紫外诱变选育米曲霉高产蛋白酶菌株

以从自然发酵黄豆酱中筛选的5株野生米曲霉为供试菌株,以这些菌株产蛋白酶(酸、中、碱)、淀粉酶(α-淀粉酶、糖化酶)活力大小为评价标准,筛选出米曲霉K61作为诱变出发菌株。采用紫外诱变对米曲霉K61菌株进行改造,最终筛选出一株蛋白酶活力高且遗传性能稳定的突变株Y29。将米曲霉Y29菌株应用于黄豆酱的生产中,并与目前工业生产中广泛应用的米曲霉沪酿3.042菌株进行比较。性能实验结果表明米曲霉Y29菌株的蛋白酶(酸、中、碱)活力明显高于米曲霉沪酿3.042菌株,但α-淀粉酶、糖化酶、生长速度和孢子数这4个指标两者的差异并不显著;制酱品质试验结果表明,米曲霉Y29菌株的酱香更浓郁一些,氨基酸态氮含量达到0.77g/100mL,高于米曲霉沪酿3.042菌株,其它指标均符合国家标准GB2718-1996。     

高产Monacolin K纯种红曲培养条件的研究

本实验对已有的十株红曲霉菌株进行纯种制曲,筛选出一株生长良好、Monacolin K含量高、不产桔霉素、产色素能力强及糖化酶活力相对较高的红曲霉菌株.以大米为原材料,采用液固联合制曲法,通过单因素实验对纯种红曲的培养条件进行优化.结果表明,固体培养基初始含水量为45％,接种量为8％,变温培养红曲时,红曲霉M-7所得纯种红曲最佳,其Monacolin K含量高达243 μg/g,色价450 U/g,糖化酶活力4 289 U/g.与优化之前比较,Monacolin K含量提高了37.2％.     

液氮保存曲霉菌种的效果

本文报告了以蒸馏水、10%甘油和5%二甲基亚砜(DMSO)为保护剂,用液氮冻结法保存5种8株曲霉的效果,并检测了这些菌分别产生的亚甲基丁二酸、柠檬酸、蛋白酶和糖化酶的生理活性。这些菌在液氮气相(接近-150℃)中保存180天全部保持着生活能力,它们的培养特征和形态特征保留原来的形状。所测定的液氮保存8株菌种的生理活性,除两株糖化酶活力稍有降低外,其它菌株没有明显的变化。     

高产糖化酶菌株的筛选与诱变育种

从土壤及霉变淀粉质材料等样品中分离获得到产糖化酶活性较高的菌株L-3,经鉴定为一种紫红曲霉;通过紫外线和硫酸二乙酯分别进行诱变,得到4株性能良好的糖化酶变异株,其中菌株7性能最好,命名为M.F7。对该菌株进行发酵性能测试,结果表明该菌株糖化酶活力比原菌株提高了20.8%;经过继代培养,证明其性状能稳定遗传。通过正交试验,确定菌株7最佳培养温度为30℃,最适pH值为5.5～6.0,最适培养时间为5 d。     

生物合成γ-氨基丁酸曲霉菌株的选育、紫外诱变和发酵条件的研究

从自然发酵的红曲中分离筛选到1株曲霉菌株,其菌丝体可以谷氨酸钠为底物转化富集γ-氨基丁酸(GABA),经初步鉴定该菌株为米曲霉(Aspergillus oryzae)。以该菌株的孢子为对象进行紫外诱变,初筛得到8株产GABA活力不同的菌株,复筛后获1株GABA的产量较高的突变菌株As-8。菌株As-8转化富集GABA的适宜条件为:pH 6.0,以蔗糖为碳源,蛋白胨为唯一氮源或蛋白胨和牛肉膏为复合氮源。产生的GABA含量最高可达4.2g/L,并能稳定遗传。     

极端嗜盐菌冷冻干燥保藏研究

本文报道了不同浓度的海藻糖和蔗糖作为保护剂对盐生盐杆菌（Ｈａｌｏｂａｃｔｅｒｉｕｍｈａｌｏｂｉｕｍ）Ｒ＿１菌株冷冻干燥存活性的影响。结果表明：６％海藻糖,１２％蔗糖和１５％ＮａＣｌ组成的混合保护剂,是冷冻干燥保藏Ｒ＿１菌株极佳的保护剂。采用该保护刑冷冻干燥２５株极端嗜盐菌,于４℃保藏１４个月,经液体培养基和斜面培养基培养检测,全部存活。     

携多拷贝glaA的重组黑曲霉过量合成糖化酶的研究

以工业生产菌株黑曲霉CICIMF0410基因组DNA为模板,扩增出糖化酶glaA基因,测序并进行表达研究。GlaA基因的核苷酸序列长为2167bp,包含4个内含子。氨基酸序列比对表明此黑曲霉糖化酶与其他曲霉属来源的糖化酶有很高的同源性。将glaA基因克隆到pBC-Hygro载体中,构建重组质粒pBC-Hygro-glaA并转化A.nigerF0410。携多拷贝glaA的转化子用150μg/mL潮霉素抗性筛选并通过荧光实时定量PCR鉴定。结果表明,在染色体整合2~3倍糖化酶基因对糖化酶的过量合成是适宜的,有助于提高糖化酶活力。对转化子进行摇瓶发酵研究,发酵终止时转化子GB0506的糖化酶活力比出发菌株F0410提高了17.5%。因此,增加黑曲霉染色体糖化酶基因的拷贝数可以显著提高糖化酶活力。     

真空冷冻干燥法保存菌种

我们测验了在室温(波动为5—36℃)贮藏4年半一8年,用脱脂牛奶作保护剂,真空冷冻干燥保存的559种20lo株细菌、放线菌、酵母菌、丝状真菌的存活力,以及其中部分菌株的主要生理特性。贮存在室温的真空冷冻干燥法保存的84种484株细菌,180种565株放线菌,59种459株酵母菌和236种502株丝状真菌的存活力分别是467(96·5％)、516(91．33％)、394(85．83％)和439(87·45％)。我们还观测了贮藏在5℃,1957年真空冷冻干燥法保存的细菌。在这种条件下保存16年的32种126株细菌,103株(81．74％)保持着活力,除个别菌株外,没有任何种的死亡。所测定的17株真空冷冻干燥保存的菌种的生理特性,与定期移植保存的同一菌株对比结果,除两株大肠杆菌,其中l株谷氨酸脱羧酶活力减退外,其他菌株都没有明显的变化。     

黑曲霉变异株UV-11-48的选育及产糖化酶条件的研究

以黑曲霉(Aspergillus niger)变异株UV-11为出发菌株,经亚硝基胍多次诱变,获得一株比亲株产糖化酶活力提高30%左右的变异株UV-11-48。对UV-11-48菌株进行了发酵条件及酶系组成等的研究。固体曲生产性试验,种曲糖化酶活力最高达12240u/g,麸曲酶活力平均8000—9000u/g,最高达11700u/g,在酿酒工业固体曲生产中,取得明显经济效益。     

中性蛋白酶高产菌株的筛选及产酶酶系分析

目的:满足水产中对中性蛋白酶的需求。方法:以实验室保藏的米曲霉ZW为出发菌株,经Co60定向诱变,通过透明圈法初筛、摇瓶发酵复筛,筛选中性蛋白酶活力高的菌株,并对其进行产酶酶系分析。结果:筛选到的米曲霉ZW-06产中性蛋白酶酶活可达15000U/g干曲,比诱变前酶活提高了74%,是目前国内报道的固体发酵产中性蛋白酶活力最高的菌株;经过10代传代之后,酶活力仍保持稳定。通过对米曲霉ZW-06进行产酶酶系分析,发现发酵产物中除了有较高的中性蛋白酶酶活,还有较高的木聚糖酶和酸性纤维素酶酶活,酶活分别达到49879U/g干曲和21099U/g干曲。结论:米曲霉ZW-06在饲料工业中有很大     

Fermentation Kinetics of Media Optimization for the Production of Alpha Amylase by a New Isolate of Aspergillus Oryzae

The starch-degrading enzyme‘α-amylase’is widelydistributedin nature .This extracellular enzyme randomlyhydrolyzesα1~4 glucosidic linkage throughout the starchmolecule in an endo-fashion producing oligosaccharidesand monosaccharides including maltose ,…     

Impact of freeze drying on drug resistance pattern of few Escherichia coli strains

Drug resistant strains of E. coli were freeze dried for long term preservation. Certain drug resistance markers were maintained after freeze drying while others were not. Streptomycin and sulphonamide resistance markers resisted freeze drying. Ampicillin, cephelaxin and neomycin resistances developed very frequently and except gentamicin all the markers were lost in varying percentage after freeze drying.     

Screening of freeze-dried protective agents for the formulation of biocontrol strains, Bacillus cereus AR156, Burkholderia vietnamiensis B418 and Pantoea agglomerans 2Re40

Aims: The effects of different freeze‐drying protective agents on the viabilities of biocontrol strains Bacillus cereus AR156, Burkholderia vietnamiensis B418 and Pantoea agglomerans 2Re40 were investigated. Method and Results: Several concentrations of protective and rehydration media were tested to improve the survival of biocontrol agents after freeze‐drying. The subsequent survival rates during storage and rehydration media of freeze‐dried biocontrol strains were also examined. Conclusions: The results indicated that cellobiose (5%) and d ‐galactose (5%) gave maximum viability of strains Bu. vietnamiensis B418 and P. agglomerans 2Re40 (98 and 54·3% respectively) while the perfect one (100%) of strain B. cereus AR156 was obtained with sucrose (5%) during freeze‐drying, and the highest survival of the three strains was reached when they were rehydrated with 10% nonfat skim milk. In the following storage, the survival rates showed that B. cereus AR156 could still reach 50% after 12 months. Significance and Impact of the study: This study showed that freeze‐drying could be used to stabilize cells of these three biocontrol strains. Further studies should focus on the scale‐up possibilities and formulation development.     

Ultraviolet induced mutations in Aspergillus wentii.

Aspergillus wentii (IMI 17295) and its three nutritional mutant strains were irradiated with UV rays. New mutants obtained differed from the parent strains in colour of the conidia, growth factor requirements and amylase activity. Arginine deficient strains showed greater amylase activity.     

Effect of culture medium and cryoprotectants on the growth and survival of probiotic lactobacilli during freeze drying

Aims:  To investigate the effects of the medium and cryoprotective agents used on the growth and survival of Lactobacillus plantarum and Lactobacillus rhamnosus GG during freeze drying.
Methods and Results:  A complex medium was developed consisting primarily of glucose, yeast extract and vegetable-derived peptone. Trehalose, sucrose and sorbitol were examined for their ability to protect the cells during freeze drying. Using standardized amount of cells and the optimized freeze drying media, the effect of the growth medium on cell survival during freeze drying was investigated. The results showed that glucose and yeast extract were the most important growth factors, while sucrose offered better protection than trehalose and sorbitol during freeze drying. When the cells were grown under carbon limiting conditions, their survival during freeze drying was significantly decreased.
Conclusions:  A clear relationship was observed between cell growth and the ability of the cells to survive during the freeze drying process.
Significance and Impact of the Study:  The survival of probiotic strains during freeze drying was shown to be dependent on the cryoprotectant used and the growth medium.     

Differentiation of <Emphasis Type="Italic">Aspergillus niger</Emphasis> by random amplification of polymorphic DNA

The randomly amplified polymorphic DNA (RAPD) patterns of whole-cell lysates from five Aspergillus niger isolates, including one reference strain, two isolated from deep freeze, and two environmental strains from soil and plant infections, were investigated. PCR-RAPD analysis of genomic DNA was performed using eight primers (Tube-A1, Tube-A6, Tube-A17, Tube-B8, Tube-B11, Tube-B15, Tube-C5, Tube-C6). The RAPD assay discriminated between all strains. Comparison of deep freeze isolates showed identical RAPD patterns in some of the reference and environmental isolates. The data indicates that the RAPD technique is useful for fingerprinting A. niger.     

Screening of marine actinobacteria for amylase enzymes inhibitors

Amylase inhibitor producing actinobacteria were isolated and characterized from terrestrial environment and there is no much report found from marine environment, hence in the present study, 17 strains isolated from the rhizosphere sediments of mangroves were tested for their amylase inhibition ability. Seawater requirement test for the growth of actinobacteria found that the strains SSR-3, SSR-12 and SSR-16 requires at least 50% and SSR-6 requires at least 25% seawater for their growth. The inhibition activity of both prokaryotic and eukaryotic amylase was tested by using Bacillus subtilis and Aspergillus niger. The maximum amylase activity (40mm) produced by the A. niger was taken as positive control, when the test actinobacteria strains grown in the medium they inhibited amylase activity and was evidenced by the reduction in inhibition zone (14–37 mm) similarly the amylase produced by the Bacillus subtilis was also recorded maximum (35 mm) amylase activity and was taken as positive control, and the test atinobacterial strains reduced enzyme action(12–33 mm) it varied levals. This indicates that the actinobacteria strains were controlled amylase enzyme activity in both the cases. The strain SSR-10 was highly effective and SSR-8 was less effective in inhibiting eukaryotic amylase produced by A. niger. The strain SSR-2 was effective and SSR-6 showed very less effect in inhibiting the prokaryotic amylase produced by the B subtilis.     

Extracellular enzyme production by haploids,heterocaryons and diploids of Aspergillus nidulans

Summary Extracellular amylase, lipase and protease produced by haploids, diploids and heterocaryons of Aspergillus nidulans were analysed. Three morphologically normal strains and 8 morphologic mutants as well as various genetic combinations of the 11 strains were examined in solid culture medium containing specific substrates. The enzyme production of each strain was determined by measuring the halo around the colony. It was observed that the colonies showing less growth also showed more alterations in enzyme production. The compact strains (BVIII and B6) and the slow-growing heterocaryons (pp+M32 and pp+M35) showed the highest enzymatic index for the three enzymes simultaneously. If colony growth is not considered, then for amylase and protease the highest values were reached by some diploid and heterocaryons and for lipase by one morphological strain. The results showed that morphological mutants and some combinations could be used for higher production of amylase, lipase and protease.     

Effects of pericarp alpha-amylase activity on wheat (Triticum aestivum) Hagberg falling number

Pericarp alpha‐amylase activity in wheat has largely been ignored as a potential cause of low Hagberg falling number (HFN) in the UK, because pericarp alpha‐amylase enzymes have been thought too temperature‐sensitive to affect HFN and are usually degraded by harvest. Nevertheless, there are several circumstances where immature grains may be present in harvested grain. Therefore, we tested the hypotheses that pericarp alpha‐amylase enzymes in immature grains could lower HFN, but also that inactivation of the enzymes during drying and storage could ameliorate the effect. The first hypothesis was tested by addition of large amounts of pericarp alpha‐amylase activity from freeze‐dried, defrosted or fresh green grains to high HFN flours of low alpha‐amylase activity, followed by HFN measurement. Enzyme assay and HFN analysis assessed the effect of drying on pericarp alpha‐amylase activity after different storage treatments. Addition of pericarp alpha‐amylase, from all three sources, to high HFN flours caused an exponential decrease in HFN. A drop in HFN from over 470 s to below 250 s resulted from addition of 5–10% by weight of green grains. Between 35–70% of the α‐AMY‐2 activity in immature grains was eliminated by air‐drying at 20°C over 8–10 days, although the residual activity could still lower HFN. Thus, pericarp alpha‐amylase activity may be a cause of low HFN in some UK wheat crops.     

Survival of Freeze Dried Bacterial Cultures

The survival of 100 strains of bacteria, representing 15 genera, on freeze drying and during storage in the freeze dried state for 10 years has been assessed. Gram-positive organisms tended to survive better than Gram-negative. Viable organisms were recovered from all but one of the cultures.