Efficient induction of inulin fructotransferase by inulin and by difructose anhydride III in Arthrobacter aurescens SK 8.001

Inulin fructotransferase (IFTase; EC 4.2.2.18) has received great attention mainly due to its application in producing difructose anhydride III (DFA III), which is a novel functional sweetener. The object of this study was to investigate the induction of IFTase in Arthrobacter aurescens SK 8.001 with various carbon sources, especially inulin and DFA III. IFTase production could be significantly promoted by the supplement of inulin (5–50 g/L) and DFA III (5–20 g/L). Inulin at high initial concentrations gave no indication of catabolite repression, whereas 30 and 40 g/L DFA III intensely inhibited cell growth and IFTase activity. No fructose was detected in broth throughout the cultivation with inulin, and inulin was converted into DFA III and minor fructooligosaccharides. And when DFA III was the carbon source, DFA III was the only sugar detected in the broth. In conclusion, both DFA III and inulin are effective for IFTase induction, and inulin with higher IFTase activity proved to be a more potent inducer.     

真菌固定床反应器发酵L(+)-乳酸的初步研究

研究了在5L发酵罐中采用纤维床固定化技术发酵生产乳酸过程不同溶氧量和固定化面积对乳酸发酵过程的影响,测定了不同固定化面积发酵过程中的氧传质系数。在通气率为1.0vvm和固定化面积为400cm2条件下,产酸速率为2.13g/(L·h),发酵液中乳酸最终浓度为73.1g/L,L(+)-乳酸光学纯度为98.9%,证明提高发酵过程中菌体层内部溶氧传质系数有助于增加乳酸产率。乳酸发酵液浊度0.49NTU,显著改善发酵液的流体力学条件,为应用膜生物反应器技术连续发酵生产乳酸奠定了基础。     

Effects of difructose anhydride III (DFA III) administration on bile acids and growth of DFA III-assimilating bacterium Ruminococcus productus on rat intestine

The growth of DFA III-assimilating bacteria in the intestines of rats fed 3% DFA III for 2 weeks was examined. Sixty-four percent of the DFA III intake had been assimilated on day 3 of ingestion, and almost all of the DFA III was assimilated at the end of the experiment. The DFA III-assimilating bacterium, Ruminococcus productus, in DFA III-fed rats was in the stationary state of 10(8)-10(9) cells/g dry feces within a week from 10(6) cells/g dry feces on day 1 of DFA III ingestion. The number of R. productus cells was associated with the amount of DFA III excreted in the feces. The acetic acid produced from DFA III by R. productus lowered the cecal pH to 5.8. In control-fed rats and DFA III-fed rats, 94% of secondary bile acids and 94% of primary bile acids, respectively, were accounted for in the total bile acids analyzed. DFA III ingestion increased the ratio of primary bile acids and changed the composition of fecal bile acids. In conclusion, R. productus assimilated DFA III, produced short chain fatty acids, and the cecal pH was lowered. The acidification of rat intestine perhaps inhibited secondary bile acid formation and decreased the ratio of secondary bile acids. Therefore, it is expected that DFA III may prevent colorectal cancer and be a new prebiotic candidate.     

Actinomycete Nonomuraea sp. isolated from Indonesian soil is a new producer of inulin fructotransferase

An actinomycete that excretes inulin fructotransferase to the culture supernatant was able to produce di-d-fructofuranose 1,2':2,3' dianhydride (DFA III) from inulin, with the greatest rate of enzyme activity at 65°C and at a pH of 5.5. Through chemotaxonomic and 16S rRNA gene analysis, this strain was identified as genus Nonomuraea in the Streptosporangiaceae family. This is the first report of an inulin fructotransferase producer in this family.     

细胞稀释强化硅橡胶膜生物反应器连续乙醇发酵

通过原位重力沉降分离酵母和渗透汽化分离乙醇构建了细胞稀释连续乙醇发酵的硅橡胶膜生物反应器。采用酿酒酵母,以0.05/h的细胞稀释率在膜生物反应器中实现了170 h的连续稳定乙醇发酵。重力沉降分离酵母对硅橡胶膜生物反应器产生的细胞稀释作用可以通过反应器内酵母自身生长得到平衡,发酵液细胞浓度稳定在5g/L。渗透汽化原位分离使发酵液内乙醇浓度维持在50 g/L。细胞稀释膜生物反应器连续发酵的乙醇体积产率达到1.63 g/(L.h),相对于同等工艺参数的细胞封闭循环膜生物反应器连续乙醇发酵细胞比产率提高了31%。     

膜生物反应器连续发酵法制取二羟基丙酮的研究

在间歇培养基础上 ,利用膜生物反应系统进行连续发酵制取二羟基丙酮。考查了不同甘油浓度、培养基组分和流速对于连续发酵中菌体生长特性和二羟基丙酮产率的影响。结果表明 :甘油浓度为 60 g/L、玉米浆和蛋白水解液浓度为 0 5g/L、稀释率为 0 0 67h-1时 ,菌体积累、甘油转化率和体积产率均较高 ,最长连续发酵持续时间为 40 0h。     

聚天冬氨酸为结合相的薄膜梯度扩散技术富集测量自来水中痕量铬(Ⅲ)

建立了以0.05mol/L聚天冬氨酸(PASP)溶液为结合相的薄膜梯度扩散(DGT)装置(PASP DGT)富集测量自来水中痕量CrIII的分析方法。DGT法测得配制水中CrIII的回收率为94.4%~105.6%,相对标准偏差(RSD)为2.78%~5.03%;测得自来水中CrIII的浓度为2.99~3.47μg/L,加标回收率为93.2%~107.7%;DGT方法对水中CrIII的检出限为0.081μg/L(采样48h),可应用于自来水中痕量CrIII的定量检测与早期预警。      

耐氨米根霉的分批补料发酵及发酵动力学初探

以氨水为中和剂,替代CaCO3,对耐氨米根霉R.oryzaeJS-N0-2-02进行15L自动发酵罐的分批和分批补料发酵及其发酵动力学的初步研究,结果表明,降低起始糖浓度,产酸期补糖可明显提高菌体L-乳酸比生产速率和耗糖产酸能力,提高L-乳酸产量和纯度,降低残糖。在发酵起始时添加1 g/L CaCO3能进一步提高补糖发酵的L-乳酸比生产速率,增强发酵后期菌体耗糖产酸能力,从而进一步提高L-乳酸产量和纯度,降低残糖。发酵结果:起始糖浓度为120 g/L,25h时补糖使最终发酵总糖浓度达137 g/L,发酵培养60 h,L-乳酸产量可达101.8 g/L,纯度97.3%,菌体耗糖转化率76%,比生产速率0.27 g/g.h,残糖降至3 g/L。     

Cloning and extracellular expression of inulin fructotransferase from Arthrobacter aurescens SK 8.001 in E. coli

BACKGROUND: Difructose anhydride (DFA) III is a natural and low‐calorie sweetener. It stimulates the absorption of calcium and other minerals. Inulin fructotransferase (IFTase; EC 4.2.2.18), catalysing inulin hydrolysis to DFA III, is considered to be the most promising enzyme for the production of DFA III. RESULTS: IFTase gene from Arthrobacter aurescens SK 8.001 was cloned and sequenced. Transformant with native IFTase signal peptide was a useful system for extracellular over‐expression of IFTase, and its extracellular IFTase activity reached 81.0 U mL^?1. This value was 4.1‐fold of that obtained with A. aurescens SK 8.001 for IFTase production. The recombinant IFTase was purified to electrophoretical homogeneity and characterized. The enzyme showed maximum activity at pH 6.0 and 55 °C, and retained 81.3% of its initial activity after incubation at 60 °C for 4 h. CONCLUSION: IFTase gene from A. aurescens SK 8.001 was cloned, sequenced and over‐expressed in E. coli. IFTase was reported for the first time to be over‐expressed extracellularly. The recombinant IFTase was purified and characterized, and shown to be a good candidate for potential application in DFA III production. Copyright © 2011 Society of Chemical Industry     

Cloning of inulin fructotransferase (DFA III-producing) gene from Arthrobacter globiformis C11-1

A gene encoding an inulin fructotransferase (DFA III-producing) [EC 2.4.1.93] from Arthrobacter globiformis C11-1 was cloned and the nucleotide sequence was determined. The cloned fragment contained a 1353 bp open reading frame. The initiation codon was estimated to be an unusual codon, GTG. The gene encoded a signal peptide (40 amino acid residues) for secretion. The molecular mass of the native enzyme was calculated as 43,400 Da from the sequencing data. The deduced amino acid sequence of the enzyme had 74.0 % homology with that of inulin fructotransferase (DFA III-producing) from Arthrobacter sp. H65-7. It also had 45.1% homology with that of inulin fructotransferase (DFA I-producing) [EC 2.4.1.200] from Arthrobacter globiformis S14-3. The enzyme produced in the culture supernatant of an Escherichia coli clone was purified to the electrophoretically homogeneous stage. The N-terminal amino acid sequence of the cloned enzyme secreted in the broth was the same as that of the native enzyme from A. globiformis C11-1. Therefore, on this enzyme, it is estimated that the cleavage sites by the signal peptidase for secretion of A. globiformis C11-1 and E. coli JM109 are the same.     

固定化酵母-膜生物反应器连续发酵生产乙醇的研究

对固定化酿酒活性干酵母-硅橡胶膜生物反应器连续发酵生产乙醇进行了实验研究。对固定化酵母和游离酵母的发酵能力进行了比较,实验研究了系统在长期运行过程中的发酵反应动力学参数和膜传质动力学参数等基本性能。结果表明,酵母细胞固定化后,其发酵速度有明显提高;膜渗透汽化分离产物乙醇使发酵反应能够长时间连续稳定地进行,葡萄糖消耗率和乙醇体积产率都较间歇发酵有明显的提高;在连续运行中,发酵液中葡萄糖浓度控制在20～50g/L的范围内,乙醇体积产率为4.0g/（L·h）,罐内乙醇浓度基本保持在45g/L左右,固定化酵母浓度维持在1.05×10^10～2.15×10^10个/克胶,发酵液中游离酵母浓度维持在0.02×10^10～0.098×10^10个/mL,膜的渗透通量和分离因子分别为300～500g/（m^2·h）和3.6～7.2;实验系统连续运行了294h,收得渗透冷凝液4841.7g,平均质量分数为21.3%。     

Characterization of membrane bioreactor for dry wine production

Characteristics of yeast growth and ethanol fermentation were examined in membrane bioreactor using a grape juice. After inoculation, batch fermentation was carried out for 24 h. When yeast growth reached the stationary phase, continuous fermentation was initiated. In continuous fermentation, a linear relationship was observed between cell concentration and dilution rate. In single-vessel membrane bioreactor, the cell concentrations of 18.7 g/l and 76.9 g/l (15 and 60 times higher than that of the batch fermentation, respectively) were observed at dilution rates of 0.1 h(-1) and 0.3 h(-1), respectively. The residual sugar concentration was higher than 10 g/l at the dilution rate of 0.1 h(-1), 0.2 h(-1) or 0.3 h(-1), therefore the single-vessel membrane bioreactor was not suitable for producing dry wine (sugar concentration: 4 g/l or less). In the double-vessel membrane bioreactor, it is most suitable to set the recycle ratio at 0.15 for keeping the sugar concentration below 4 g/l. The productivity of dry wine in the double-vessel membrane bioreactor was 28 times higher than that in the batch fermentation.     

利用TGase 结合微滤技术从乳清粉中分离纯化C G M P 的研究

利用谷氨酰胺转氨酶(TGase)结合微滤技术从乳清中分离纯化酪蛋白糖巨肽(CGMP)。结合微滤技术考察底物浓度、TGase浓度、pH值、温度和反应时间对分离纯化的影响。通过实验得出最佳酶反应条件为底物浓度8%、TGase酶浓度7U/g、pH6.5、温度39℃、反应时间80min、微滤浓缩循环6次,最后经微滤可得到纯度为70%左右的CGMP。     

Cr(III)在钝顶螺旋藻中的生物富集及其对螺旋藻生长的影响

本实验研究了钝顶螺旋藻对Cr(III)的吸收和生物转化以及Cr(III)对钝顶螺旋藻的生长影响,用ICP-MSHPLC对无机Cr(III) 经钝顶螺旋藻吸收后的存在价态进行了分析。结果表明,钝顶螺旋藻对Cr(III)具有良好的富集和生物转化能力,在本实验中总铬富集量可达到173.17mg/g,有机化程度可高达96.99%。ICP-MS-HPLC 分析结果表明没有有毒的Cr(VI)的产生。此外,干重测定结果显示低浓度的Cr(III) (< 234.38 × 10-6g/g)促进钝顶螺旋藻的生长,高浓度的Gr(Ⅲ)(> 234.38 × 10-6g/g)则抑制共生长,并导致钝顶螺旋藻形态异常。在一定范围内钝顶螺旋藻能高效富集Cr(III),可作为安全营养的保健食品;钝顶螺旋藻抗高Cr(III)压,吸附高浓度Cr(III)的能力使其可用于环境中Cr(III)污染的去除。     

Short communication: Effect of difructose anhydride III on serum immunoglobulin G concentration in newborn calves

Difructose anhydride (DFA) III is an indigestible disaccharide that promotes paracellular absorption of calcium, magnesium, and other minerals in the intestine by acting on epithelial tight junctions. This study aimed to elucidate the effect of DFA III on serum IgG concentration. One hundred and twenty Holstein and Holstein/Japanese Black crossbred calves were randomly divided into 4 groups of 30 to receive untreated colostrum (DFA0) or colostrum containing 3, 6, or 18 g of DFA III (DFA3, DFA6, or DFA18, respectively). At 24h after birth, both serum IgG (ranging from 16.4 to 21.2mg/mL) and apparent efficiency of absorption (26.0 to 37.2%) showed increases with the amount of DFA III intake. By multiple regression analysis, the standardized partial regression coefficient for DFA III was 0.25, the second highest following that for the colostrum IgG concentration (0.80), indicating a positive effect of DFA III on serum IgG. A positive linear regression was found between colostrum IgG and serum IgG concentrations at 24h of age. These results indicate that IgG absorption occurred as a nonsaturable process, which might be characteristic of gradient-dependent paracellular transport. Thus, it was concluded that DFA III improves not only minerals but IgG absorption in calves.     

生物法制造丁二酸研究进展

为了优化产琥珀酸放线杆菌（Actinobacillus succinogenes）发酵甘薯粉生产丁二酸的发酵培养基,提高丁二酸产量,降低生产成本,本研究首先通过单因素实验对甘薯粉发酵产丁二酸的甘薯粉、MgCO₃、液化酶、糖化酶、氮源浓度和发酵时间进行了优化,再利用正交试验确定重要参数的最佳水平,最后利用2 L发酵罐对获得最佳发酵工艺进行放大实验。结果表明,混合氮源（酵母粉:玉米浆=1:2）可满足甘薯粉丁二酸发酵营养需求,影响丁二酸产量的重要参数是甘薯粉、MgCO₃、液化酶、糖化酶、混合氮源（酵母粉:玉米浆=1:2）的浓度。各因素最佳水平为：甘薯粉115 g/L、MgCO₃ 60 g/L,液化酶0.008 KUN-S/g底物,糖化酶3.09 AGU/g底物,混合氮源33 g/L。优化后丁二酸产量可达69.89 g/L,与优化前相比（42.46 g/L）,丁二酸浓度提高了64.6%。2 L发酵罐发酵72 h,丁二酸可达71.42 g/L,丁二酸产率为79.87%,生产强度为0.99 g/（L·h）。因此,利用 A. succinogenes发酵甘薯粉产丁二酸具有较好的工业化应用前景。     

Effective onion vinegar production by a two-step fermentation system

A two-step fermentation system combining a repeated batch process using a flocculating yeast with a charcoal pellet bioreactor was developed for onion vinegar production. Juice from the red onion R-3, which contained 67.3 g/l total sugar, was smoothly converted to onion alcohol containing 30.6 g/l ethanol by repeated batch operation using the flocculating yeast Saccharomyces cerevisiae strain IR-2. Stable operation was possible and the maximum productivity was about 8.0 g/l/h. A packed bed bioreactor containing charcoal pellets produced from waste mushroom medium was then applied to continuous onion vinegar production from the onion alcohol. Onion vinegar was successfully produced, with a maximum productivity and acetic acid concentration of about 3.3 g/l/h and 37.9 g/l, respectively. The total acetic acid yield calculated from the amount of sugar consumed was 0.86. The two-step system was operated for 50 d and proved to be competitive with other systems in terms of its high productivity, high acetic acid yield, operational stability and low production costs.     

High gravity primary continuous beer fermentation using flocculent yeast biomass

The current work assessed a new immobilized cell reactor system throughout a long‐term (54 days) continuous primary fermentation of lager‐type wort of high specific gravity. The experiment was performed in a 4 L airlift bioreactor and immobilization of biomass was attained solely by flocculation. Despite the constant liquid agitation and washout of biomass, up to 53 g dry wt/L of yeast remained immobilized in the system. Two types of beer were produced without interrupting the reactor, based on two types of wort: a Pilsener type with high specific gravity of 15.6 ± 0.3°P; and a dark lager wort with specific gravity of 14.4 ± 0.03°P. Even during the inlet of high gravity wort, the desired attenuation was achieved without the need for either recirculation or an auxiliary second stage bioreactor. The specific saccharide consumption rate was kept around 7.9 ± 0.4 g/L/h and ethanol productivity oscillated at 3.36 ± 0.2 g/L/h for nearly a month. During this period the volumetric productivity of the current bioreactor reached 1.6 L beer/L/day. The green beers produced from the Pilsener and dark lager worts met the standards of regular finished primary beer fermentation. The productivity of diacetyl through the entire experiment could be correlated to the free amino nitrogen consumption rate. Copyright © 2014 The Institute of Brewing & Distilling     

填充床反应器固定化菊糖果糖转移酶催化合成双果糖酐Ⅲ的研究

本文建立了填充床反应器固定化菊糖果糖转移酶水解菊糖的工艺。以菊糖为底物,探究该工艺水解菊糖的条件,以单因素实验为基础,依据正交实验优化,考察菊糖浓度、反应时间、反应温度及底物流速等因素对双果糖酐Ⅲ含量的影响,获得最佳的工艺条件。该工艺采用填充床反应器的容积为20m L,固定化酶的装载量为15m L。结果表明:菊糖浓度为100g/L,反应时间为1h,温度为60℃,底物流速为20m L/h,在该工艺条件下制取双果糖酐Ⅲ的浓度为67.38g/L。该工艺能持续操作48d以上,半衰期为48d,为该工艺大规模生产提供理论与操作的基础。