Screening of multi enzyme producing strain and its application to tobacco aging

从进口烟叶表面分离筛选出5株产复合酶的菌株,其中菌株HY-2能产多种复合酶,且活性相对较高,通过生理生化及16S rDNA序列分析,鉴定该菌株为解淀粉芽孢杆菌(Bacillus amyloliquefaciens).利用该菌株配制成的生物制剂(编号MR-HY),分别采用回潮控温控湿及直接添加的方式在烟叶醇化中进行应用.结果表明,采用回潮控温控湿方式,生物制剂能在短时间内快速作用烟叶中组分,可使得烟叶糖氮比、糖碱比更趋于协调,品质得到一定的提升;采用直接添加方式也能有效的加速烟叶醇化过程,同时烟叶的香气量增加,香气质感变好,杂气及刺激降低.可见,通过添加该菌株配制的生物制剂能有效的改善烟叶醇化过程,有较好的应用空间.     

产香细菌的筛选及产香特性分析

醇化烟叶作为巨大的微生物库,具有极其可观的研究和应用价值。本研究应用模拟烟叶生长环境的烟叶浸提物培养基筛选产香细菌,并分析所筛选菌株发酵烟叶的产香特性,用于研究其在烟叶醇化中提升烟叶香味的可行性,同时探究该微生物用于生产天然香料的可能性。实验结果表明:通过烟叶浸提物培养基筛选出一株细菌HD-40,该菌可发酵烟叶可产生明显奶香味;经生理生化和分子生物学鉴定,初步表明该菌属于甲基营养型芽孢杆菌;进一步,利用该菌株发酵处理烟叶,并将发酵液萃取后GC-MS检测,与未经菌株处理的发酵液对比,其所含的酯类、醛类、酸类等对香气有贡献的成分明显增加,特别是其中3-羟基-2-丁酮的比例可高达60%以上。HD-40可发酵烟叶产生明显的奶香味,其不仅在烟叶醇化方面,用于提高烟叶香气、改善烟叶吸味等,还可在制备天然香料香精方面均有着很好的应用前景。     

烟叶微生物及其在烟叶发酵和醇化中的作用研究进展

微生物在烟叶发酵和醇化过程中具有十分重要的作用。本文综述了烟叶微生物概况及其在烟叶发酵和醇化中的应用和研究进展。主要介绍了烟叶微生物的区系划分、烟叶发酵和醇化过程中微生物动态变化以及外源添加微生物的应用方法。阐述了微生物在缩短烟叶发酵和醇化周期、改善烟叶品质、降低烟叶有害物质和提高烟叶安全性等方面的研究应用成果。最后,对该领域今后的研究方向提出了展望。     

产糖乳酸菌的筛选、鉴定及其产糖条件的优化

微生物胞外多糖是一类由微生物产生的,有一定加工性能和/或人体健康增益效果的高分子聚合物。为了筛选胞外多糖高产的菌株并提高其多糖产量,以多糖含量为衡量指标,首先通过比较实验筛选目标菌株,然后采用生理生化和分子生物学的方法对该菌株进行菌种鉴定,最后运用单因素分析和响应面实验确定该菌株发酵脱脂乳产糖的最佳条件。结果表明,菌株B6的产糖能力显著高于其他常规乳酸菌,经鉴定为鼠李糖乳杆菌（CGMCC No.13310）。该菌株最优的产糖条件为发酵时间41 h,发酵温度39 ℃,脱脂乳浓度91 mg/mL,接种量5%（体积分数）。在此条件下获得的发酵乳中多糖含量可达354.5 mg/L,比优化前提高35.8%。     

土壤中选育产壳聚糖酶菌株的研究

采用酶法降解壳聚糖具有反应条件易于控制、产物安全性高和环境污染少等独特的优越性。因此,筛选出高活力产壳聚糖酶菌株有着重要意义。该研究以不同地区采集土样分离出的1株细菌为出发菌株S,采用紫外线诱变（30W,20cm,5min）,经初筛和复筛及控温培养处理,获得了一株产壳聚糖酶较好的突变菌株,结果表明：所产酶活力达到3．47U/ml,酶活力提高近2倍,并具有较好的遗传稳定性,明显优于出发菌株,为发酵产壳聚糖酶的进一步研究提供了高产菌株。     

一种产邻苯二酚菌株高通量筛选方法

目的：对产邻苯二酚菌株进行筛选。方法：采用前期筛得的产邻苯二酚菌为出发菌株,通过硫酸二乙酯诱变的方法使该菌株突变,同时建立96孔板培养和酶标仪检测方法对产邻苯二酚菌株进行高通量筛选。结果：硫酸二乙酯的终浓度为0.1%,诱变时间为8 min的条件下,突变菌致死率为84.5%,突变效果最好。筛选培养基中吸光值（495nm）和富集培养基中菌液浊度值（OD630）的加和值较大的突变菌株产邻苯二酚能力高。通过诱变和筛选得到的菌株,产邻苯二酚浓度可达0.87mg/ml,比出发菌株的提高了262.5%。经过形态学和生理生化反应,初步鉴定该菌株属于假单胞菌属（Pseudomonas sp.）。结论：硫酸二乙酯诱变和96孔板筛选的方法能以高通量方式快速筛选出产邻苯二酚菌株。     

镉污染对烤烟光合特性、产量及其品质的影响

采用盆栽方法,研究了cd污染对烤烟叶片光合特性、烟叶品质及其产量的影响。结果表明,随着Cd处理浓度的增加,烟叶净光合速率（砌）和气孔导度（Gs）逐渐降低,胞间CO2浓度（Ci）随Cd浓度的增加逐渐增大;光系统Ⅱ活性（Fv/Fo）、最大光能转换效率（Fv／Fm）、光化学猝灭系数（qP）、非光化学猝灭系数（NPQ）、电子传递的量子产率（φps Ⅱ）和有效电子传递速率（ETR）则随Cd浓度的增加而降低;烤烟烟叶中的糖,碱比和氮,碱比升高,化学成分组成趋于不协调,不利于烟叶香吃味的形成。     

外源热稳定葡萄糖淀粉酶处理对初烤烟叶的影响

采用Thermomyces lanuginosus YNUCC4154生产的热稳定葡萄糖淀粉酶处理新鲜烟叶后进行烘烤.结果表明：处理后上部烟叶（B2K、B3F和B4F）的平均总糖和还原糖分别比对照增加2.8%和7.9%,中部烟叶（C3F、C4F和CX1K）的平均总糖和还原糖分别比对照增加20.9%和40.7%,但对不同部位和不同等级烟叶的作用效果不同.葡萄糖淀粉酶处理样在香气质方面较未处理样效果提升明显,上部低次烟叶的整体改善程度较其他部位的烟叶明显.     

等离子体-紫外线复合诱变选育高产谷胱甘肽酵母菌

以产还原型谷胱甘肽（glutathione,GSH）的啤酒酵母（Sacchromyces cerevisiae）SC-20为出发菌株,采用氩气等离子体射线、紫外照射及两者的复合诱变处理,获得一株高产GSH的酿酒酵母优良菌株（S．cerevisiae）DU-20．结果表明该菌株具有稳定的遗传性能,GSH含量与产量分别比出发菌株提高了78.33％和118．4％．     

醇化烟叶产蛋白酶菌株的分离鉴定及其对烟叶内在品质影响

目的从采自云南玉溪的醇化烤烟中分离得到15株产蛋白酶菌株,通过16SrRNA基因分析鉴定为芽胞杆菌属的菌株。方法为改善玉溪低次等烟叶的内在品质,利用分离到的15株产蛋白酶细菌进行发酵,发酵液经超滤制备得到的粗蛋白酶液,在室温条件下处理云南初烤烟叶,然后通过感官评吸进行评价。结果不同菌株来源的蛋白酶对烟叶感官质量改善效果不同,酶液P7、P9和P10可明显改善烟叶感官质量,烟叶香气质改善,杂气减轻,均衡性好;而酶液P5和P11则使烟叶的感官质量下降。结论研究为蛋白酶应用于烟草生产进行了有益的探索。     

不同酶制剂对烤烟上部叶化学成分、游离态和糖苷结合态中性香气成分的影响

为研究酶制剂对烤烟上部叶品质提升的影响,以中烟100为材料,采用烘烤前叶面喷施酶制剂的方法,分析了不同酶制剂对烤烟上部叶化学成分、游离态和糖苷结合态中性香气成分的影响。结果表明：不同酶制剂能不同程度的降低烤烟的蛋白质含量,改善烟叶化学成分,提高游离态和糖苷结合态中性香气成分的含量;喷施酶制剂对烟叶蛋白质、总糖、还原糖、总氮均有显著的影响,而对烤烟钾、氯的影响则未达到显著水平;混和喷施酶制剂相对于单独喷施酶制剂对烟叶品质的影响更显著。     

Application of exogenous substances reduces tobacco-specific nitrosamines content by regulating biosynthesis of nicotine and nitrite in burley tobacco

Tobacco-specific nitrosamines (TSNAs) are carcinogenic chemicals found in tobacco plants. The increasing health consciousness of individuals had led to an increased interest in research on reducing TSNAs content. The aim of this study was to use a pot experiment in which exogenous substances were applied to burley tobacco to dissect the mechanism of TSNAs production. The results indicated that spraying the exogenous substances IAA, NAA, SA and combination thereof on burley tobacco after topping decreased TSNAs content by 2.69–29.4 % in upper leaves and 0.23–39.3 % in middle leaves without affecting total sugar, total nitrogen, potassium and chlorine contents. The application of exogenous substances could down-regulate expression of the NR gene and the activity of the NR enzyme, resulting in less accumulation of the TSNAs precursor nitrite. The exogenous substances significantly reduced nicotine accumulation, which was consistent with low enzyme activities and the down-regulated expressions of genes involved in nicotine biosynthesis, especially significant in the case of quinolinate phosphoribosyltransferase. These results suggested that the application of exogenous substances on burley tobacco after topping could reduce TSNAs content which may be attributed to the regulation of exogenous substances on nitrite and nicotine. This also implies one potential improvement to agronomic practices aimed at controlling the accumulation of TSNAs in burley tobacco.     

Production of tannase from Aspergillus ruber under solid-state fermentation using jamun (Syzygium cumini) leaves

Tannase producing fungal strains were isolated from different locations including garbages, forests and orchards, etc. The strain giving maximum enzyme yield was identified to be Aspergillus ruber. Enzyme production was studied under solid state fermentation using different tannin rich substrates like ber leaves (Zyzyphus mauritiana), jamun leaves (Syzygium cumini), amla leaves (Phyllanthus emblica) and jawar leaves (Sorghum vulgaris). Jamun leaves were found to be the best substrate for enzyme production under solid-state fermentation (SSF). In SSF with jamun leaves, the maximum production of tannase was found to be at 30 °C after 96 h of incubation. Tap water was found to be the best moistening agent, with pH 5.5 in ratio of 1:2 (w/v) with substrate. Addition of carbon and nitrogen sources to the medium did not increase tannase production. Under optimum conditions as standardized here, the enzyme production was 69 U/g dry substrate. This is the first report on production of tannase by A. ruber, giving higher yield under SSF with agro-waste as the substrate.     

钙对烟草叶片热激忍耐和活性氧代谢的影响

热胁迫导致烟草叶片细胞膜系统显著受损,表现为SOD活性降低和MDA含量明显升高,叶片叶绿素含量下降,活性氧增加。10 mmol/L CaCl2溶液处理烟草幼苗后,能有效降低热胁迫下叶片细胞膜透性,维持较高的SOD和CAT等抗氧化酶活性,减缓 O-·2 形成和膜脂过氧化反应。研究结果表明,CaCl2处理提高了烟草叶片膜稳定性和膜保护酶活性,有利于保护细胞膜结构,降低高温对烟草幼苗的伤害。钙离子螯合剂EGTA能在一定程度上降低烟草叶片的抗热性。     

Field evaluation of resistance to pyriproxyfen in Bemisia tabaci (B biotype)

We determined effects of aerial sprays of the insect growth regulator pyriproxyfen on sweetpotato whitefly, Bemisia tabaci (Gennadius) (B biotype), in Arizona cotton (Gossypium spp.) fields. We measured survival for males and females from a susceptible strain and a laboratory-selected resistant strain, as well as for hybrid female progeny from crosses between the strains. Insects were exposed directly to pyriproxyfen sprays in the field or indirectly in the laboratory by rearing them on sprayed leaves collected from the field. In all tests, survival was higher for the resistant strain than the susceptible strain, but did not differ between sexes in each strain. Survival to the adult stage did not differ between eggs and nymphs directly exposed to sprays. For susceptible and hybrid individuals, survival was lower on leaves collected the day of spraying than on leaves collected 2 wk after spraying. In contrast, survival of resistant individuals did not differ based on the timing of exposure. Dominance of resistance to pyriproxyfen depended on the type of exposure. Resistance was partially or completely dominant in direct exposure bioassays and on leaves collected 2 wk after spraying (h > 0.6). Resistance was partially recessive on leaves collected the day of spraying (mean h = 0.34). Rapid evolution of resistance to pyriproxyfen could occur if individuals in field populations with traits similar to those of the laboratory-selected strain examined here were treated intensively with this insecticide.     

Moisture Diffusion within Shredded Tobacco Leaves

The mechanism of moisture transfer into shredded tobacco was investigated in connection with moistening process control in cigarette manufacturing. For shredded tobaccos of less than about 1.0 mm shred width, the moisture was sorbed mainly through the cut faces rather than through the leaf epidermis, indicating that the moisture diffusion within the leaf was the rate limiting step of moisture transfer. The moisture diffusion coefficient values within the cured tobacco leaf ranged from 8.80 × 10^?7 to 64.2 × 10^?7cm²-s^?1 depending on the type of tobacco, the shred width of tobacco and the temperature at sorption. But these values were not affected by the relative humidity at sorption. The entire moisture transfer into the shredded tobacco leaves was predictable with the diffusion theory.     

Nicotine degradation enhancement by <Emphasis Type="Italic">Pseudomonas stutzeri</Emphasis> ZCJ during aging process of tobacco leaves

Nicotine is a key harmful component of tobacco and cigarettes, and the development of low-nicotine cigarettes is of increasing importance in the market. The objectives of this study are to isolate native nicotine-degrading strains and evaluate their feasibility for nicotine reduction during the aging (or fermentation) of tobacco leaves. A novel nicotine-degrading strain was isolated and identified as Pseudomonas stutzeri ZCJ based on its 16S rDNA sequence and morphological-biochemical characteristics. In submerged cultures, P. stutzeri ZCJ could tolerate 4.5 g/L nicotine and completely degrade 1.5 g/L nicotine within 24 h at 37°C and pH 7.4. The addition of glucose (1 g/L) could improve nicotine degradation by P. stutzeri ZCJ in submerged cultures. After submerged culturing, the cell suspension of P. stutzeri ZCJ could be utilized to improve nicotine reduction in tobacco leaves during solid-state fermentation. The nicotine content of tobacco leaves decreased by as much as 32.24% after 7 days of solid-state fermentation by P. stutzeri ZCJ, suggesting the industrial application potential of the native strain to enhance nicotine degradation during the aging of tobacco leaves.     

外源植物生长调节物质对烟叶中腐胺和烟碱含量的影响

水培的烟草打顶和打顶后喷施腐胺（Put）,烟叶中Put和烟碱含量均增加,钾含量下降,Put含量与烟碱含量之间呈显著正相关;打顶喷施吲哚乙酸（IAA）和赤霉素（GA3）的烟叶中Put和烟碱含量下降,钾含量上升;喷施茉莉酸（JA）的烟碱含量提高,而Put含量变化不大;喷施脱落酸（ABA）和6-BA的叶中Put含量下降。