试验旨在实时监测豆粕发酵过程中抗营养因子的降解效果.以β-甘露聚糖酶高产菌株HTGC-10为发酵菌株,降解甘露聚糖,使用薄层层析及实时电泳检测菌株HTGC-10的降解效果.结果显示,菌株HTGC-10可将甘露聚糖降解为小分子的甘露寡糖;使用HTGC-10发酵10％的豆粕,在发酵的0～24 h内,菌株HTGC-10可以很好地降解豆粕中的大分子蛋白.在发酵的0～24 h内,菌株HTGC-10可以很好地降解抗营养因子,将豆粕中的大豆球蛋白、β-伴大豆球蛋白和KTI抗营养因子分别降解至原含量的11.9％、18.5％和2.0％,继续发酵24～48 h对豆粕抗营养因子的降解作用不大.研究表明,豆粕发酵以24 h为宜,可以更好地利用豆粕资源.

该研究在对六妹羊肚菌(Morchella sextelata)原生质体单核化再生菌株分离及交配型鉴定的基础上,对单交配型原生质体再生菌株进行对峙培养杂交实验制备杂交菌株.以菌株交配型、生长势、抗鞣酸能力和菌核产生能力为技术指标,对单核化再生菌株及杂交菌株的多态性进行分析,并对杂交菌株的交配型进行检测.结果表明,20株MAT1-1-1单交配型及17株MAT1-2-1原生质体再生菌株表现较好,其菌丝带宽度均≤10 mm,褐变圈直径分别为25～34 mm、23～34 mm;从63组杂交菌株中得到40株(占63.50％)双交配型(MAT1-2-1/MAT1-2-1)杂交菌株,杂交菌株的菌丝带宽度均≥10 mm,褐变圈直径为32～54 mm,杂交菌株的交配型电泳结果表明,成功获得40株具有双交配型的杂交菌株(两条扩增带).因此,双交配型杂交菌株在生长势、抗鞣酸能力方面存在杂交优势,而菌核产生能力与菌株交配型没有相关性.

该研究以采集的河南南阳魔芋种植土壤样品为试验材料,通过富集培养、测定产β-甘露聚糖酶酶活力,分离筛选高产β-甘露聚糖酶的菌株,并通过形态观察、内转录间隔区(ITS)基因和β-微管蛋白基因BenA序列分析对其进行菌种鉴定.结果表明,筛选得到一株高产β-甘露聚糖酶真菌菌株HKS016,其β-甘露聚糖酶酶活力为2.67 U/mL,该菌株被鉴定为黑曲霉(Aspergillus niger),保藏于中国普通微生物学菌种保藏中心(CGMCC),保藏编号为CGMCC No.22413.

为了改造黑曲霉(Aspergillus niger)来源的β-甘露聚糖酶ManA,获得耐热性高的突变体.利用易错聚合酶链式反应(PCR)技术对构建的表达质粒pGAPZαA-manA进行随机突变,将突变文库转化至毕赤酵母(Pichia pastoris)GS115,表达筛选耐热性高的突变体,并进一步对突变位点进行定点突变,筛选其他耐热性高的突变体.结果表明,ManA的H283R与H283K突变体相对于野生型在耐热性方面有显著提升,75℃加热30 min其相对酶活由26.66％分别提升至76.95％和83.57％;在pH 3.0～9.0的条件下,50℃保存24 h,H283K突变体与野生型的相对酶活均在90％以上,H283R突变体在pH 3.0时的相对酶活下降到了84.72％,两个突变体的最适温度、pH及比酶活与野生型没有明显差异.说明β-甘露聚糖酶ManA的283位组氨酸(H283)对其耐热性较为关键,将该位点突变为精氨酸(R)和赖氨酸(K)时,ManA的耐热性得到显著提升.

通过初筛和复筛从魔芋种植基地土壤样品中分离筛选高产β-甘露聚糖酶菌株.通过形态学观察、生理生化试验及16S rDNA序列分析对菌株进行鉴定,并对其产β-甘露聚糖酶酶学性质进行研究.结果表明,筛选出一株高产β-甘露聚糖酶的菌株,编号为HTGC-10,被鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens).菌株HTGC-10在30℃、180 r/min条件下液体发酵24 h后,发酵液β-甘露聚糖酶活力为61.75 U/mL.酶学性质的研究结果表明,该菌株所产酶的最适反应pH值为6.0,在中性偏酸环境下稳定性较好,属于偏酸性酶;最适反应温度为55℃,热稳定性相对较差;乙二胺四乙酸(EDTA)和金属离子Na+、K+、Mn2+、NH4+、Mg2+、Fe2+、Cu2+、Ca2+对酶活力均有不同程度的抑制作用,其中Cu2+对酶活力的抑制作用最显著.

从市售豆豉和保藏的固体发酵产纳豆激酶(NK)高活性的菌株中,筛选出5株诱变育种出发菌株.经紫外-亚硝基胍-Co60复合诱变后,获得5株液体发酵产NK高活性的菌株LJJ-1-13-12-4、PXDBJ-13-9-7-18、DNS-17-3-15-7、CBL-2-7-10-6和L-5-8-15,其产NK活性,分别为1846.15、1956.48、2184.32、1865.38和2095.23 U/mL,分别是诱变前出发菌株的1.62、1.81、1.98、1.61和1.62倍.高产纳豆激酶菌株的选育为纳豆激酶液体发酵的进一步工业化奠定了良好的基础.

用实验室复合诱变选育的5株产纳豆激酶正向突变菌株,通过随机组合混菌发酵试验,确定了混菌发酵产NK活性最高的菌株组合为CBL-2-7-10-6、DNS-17-3-15-7和L-5-8-15.随后经过一系列正交试验和单因素试验,确定了这3个突变菌株混合发酵的最佳发酵工艺:发酵培养基中葡萄糖质量分数为4％、大豆蛋白胨4％、MgSO40.04％、CaCl20.06％、K2HPO40.2％、KH2PO40.1％;CBL-2-7-10-6、DNS-17-3-15-7和L-5-8-15这3个突变株接种量分别为670、670和1000μL/100 mL;初始pH 7.0,发酵温度31℃,摇床转速160 r/min,发酵时间50 h.在该条件下,发酵NK酶活性可以达到4952.33 IU/mL,是本研究单菌株发酵最高酶活(DNS-17-3-15-7的NK活性2096.57 IU/mL)的2.36倍.

在体外培养的人非小细胞肺癌A549细胞、人胃腺癌BGC-823细胞、人结肠癌HCT 116细胞、人肝癌Hep 3B细胞、人宫颈癌Hela细胞中加入不同浓度的酵母异源表达的LZ-8或FIP-tvc,培养24 h后显微镜下观察细胞形态变化,并用CCK8试剂盒检测细胞存活率,以用来检测灵芝免疫调节蛋白LZ-8及云芝免疫调节蛋白FIP-tvc对几种癌细胞生长抑制能力.结果显示,LZ-8和FIP-tvc在5～10μg/mL浓度时使A549、BGC-823、Hela和Hep 3B细胞贴壁性变差,且CCK8结果显示前3种细胞的存活率有不同程度的降低(80％～95％存活率),而在20μg/mL浓度时FIP-tvc对Hep 3B细胞的存活率开始有明显抑制效果(89％存活率),但对HCT 116细胞的形态无影响,对其存活率也无抑制作用.

纳豆红曲胶囊是以保健红曲和纳豆冻干粉为原料制成的保健食品,不仅含有红曲的活性物质,如红曲色素、Monacolin K、麦角甾醇、γ-氨基丁酸等,还含有纳豆的活性成分如纳豆激酶、纳豆菌、维生素K2、异黄酮等,因此纳豆红曲胶囊具有传统红曲和纳豆的双重保健功效,具有降血脂、溶栓、抗心脑血管疾病、抗菌、抗癌等功能.本文对红曲和纳豆的主要活性成分及其保健功效作了简单的介绍,并对其发展前景进行了展望.

为了优化绣球菌液体菌种制备和栽培方法,对绣球菌液体菌种的发酵培养基pH和发酵时间进行了优化,并比较了不同接种量对发酵时间的影响.结果表明:绣球菌液体菌种发酵培养基的最佳pH为3.5,发酵13 d时生物量达到最大值,最佳接种比例为30 mL菌液/550 g固体栽培湿料,液体菌种接种至栽培袋4个月后每袋可收获鲜重100 g左右的绣球菌子实体.

To further ameliorate the cultivation characteristics and improve the yield of Sparassis crispa,the condition for preparation and regeneration of protoplasts from Sparassis crispa were preliminary studied.Factors including isotonic solutions,enzyme temperature,pH,enzymolysis time were optimized based on the regeneration amount.Results showed that 2％ lysozyme was used to dissolve the mycelium cell wall of mycelium of S.crispa,and the optimal condition for preparing protoplasts were 0.6 M sucrose as isotonic solution,30 ℃,pH 6.0,3 h.Under this condition,the regeneration amount was 11 850 per mL.

本研究从实验室保存的15株枯草芽孢杆菌中选育出适合鹰嘴豆(Cicer arietinum)发酵生产纳豆激酶(Nattokinase，NK)的优良菌株，并开展了菌株固体发酵产NK的工艺参数优化研究；NK酶活检测采用血纤维蛋白平板透明圈法。结果发现，培养基含水量、pH值、发酵温度及发酵时间均对鹰嘴豆发酵产生纳豆激酶有较大影响，培养基中添加适量的无机盐和表面活性剂有利于纳豆激酶的产生。在优化条件下（培养基含水量64%，PH7.0，31℃，培养36h），鹰嘴豆固态发酵产纳豆激酶的单位酶活可达到6731FU/g，获得了较高的单位酶活。 In this study, fifteen laboratory stored Bacillus subtilus strains were tested for their NK activity, and several parameters of the solid fermentation with chickpea (Cicer arietinum) media for NK production were optimized. The NK activity was decided with the transparent zones method on the fibrin-thrombin plate. The results showed that the NK secretion ability of strains was different; and the water content, pH value, cultural temperature and cultural duration have obviously influence on NK production. The optimum condition for NK production was 31 ˚C, pH 7.0, 64% water content and 36 hrs cultural duration and the addition of inorganic salts and surface active agents was helpful for NK production with chickpea media. At the optimum condition, the NK activity of the solid fermentation could reach 6731 FU/g.

Vectors pGAPZαA and pPICZαA were utilized to construct the multi-copies expression plasmid that can be linearized to maintain the transformation rate at a high level and have a higher yield of interesting protein than the single-copy plasmid.The gene of the interesting protein was originally constructed in the constitutiveexpression vector pGAPZαA,then the expression cassette is digested out with the isocaudamers Bgl Ⅱ and BamH Ⅰ and constructed in the methanol inducible expression vector pPICZαA at BamH Ⅰ site.The multi-copies expression plasmid was constructed after several times repeating.The LZ8 which is a fungal immunomodulatory protein from Lingzhi is used as an example.Its multi-copies expression plasmid pPICZαA-[GAPZαA-LZ8] 4 is constructed after repeating 4 times.Then the plasmid is linearized at the unique Sac Ⅰ site of pPICZαA and transformed in GS115.The yield of LZ8 expressed in the clones is transformed with pPICZαA-[GAPZαA-LZ8]4 reaches 2 or 3 times of that in the clones which is transformed with pGAPZαA-LZ8.We find the linearizable multi-copies expression plasmid bases on combination utilization of pGAPZαA and pPICZαA can express the interesting protein in GS115 at a higher level than the single-copy plasmid.

Deinococcus radiodurans is one of the most radiation-resistant organisms that was found on the earth,and has very high resistance to UV,drought,and mutagenic agents. D. radiodurans has drawn attentions from scientists around the world,and there are many reports about its radiation-resistant mechanisms. In this paper the recent progresses on radiation-resistant mechanisms of D. radiodurans are summarized from DNA-repair pathway,antioxidative defense system,special survival approaches,and cellular purification system. The development trends and future prospects of radiation-resistant mechanisms of D. radiodurans are also discussed,the aim of this paper is to provide some foundations for further study of radiation-resistant mechanisms of the bacteria.

In this paper,a strain named Adt was isolated from soil.The DNA sequences of 16S rDNA and F420-dependent glucose 6-phosphate dehydrogenase (FGD) were utilized to identify the strain.TLC and ELISA were analyzed for the degradation of coumarin and AFB1.The DNA sequence alignments of the Adt by 16S rDNA and FGD showed that it had high identity to Rhodococcus aetherivorans.The results of TLC and ELISA showed that the strain Adt could degrade coumarin and AFB 1,and a reductase belonged to FDR family which isolated from its genome was probably responsible for part of this activity.

This work aims to analyze the reason that resulted in the failure of cloning and to check if there are some microorganisms strongly degrading DNA in the environment of electrophoresis chamber. The colony morphology,gram staining and the 16S rDNA sequence were utilized to identify the strain degrading DNA,and agarose gel electrophoresis was for analyzing the DNA-degrading activity. As results, a strain was isolated from DNA electrophoresis chamber,and identified as gram-negative bacterium. Then it was cultured in YPD medium, and the capacity of the fermentation broth degrading DNA was detected using plasmid pUC19 as substrate. The strain quickly and sufficiently degraded DNA at the optimal temperature 45℃,thus this strain was designated DD(DNA degrading). The sequence alignment of the DD by the 16S rDNA showed that this DD had 100% identity with the that of Serrtia marcescens. Conclusively,the bacterial strain DD isolated from DNA electrophoresis chamber is a Serrtia marcescens strain possessing significant DNA-degrading activity based on the results of colony morphology,gram staining,and 16S rDNA sequence.

The objective of this work is to identify an isolate Td of yielding high nattokinase, and to analyze the molecular characteristics of its nattokinase. The morphologic, biochemical and physiological characterizations, and molecular biological methods were used to identify the taxonomic position of isolate Td ;mass spectrometric determination and analysis of MALDI-TOF, SDS-PAGE and fibrinolytic activity were employed to measure the characteristics of nattokinase, and PCR method was adopted to clone the full length of nattokinase gene. The bacterial strain Td was identified as Bacillus subtilis subsp. subtilis according to the results of morphologic, biochemical and physiological characterizations, as well as 16S rDNA, the sequences of 16S rDNA gyrA, hybrid rate of DNA-DNA. The yield of nattokinase produced by strain Td reached 300 mg/L, accounted for over 40% of the total protein. Fibrinolytic activity was over 230 U/mL. Amino acid sequence of nattokinase produced by Td showed the highest similarity to the subtilisin E. and the full length of the gene was 1143 bp. Conclusively, as a high-yield and high-activity strain for nattokinase, B. subtilis subsp. subtilis Td is a promising candidate for industrial development and utilization.

It was to assay theantioxidant activities of the Chickpea protein isolates(CPI)and its peptide fractionsin vitro. CPI were hydrolyzed with alcalase, and the hydrolysates were further separated into peptide fractions of <3 kD, 3-5 kD, 5-10 kD and >10 kD, respectively, using membrane ultrafiltration. Results showed that, the <3 kD peptides exhibited better ferric reducing power and radicals scavenging activities when compared to peptide fractions of higher molecular weights. CPI and its peptide fractions had significant ability to chelate metal ions compared to glutathione(GSH), and also had some ferric reducing powers. The remarkable antioxidant properties indicate that CPI and its peptide fractions have the potential to be used in manufacturing antioxidant functional foods and healthy foods.

为对比观察国产蛋白酶酶解和益生菌发酵降解鹰嘴豆浆液蛋白的效果,比较酶解法、发酵法降解鹰嘴豆浆液蛋白为小分子蛋白肽的能力,采用碱性蛋白酶、木瓜蛋白酶分别酶解,纳豆芽孢杆菌、植物乳酸菌、蛹虫草分别发酵鹰嘴豆浆液蛋白,测定不同处理时间时的可溶性氮、DH、电泳条带等的变化情况.研究结果发现,国产碱性蛋白酶酶解鹰嘴豆浆液蛋白240 min,上清液的可溶性氮含量可达到0.1389％,DH为6.43％;木瓜蛋白酶酶解240 min时的上清液可溶性氮含量达到0.0972％,DH为4.41％;而发酵法处理以纳豆芽孢杆菌效果最显著,纳豆芽孢杆菌发酵处理鹰嘴豆浆液蛋白8h时的上清液可溶性氮含量为0.0895％,DH为4.03％.本研究表明,酶解法降解鹰嘴豆浆液蛋白的效果要比微生物发酵法处理时间短,降解效果更好.

A bacterial strain(JQ-6)capable to degrade and metabolize formaldehyde as a sole carbon and energy sources was isolated from the collected activated sludge of leather wastewater treatment plants. The results of morpho-logical features,physiological and biochemical characters as well as 16S rDNA sequence analysis of the bacterium showed that,the JQ-6 strain was Pseudomonas putida. Single-factor and orthogonal experiments were employed to study the capability of degradation characteristics of strain JQ-6. The optimum conditions of formaldehyde degradation were as follow:formaldehyde concentration of 1000 mg/L,incubation temperature of 30℃,pH 6.0,rotating rate of 150 r/min,100 mL fresh medium seeded with 3 mL JQ-6 bacterial suspension. The formaldehyde degradation rate reached 70%within 24 h by this strain.