S. xylosus could hydrolyze Tween 20 against 33.3% that could hydrolyze tributyrin. Tween 80 was hydrolyzed by only 10% of strains. Strains of S. xylosus hydrolyzed pork fat better than beef and lamb fat. The majority of strains had antibacterial activity against Salmonella arizonae, Staphylococcus aureus, Pseudomonas aeuroginosa, Escherichia coli and Enterococcus faecalis.     

The ability of biogenic amines and ammonia production by single bacterial cultures

The amino acid decarboxylating activity and production of biogenic amines, trimethylamine and ammonia by Morganella morganii (two strains), Klebsiella pneumoniae (three strains), Hafnia alvei (two strains), Enterococcus faecalis, Photobacterium phosphoreum, Micrococcus sp., Psychrobacter immobilis, Corynebacterium sp., Vibrio fischeri, Vibrio harveyi and Pseudomonas putida were investigated using a rapid HPLC method. In a laboratory medium containing amino acid (histidine, ornithine, lysine, tyrosine and arginine), not all bacterial strains produced the biogenic amines but most of them produced histamine, putrescine, cadaverine and ammonia. Cadaverine production by Klebsiella pneumoniae (8152), Klebsiella pneumoniae (673), Klebsiella pneumoniae (2122), Hafnia alvei (6578), Hafnia alvei (11999), Vibrio fischeri (25) Vibrio harveyi (42) and Pseudomonas putida (10936) was 531, 422, 532, 485, 472, 343, 547 and 343 mg/l, respectively in lysine decarboxylase broth. Tyramine was produced in highest concentration (526 mg/l) by Enterococcus faecalis (775). Agmatine was not produced apart from Psychrobacter immobilis (100) in an arginine decarboxylase broth.     

高抗氧化活性肉用乳杆菌的筛选鉴定及其在发酵香肠中的应用

目的 筛选高抗氧化活性肉用乳杆菌,并应用到发酵香肠中。方法 以肉品发酵剂标准、自由基清除率、耐过氧化氢能力和抗氧化酶活性为指标进行菌株筛选,经形态学、生理生化及16S rDNA对菌株进行鉴定,通过硫代巴比妥酸值、羰基和巯基值探究菌株对发酵香肠的抗氧化作用。结果 菌株CC-3符合硝酸盐还原酶阳性、氨基酸脱羧酶阴性等肉用发酵剂标准；该菌株具有较高的自由基清除能力,其发酵上清液DPPH.和OH.清除率分别为98.43%、42.75%,菌体细胞.清除率为75.56%；具有较高的抗氧化酶活性,其细胞破碎液T-SOD活力和CAT活力分别为45.17±0.29、58.50±0.28 U. mgprot_^-1,发酵上清液GSH-Px活力为98.16±0.65 U.mgprot_^-1。菌株CC-3鉴定为植物乳杆菌。接种菌株CC-3的发酵香肠与其他组比较在发酵结束时TBARS值和羰基值最低,分别为0.22±0.01、1.57±0.06 nmol.mg_^-1；巯基值最高,为0.05±0.01 nmol.g_^-1。结论 菌株CC-3符合肉用发酵剂标准且具有高抗氧化活性,可以抑制发酵香肠脂肪氧化和蛋白氧化,具有重要的开发潜力。     

Relationship between nitrate/nitrite reductase activities in meat associated staphylococci and nitrosylmyoglobin formation in a cured meat model system

Quantitative determination of catalase, nitrate reductase, nitrite reductase and nitric oxide synthase activities (NOS) was performed on 11 different bacterial strains, mainly staphylococci, isolated from fermented sausages, bacon brine or cured meat products. All except one strain possessed catalase activity in the range from 1.0 to 6.1 μmol min^− 1 ml^− 1. Ten out of 11 bacteria strains showed nitrate reductase activity in the range between 50 and 796 nmol min^− 1 ml^− 1 and nine showed nitrite reductase activity in the range between 6 and 42 nmol min^− 1 ml^− 1. No evidence of NOS activity of the selected strains was detected. In a colour formation assay containing myoglobin all strains affected nitrosylmyoglobin (MbFe^IINO) formation in assays containing nitrite, whereas only strains having nitrate reductase activity generated MbFe^IINO in assays containing nitrate as the sole nitrosylating agent. The quantitative nitrate and nitrite reductase activity did not fully explain or correlate well with the observed rate of formation of MbFe^IINO, which seemed to be more affected by the growth rate of the different strains. The mechanism of the reduction of nitrite into NO of strains not having nitrite reductase activity remains to be fully elucidated, but could be due to a dual-mode action of nitrate reductase capable of acting on nitrate.     

冷却猪肉中特定腐败菌的靶向筛选

根据有氧包装冷却猪肉中特定腐败菌的种类特点和致腐特性,以假单胞菌（Pseudomonas sp.）和肠杆菌（Enterobacteriaceae）为目标菌建立筛选模型,同时结合简单的生理生化鉴定确保筛选方向。以托盘包装冷却猪肉贮藏末期腐败菌为菌源,用铬天青（chromeazurol S,CAS）嗜铁素检测平板法,从假单胞菌CFC选择性培养基上分离的116 株菌中筛选到86 株产嗜铁素的腐败菌;用氨基酸脱羧酶检测管从双层结晶紫中性红胆盐葡萄糖琼脂（VRBDA）平板上分离的66 株菌中筛选到21 株产氨基酸脱羧酶腐败菌。采用根癌农杆菌（Agrobacteriumtumefaciens）为报告菌再从上述腐败菌中筛选出产N-酰基高丝氨酸内酯（N-acyl homoserine lactones,N-AHLs）的菌株（产嗜铁素的51 株,产氨基酸脱羧酶的19 株）。根据实验需要,选取其中部分菌株进行16S rDNA鉴定,最后得到产嗜铁素和AHLs的假单胞菌7 株,产氨基酸脱羧酶和AHLs的肠杆菌科菌4 株,为靶向抑菌防腐提供了研究基础。     

Technological and safety characteristics of Staphylococcaceae isolated from Spanish traditional dry-cured sausages

The aim of this study was to determine the technological properties (nitrate reductase, proteolytic and lipolytic activities; and the ability to grow at the temperature and pH values of fermenting sausage, and at high NaCl concentrations) and safety characteristics (amino acid decarboxylase and enterotoxigenic activities) of 38 strains of Staphylococcaceae (11 of Staphylococcus epidermidis, 15 of Staphylococcus equorum, 5 of Staphylococcus pasteuri and 7 of Staphylococcus saprophyticus) isolated from Androlla and Botillo, two Spanish traditional sausages, in order to evaluate their suitability as potential starter cultures in the manufacture of these sausages. Most strains were able to grow at 10 °C, in the presence of 10% and 15% NaCl and at pH values of 5.5 and 5.0, except for S. equorum strains, growth of which was reduced at these pH values. The proteolytic activity assessed by the agar plate method showed that 89.5% and 52.6% of the strains were able to hydrolyze sarcoplasmic and myofibrillar proteins, respectively. These results were not confirmed by electrophoretic assays as only 47.2% of the strains changed the SDS-PAGE profile of actin, myosin and/or sarcoplasmic protein extracts. The assessment of the lipolytic activity by titration showed that only 21.0% of the strains can hydrolyze pork fat to any extent; whereas the profiles of the freed fatty acids were different in the different strains. Most of the strains showed decarboxylase activity against histidine, lysine, ornithine and tyrosine, but the quantities of biogenic amines produced were in most cases <25 ppm and <5 ppm for putrescine and cadaverine, respectively. Only four strains (10.5%), of S. epidermidis, produced enterotoxin C.     

Characterization and expression analysis of a gene cluster for nitrate assimilation from the yeast Arxula adeninivorans

In Arxula adeninivorans nitrate assimilation is mediated by the combined actions of a nitrate transporter, a nitrate reductase and a nitrite reductase. Single‐copy genes for these activities (AYNT1, AYNR1, AYNI1, respectively) form a 9103 bp gene cluster localized on chromosome 2. The 3210 bp AYNI1 ORF codes for a protein of 1070 amino acids, which exhibits a high degree of identity to nitrite reductases from the yeasts Pichia anomala (58%), Hansenula polymorpha (58%) and Dekkera bruxellensis (54%). The second ORF (AYNR1, 2535 bp) encodes a nitrate reductase of 845 residues that shows significant (51%) identity to nitrate reductases of P. anomala and H. polymorpha. The third ORF in the cluster (AYNT1, 1518 bp) specifies a nitrate transporter with 506 amino acids, which is 46% identical to that of H. polymorpha. The three genes are independently expressed upon induction with NaNO₃. We quantitatively analysed the promoter activities by qRT–PCR and after fusing individual promoter fragments to the phytase (phyK) gene from Klebsiella sp. ASR1. The AYNI1 promoter was found to exhibit the highest activity, followed by the AYNT1 and AYNR1 elements. Direct measurements of nitrate and nitrite reductase activities performed after induction with NaNO₃ are compatible with these results. Both enzymes show optimal activity at around 42°C and near‐neutral pH, and require FAD as a co‐factor and NADPH as electron donor. Copyright © 2009 John Wiley & Sons, Ltd.     

Characterization of petroleum-degrading bacteria from oil-contaminated sites in Vietnam

Four petroleum-degrading bacterial strains, 2TN-NB, 6TBX-CL, MVK2-5, and XCK, were isolated from various oil-contaminated sites in Vietnam. Determination of the nucleotide sequence of the gene encoding 16S rRNA allowed 2TN-NB to be identified as Acinetobacter sp. and the other three stains as Pseudomonas sp. Among the four isolates, 2TN-NB was most effective in degrading crude oil: in 1 d, it degraded 95% of the crude oil in the culture medium (5%, v/v). The isolated strains could also degrade a sulfur-containing aromatic hydrocarbon, dibenzothiophene (DBT), with low efficiency. Except for MVK2-5, which degraded crude oil least efficiently, the isolates produced biosurfactants in amounts sufficient for structural analysis. FT-IR measurement suggested that strains 6TBX-CL and XCK produced glycolipid-type biosurfactants while that produced by 2TN-NB was of the polysaccharide type.