弗氏柠檬酸杆菌富硒发酵工艺优化

纳米硒作为一种安全高效的材料，在食品、医药、半导体等领域受到广泛应用。由于生物相容性高的优点，生物合成纳米硒也成为研究热点。从福建省富硒土壤中筛选出一株具有较高还原亚硒酸钠能力的菌株，转化率48 h高达75%，并对该菌株进行理化性质等分析，结果表明该菌为弗氏柠檬酸杆菌(Citrobacter freundii)。为增加菌丝生物量，提高转化率，以柠檬酸杆菌光密度为指标，通过单因素试验和Box-Behnken试验，对菌株的培养工艺进行优化。结果表明，最佳培养条件为酵母提取物24.49 g/L、酪蛋白胨13.15 g/L、K₃PO₄ 1 g/L、NaCl 10 g/L、pH6.4，在此优化条件下，菌丝生物量达到1.51 g/L，转化速率提高2.40倍，24 h可转化89%的亚硒酸钠。     

乳酸菌LA4还原亚硒酸钠形成红色单质硒

通过从实验室保藏的乳酸菌中筛选出1株对亚硒酸钠耐受性较高的菌株LA4,经分光光度法测定得知在69.28 mg(4 mmol/L)亚硒酸钠浓度下,亚硒酸钠的还原量为32.42 mg(1.87 mmol/L),还原效率为46.79%。并将其亚硒酸钠还原产物通过X射线光电子能谱仪(XPS)和透射电子显微镜(TEM)测试,发现乳酸菌LA4能将亚硒酸钠还原为红色单质硒,观察透射电镜(TEM)图发现,添加亚硒酸钠的发酵液中细胞表面以及培养基中出现大量纳米尺寸的球状颗粒,粒径基本在50²00 nm。     

纳米硒合成细菌Lxz-41的鉴定、培养条件优化及其在富硒猕猴桃栽培中的应用

本实验利用16S rDNA测序分析和Biolog GenⅢ细菌鉴定板对一株具有纳米硒合成功能的细菌lxz-41进行鉴定,通过单因素和响应面试验优化了该菌株以亚硒酸钠为原料合成纳米硒的培养基组分和培养条件,并将合成的纳米硒在猕猴桃栽培中进行初步应用实验。结果表明:lxz-41菌株被鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens),且对亚硒酸钠具有较高的耐受性。该菌株合成纳米硒的最佳条件为蔗糖4.5%,尿素0.1%,酵母粉1%,磷酸氢二钾0.3%,磷酸二氢钾0.1%,氯化钠0.5%,亚硒酸钠0.15%,初始pH为7.0,培养温度40.5 ℃,转速160 r/min,在此条件下培养18 h,纳米硒的最高转化率为86.8%±4.7%。将该纳米硒应用在猕猴桃栽培中确实增加了果肉中硒的含量,翠香品种果肉中硒含量达到0.050 mg/kg,华优品种果肉中硒含量达到0.035 mg/kg,且富硒效果要好于亚硒酸钠。本实验为微生物源纳米硒的制备及富硒作物的栽培提供了新的途径和技术依据。     

富硒益生菌的筛选及其富硒条件的优化

为了获得能耐受较高亚硒酸钠质量浓度和具有富硒能力的益生菌菌株,对7株酵母菌和12株乳酸菌进行了筛选。结果表明,所有菌株均能在亚硒酸钠质量浓度为20~80μg/m L的平板上生长,乳酸菌YQRS菌株和酵母菌FJYJM3菌株具有较高的耐受性和富硒能力。对它们进行发酵条件的优化,表明YQRS菌株在亚硒酸钠添加量为15μg/m L、添加硒的时间为对数期前期时,富硒效果最好,菌体生物量为2.66 g/L,总硒含量能达到2 300.26μg/L。而FJYJM3菌株在亚硒酸钠添加量为20μg/m L,添加硒的时间为对数期前期时,富硒效果最显著,生物量能达到4.86 g/L,总硒含量能达到5 790.99μg/L。     

富硒短小芽孢杆菌D1-019的筛选与鉴定

将无机硒耐受性筛选和“红硒法”筛选相结合,经过4 轮筛选,从前期分离的119 株来源于鸭食的菌株中,筛选到1 株富硒菌株D1-019。基于菌落形态观察、革兰氏染色、16S rDNA序列分析和系统发育树分析,D1-019菌株被鉴定为益生菌短小芽孢杆菌。采用正交试验设计方法,获得的最佳富硒条件为培养基初始pH 5,30 ℃培养48 h,培养6 h时加入质量浓度为20 μg/mL的亚硒酸钠。在该富硒条件下,短小芽孢杆菌D1-019对培养基中亚硒酸钠的转化率为100%,菌体有机硒含量为（1 327±113）mg/kg,优于已报道富硒微生物。结果表明,短小芽孢杆菌D1-019有潜力开发成为一个可应用于食品领域的新富硒益生菌源。     

常压室温等离子体诱变选育富硒纳豆芽孢杆菌

纳豆芽孢杆菌可以转化亚硒酸钠为有机硒。对一株纳豆芽孢杆菌的生长曲线进行测定,确定了亚硒酸钠适宜的添加时间和添加量。以纳豆芽孢杆菌BSN424为出发菌株,采用常压室温等离子体诱变系统进行诱变,根据耐硒和富硒能力筛选,经连续传代培养后筛选出了富硒纳豆芽孢杆菌。结果表明,适宜加硒时间为培养后3 h,培养时间为24 h,培养基适宜硒质量浓度为6μg/mL,常压室温等离子体诱变系统功率为100 W,诱变时间为25 s。诱变后筛选得到一株具有较高富硒能力的诱变菌株BN-44,经摇瓶发酵后的富硒量为1136.43μg/g,相比出发菌株的742.12μg/g提高了53.13%。研究表明常压室温等离子体诱变育种能有效地对纳豆芽孢杆菌BSN424进行诱变,旨在为有机硒生物转化法中寻找益生菌富硒载体及其诱变育种提供一定依据。     

保加利亚乳杆菌制备有机纳米硒的研究

利用保加利亚乳杆菌制备有机纳米硒。对保加利亚乳杆菌进行筛选和鉴定。在分析亚硒酸钠浓度、培养温度、培养时间、保加利亚乳杆菌接种量和起始培养基pH的单因素试验基础上,以硒含量为优化指标,采用正交试验优化有机纳米硒的制备工艺。结果表明最佳制备工艺为亚硒酸钠浓度0.6 mg/mL、培养温度46 ℃、培养时间60 h、保加利亚乳杆菌接种量6%和起始培养基pH值为6.2,此条件下产品的硒含量为68.23%。有机纳米硒呈现规则颗粒状,表面较为光滑,粒径为300~400 nm。     

不同红曲菌菌株耐硒能力和富硒能力的初步研究

为比较不同红曲菌菌株的耐硒能力和富硒能力，以5株红曲菌菌株为研究对象，通过接种于亚硒酸钠浓度为0、10、20、50、100、200μg/mL的PDA培养基中培养，测量其生长曲线和菌丝体中硒含量和总硒产量。结果显示：不同亚硒酸钠浓度对红曲菌菌株生长的影响与硒的富集均存在差异，其中耐硒能力最强的菌株为紫色红曲菌CICC 5046，其在亚硒酸钠浓度为50μg/mL的PDA培养基平板上培养15 d后菌落直径可达(53.8±1.5)mm；富硒能力最强的菌株为红色红曲菌M7，其在亚硒酸钠浓度为50μg/mL的PDA培养基平板(25 mL培养基)上培养15 d后总硒产量达到最高，为(124.43±2.01)μg；而当PDA培养基中亚硒酸钠浓度为200μg/mL时，红色红曲菌M7菌丝体中的硒含量达到最高，为(3120.59±193.63)μg/g。因此，不同红曲菌对亚硒酸钠的耐受能力和富集能力均不同，这种差异性可能与菌株的基因型或其抗氧化能力有关。     

耐硒菌株Cytobacillus firmus N4的筛选及其对α-葡萄糖苷酶抑制活性的影响

微生物制备纳米硒具有低毒、温和等优点,具有广阔的应用前景。从武汉施加硒肥的土壤中分离纯化得到一株能将亚硒酸钠还原成纳米硒的菌株,对其进行菌种鉴定,测定其耐硒能力和生长动力学曲线,利用扫描电镜对制备的纳米硒颗粒进行表征,并将转化得到的纳米硒及菌株次生代谢产物对α-葡萄糖苷酶的抑制活性进行初步探究。结果表明,在分离纯化的16株菌株中,坚强芽孢杆菌N4（Cytobacillus firmus N4）对亚硒酸钠的最大耐受性为36 mg/mL,产生的纳米硒粒径为150~220 nm。纳米硒质量浓度为0.625 mg/mL时对α-葡萄糖苷酶的抑制率为25.42%±0.69%;次生代谢产物质量浓度为2.500 mg/mL时,抑制率达到73.83%±4.63%。Cytobacillus firmus N4具有高耐硒性质,可为纳米硒的生物转化提供基础,且其次生代谢产物有一定的α-葡萄糖苷酶抑制活性,值得进一步深入研究。     

烟草Rubisco还原亚硒酸钠制备纳米硒

谷胱甘肽为含半胱氨酸的小肽,已知它能还原亚硒酸钠形成零价硒。蛋白质为含半胱氨酸大分子化合物,烟草1,5二磷酸核酮糖羧化酶(Rubisco)蛋白富含半胱氨酸,推测烟草Rubisco与亚硒酸钠反应也能形成零价硒。对比谷胱甘肽和烟草Rubisco形成的零价硒的区别,研究发现,谷胱甘肽还原亚硒酸钠形成浑浊态红色零价硒,不稳定,易变为黑色零价硒沉淀,从而失去生物活性;烟草Rubiscon能还原亚硒酸钠成纳米尺寸零价硒,稳定,不易变为黑色零价硒沉淀,具有生物活性,能降低癌症化疗药阿霉素的心脏毒性。     

乳酸菌富硒及产胞外多糖条件的研究

试验以实验室保藏的6株乳酸菌为研究对象,筛选出最佳试验条件下富硒率和产胞外多糖含量最优的菌株,以期为生产富硒调味品提供一定的理论依据.通过测定其生长曲线确定各菌的加硒时间,对比6株乳酸菌在不同亚硒酸钠质量浓度下的颜色变化、富硒率变化和胞外多糖含量变化,选定鼠李糖乳杆菌作为最佳试验菌株;通过测定鼠李糖乳杆菌在不同培养时间...     

Factors affecting soluble selenium removal by a selenate-reducing bacterium Bacillus sp. SF-1

High concentrations of soluble selenium, selenate and selenite, have acute and chronic toxicity toward living things. With the aim of developing a biological process for selenium removal, the effects of a variety of parameters on the reduction of soluble selenium by a Bacillus sp. strain SF-1, which is capable of reductively transforming selenate into selenite and, subsequently, into nontoxic insoluble elemental selenium, were studied. The bacterial strain could effectively reduce 20 mM of selenate to selenite and 2 mM of selenite to elemental selenium in the presence of an appropriate carbon source and in the absence of oxygen. The reduction rate of selenate to selenite was much higher than that of selenite to elemental selenium, resulting in the transient accumulation of selenite during selenate reduction. The selenate reduction rate increased with increases in the selenate concentration up to 20 mM, while the rate of selenite reduction decreased sharply at selenite concentrations of more than 2 mM. The elemental selenium transformed from selenate via selenite was found both inside and outside the cells. Bacillus sp. SF-1 was able to utilize a variety of organic acids or sugars as a carbon source in selenate reduction. Although the copresence of sulfate did not inhibit selenate reduction, it was completely inhibited by some other oxyanions, including nitrate. A model sequencing batch system using the bacterial strain was developed and exhibited good performance in the treatment of wastewater containing high concentrations of selenate.     

Factors affecting soluble selenium removal by a selenate-reducing bacterium Bacillus sp. SF-1

High concentrations of soluble selenium, selenate and selenite, have acute and chronic toxicity toward living things. With the aim of developing a biological process for selenium removal, the effects of a variety of parameters on the reduction of soluble selenium by a Bacillus sp. strain SF-1, which is capable of reductively transforming selenate into selenite and, subsequently, into nontoxic insoluble elemental selenium, were studied. The bacterial strain could effectively reduce 20 mM of selenate to selenite and 2 mM of selenite to elemental selenium in the presence of an appropriate carbon source and in the absence of oxygen. The reduction rate of selenate to selenite was much higher than that of selenite to elemental selenium, resulting in the transient accumulation of selenite during selenate reduction. The selenate reduction rate increased with increases in the selenate concentration up to 20 mM, while the rate of selenite reduction decreased sharply at selenite concentrations of more than 2 mM. The elemental selenium transformed from selenate via selenite was found both inside and outside the cells. Bacillus sp. SF-1 was able to utilize a variety of organic acids or sugars as a carbon source in selenate reduction. Although the copresence of sulfate did not inhibit selenate reduction, it was completely inhibited by some other oxyanions, including nitrate. A model sequencing batch system using the bacterial strain was developed and exhibited good performance in the treatment of wastewater containing high concentrations of selenate.     

富硒冬虫夏草发酵工艺优化

采用冬虫夏草作为富硒的载体,啤酒糟为基质,进行富硒冬虫夏草液态深层发酵试验,研究冬虫夏草对无机硒的生物转化能力.利用正交试验设计,对富硒冬虫夏草液态深层发酵条件如摇床转速、接种量、pH等进行了优化.结果表明,冬虫夏草能在含有低质量浓度亚硒酸钠（10～25mg/L）的培养基中生长,并在菌丝体内富集硒,最佳发酵工艺为摇床频率180r/min、培养10d的条件下,装液量50mL/250mL,pH值为7.0,接种量15％,温度20℃,亚硒酸钠质量浓度25 mg/L,啤酒糟质量浓度20 mg/L.在此优化的发酵条件下,富硒冬虫夏草菌丝体总富硒量为5808.20 μg/L.     

Preparation of elemental selenium-enriched fermented milk by newly isolated Lactobacillus brevis from kefir grains

A Lactobacillus brevis strain CGMCC No. 6683 that can survive at high selenium concentrations was isolated from kefir grains. Using a scanning electron microscope equipped with an energy dispersive X-ray spectroscope, it was shown that L. brevis CGMCC No. 6683 reduced sodium selenite into elemental selenium. L. brevis CGMCC No. 6683 was then pre-treated with sodium selenite to prepare selenium-enriched L. brevis. To study the selenium-enriched yoghurt, selenium-enriched L. brevis was co-fermented in skimmed milk with traditional yoghurt starter culture (Streptococcus thermophilus and Lactobacillus bulgaricus). Results showed that it enhanced selenium concentration in the selenium-enriched yoghurt, and more importantly, elemental selenium was detected in the selenium-enriched yoghurt. The isolated L. brevis provides a new way of preparing a functional dairy product: elemental selenium-enriched fermented milk.     

沪酿3.042米曲霉富硒条件的探索

目的：探索沪酿3．042米曲霉的富硒能力并找到最佳的富硒条件。方法：于豆芽汁液体培养基中添加一系列浓度的亚硒酸钠，培养一段时间，研究亚硒酸钠浓度对米曲霉生物量、富硒能力，蛋白酶活力的影响，找到适合的亚硒酸钠浓度，测定此浓度下米曲霉的生长曲线、富硒量曲线，找到最佳的富硒条件，分析此条件下富硒米曲霉胞内硒的存在状态。结果：亚硒酸钠浓度为100mg／L时米曲霉的生物量最大，富硒能力最高，50mL液体培养基可以收获0．454g湿茵体，含有胞内硒2mg，其中有机硒含量达到95％以上，最适合的茵体收获时间是48h。但是此时蛋白酶活力降低明显，亚硒酸钠浓度为50mg／L时蛋白酶活力比不加亚硒酸钠时活力更高，36h达到活力峰，此时富硒能力也较强。结论：米曲霉是个很好的富硒载体，亚硒酸钠浓度为100mg／L时富硒能力最高，应用前景很好。     

Response of sediment bacterial assemblages to selenate and acetate amendments

We followed the response of bacterial assemblages in slurries of estuarine sediments to amendments of 100 microM sodium selenate and 10 mM sodium acetate. Selenate was removed from the dissolved phase in all slurries after an initial lag that varied depending on the source of the sediment used in the slurry. Subsequent selenate amendments were removed without a lag but with the appearance of transient peaks of selenite. We documented changes in the composition of bacterial assemblages in the slurries using PCR/DGGE and RT-PCR/DGGE of 16S rDNA and rRNA. Bands of interest were sequenced to identify organisms responding to selenate amendments. The composition of communities from the two sampling sites was different and changed but did not converge during incubations with selenate. Selenate-reducing assemblages had broad phylogenetic diversity. Most bands were related to groups of bacteria known to contain organisms capable of selenate or selenite reduction, except for Acinetobacter species which dominated one of the samples and has not previously been associated with selenium oxyanion reduction.