利用米曲霉制作腊八豆的研究

以酿造酱油用的米曲霉经紫外线诱变选育后，人工接种于煮熟的黄豆制作腊八豆。结果表明，接种量为熟豆重量的0．5％(孢了含量10^10个／g菌剂)，经32～35℃、48h前发酵便可得优质霉豆胚，加适量食盐、食用白酒及生姜等拌匀入坛进行10～15d的后发酵，便可获得品质极鲜美，食后余味甘甜的腊八豆，优于毛霉型腊八豆，更优于传统方法制作的腊八豆。比传统腊八豆的制作周期缩短近1个月，便于工业化生产。     

双亲灭活米曲霉原生质体融合中原生质体制备的研究

对双亲灭活米曲霉原生质体融合育种中原生质体制备的条件进行了研究,将菌丝的预处理与细胞壁的酶解合为一步,并讨论了DTT的加入量。结果表明,原生质体制备的最佳破壁酶为纤维素酶、溶壁酶、蜗牛酶3种酶混合浓度比为5:3:1;菌丝培养15h;酶解时加入3mol/L DTT;酶解2.5h;0.8mol/L NaCl作为渗压稳定剂。所得双亲菌株原生质体的融合率为3.31%。     

珍珠贝肉制曲过程中物质代谢与酶活之间的关系

以马氏珍珠贝肉为原料固态发酵制备成曲,通过监测发酵过程各营养底物及中间代谢产物变化趋势,探究了制曲过程中物质代谢与酶活力的相互关系。研究表明:制曲过程中,曲质量和水分含量均呈现下降趋势,曲中的干物质大体也是呈现下降的变化趋势,曲温则是呈现先上升后下降的变化趋势。营养底物的代谢变化分为4个阶段,24 h是物质变化突变点,此后,柠檬酸循环是糖代谢的主要途径。24~36 h,小分子糖和游离氨基酸吸收利用量达到最高值,而40~46 h为蛋白酶活力最高值,酶系分泌滞后于代谢中产物的消耗。糖代谢对蛋白酶活力影响最显著,与中性蛋白酶活力呈显著负相关性(皮尔逊指数P依次为:-0.943);发酵前期提高淀粉酶活力,成曲蛋白酶活力提高了26.81%。     

有机溶剂对米曲霉及荧光假单胞菌细胞毒性的研究

本文以米曲霉以及荧光假单胞菌为代表性霉菌及细菌型微生物,通过对菌体生长量、菌丝球大小以及显微观察,研究了常见有机溶剂对具有催化活性的微生物细胞的生长毒性。研究发现有机溶剂对真菌和细菌的毒性与其lgP值有密切关联关系,且与具体的溶剂种类有关。在针对米曲霉的研究中,正丙醇、叔丁醇和甲苯(lgP值分别为0.55、0.80、2.5)对菌丝球的形成有一定的抑制作用;当正丁醇与氯仿(lgP值分别为0.82、2.00)存在时菌丝质量为0,即能完全抑制菌丝生长;丙酮,短链醇,长链烷烃等的存在对菌丝体总质量(0.81±0.07)和菌丝形态没有明显改变,但对生殖期以及孢子形态有显著影响。对荧光假单胞菌而言,正丁醇、氯仿、甲苯和辛烷(4.6lgP0.8)可以完全抑制菌体生长(OD5601.17±0.05),其他溶剂存在时菌体生长与对照(OD560=2.27±0.05)无显著差异;此外,有机溶剂会影响培养过程中细胞的形状和细胞间的排列方式。     

Multiplex Real‐Time PCR Method for Detection and Quantification of Mycotoxigenic Fungi Belonging to Three Different Genera

Abstract: Cereal crop plants are colonized by many fungal species such as Aspergillus ochraceus and Penicillium verrucosum, which produce ochratoxins, and Fusarium graminearum, which produces trichothecene mycotoxins. A multiplex real‐time PCR method using TaqMan probes was developed to simultaneously detect and quantify these mycotoxigenic Fusarium, Penicillium and Aspergillus species in cereal grains. Primers and probes used in this method were designed targeting the trichothecene synthase (Tri5) gene in trichothecene‐producing Fusarium, rRNA gene in Penicillium verrucosum, and polyketide synthase gene (Pks) in Aspergillus ochraceus. The method was highly specific in detecting fungal species containing these genes and was sensitive, detecting up to 3 pg of genomic DNA. These PCR products were detectable over five orders of magnitude (3 pg to 30 ng of genomic DNA). The method was validated by evaluating sixteen barley culture samples for the presence of deoxynivalenol (DON) and ochratoxin A (OTA) producing fungi. Among the barley culture samples tested, 9 were positive for Fusarium spp, 5 tested positive for Penicillium spp, and 2 tested positive for Aspergillus spp. Results were confirmed by traditional microbiological methods. These results indicate that DON‐ and OTA‐producing fungi can be detected and quantified in a single reaction tube using this multiplex real‐time PCR method. Practical Application: This method would be helpful in detecting and quantifying the mycotoxin producing fungi such as Fusarium, Aspergillus, and Penicillium in cereal grains and cereal‐based foods.     

宇佐美曲霉羧肽酶成熟肽基因的表达及鉴定

目的克隆、表达宇佐美曲霉(Aspergillus usami)羧肽酶成熟肽基因,并检测其酶学性质。方法根据前期克隆的宇佐美曲霉羧肽酶基因序列设计引物,克隆羧肽酶成熟肽基因AucpA,构建重组表达质粒pPIC9k-AucpA,电击转化毕赤酵母GS115,甲醇诱导重组蛋白表达。表达的重组蛋白经Sephadex-50层析纯化后,进行酶活性及其影响因素的检测。结果重组表达质粒pPIC9K-AucpA经双酶切及测序证明构建正确;表达的重组蛋白相对分子质量约81 000,纯化的重组羧肽酶最高比酶活为57.15 nkat/mg,最适反应温度为45℃,最适反应pH为3.5,金属离子、EDTA对重组羧肽酶的酶活性无影响,而PMSF对重组羧肽酶酶活性的抑制率达50%以上。结论已成功在毕赤酵母中表达了具有较高酶活的宇佐美曲霉羧肽酶,为进一步研究该重组酶的应用奠定了基础。     

Biodegradation study on poly(ε‐caprolactone) with bimodal molecular weight distribution

Poly(ε‐caprolactone) (PCL) of bimodal molecular weight distribution was exposed to the action of enzymes‐lipases from Aspergillus oryzae in phosphate buffer at pH 7 and 37°C, and those produced in situ by Bacillus subtilis in nutrient medium at 30°C for 42 days. The occurrence of biodegradation is proved on the basis of the weight loss, decrease of molecular weight, carbonyl index, crystallinity, and development of cracks on the PCL surfaces. In the case of Bacillus subtilis, the degradation (10 wt % loss of PCL) proceeds faster in comparison with lipase from Aspergillus oryzae (2.6 wt % loss of PCL), where the degradation process seems to stop during 14 days of experiment. The gel permeation chromatography results reveal that preferential degradation of lower molecular portion did not occur but it is assumed that PCL chains were cleaved in accordance with particular degradation mechanism that depends significantly on biological agent. © 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013     

Expression and characterization of β-1,3-1,4-glucanase of Aspergillus usamii in Escherichia coli and its application in sourdough bread making

A β-1,3-1,4-glucanase gene (Auglu12A) from Aspergillus usamii was successfully expressed in Escherichia coli BL21(DE3). The recombinant enzyme, reAuglu12A was efficiently purified using the one-step nickel-nitrilotriacetic acid affinity chromatography. The specific activity of reAuglu12A was 694.8 U/mg, with an optimal temperature of 55°C and pH of 5.0. The reAuglu12A exhibited stability at temperatures up to 60°C and within the pH range of 4.0–5.5. The reAuglu12A hydrolytic activity was increased in the presence of metal ions, especially K⁺ and Na⁺, whereas it exhibited a K_m and V_max of 8.35 mg/mL and 1254.02 µmol/min/mg, respectively, toward barley β-glucan at pH 5.0 and 55°C. The addition of reAuglu12A significantly increased the specific volume (p < 0.05) and reduced crumb firmness and chewiness (p < 0.05) of wheat–barley sourdough bread during a 7-day storage period compared to the control. Overall, the quality of wheat–barley sourdough bread was improved after incorporation of reAuglu12A (especially at 3000 U/300 g). These changes were attributed to the synergistic effect of acidification by sourdough and its metabolites which provided a conducive environment for the optimal action of reAuglu12A in the degradation of β-glucans of barley flour in sourdough. This stabilized the dough structure, thereby enhancing the quality, texture, and shelf life of the bread. These findings suggest that reAuglu12A holds promise as a candidate for β-glucanase application in the baking industry.     

新型脂肪酶基因的克隆与表达

目的 通过同源比对筛选黑曲霉中新型活性脂肪酶.方法 抽提黑曲霉基因组,依据从烟曲霉中获得的高活性脂肪酶基因序列,NCBI网站序列比对后搜索到与烟曲霉af1-1同源性较高的黑曲霉脂肪酶基因序列an1-1,设计引物进行PCR扩增,产物胶回收后连接至PMD 18T载体,测序验证正确后将目的序列连接至表达载体PET28a,转化Escherichia coli BL21 (DE3)中表达和测定活性.结果 克隆了一种来源于黑曲霉新型脂肪酶基因an1-1,15℃表达条件下主要为可溶性表达,对C4底物(对硝基苯酚丁酸酯)活性达2365 U/L.结论 同源序列比对能有效提高筛选效率,并从黑曲霉中筛选到一种新的脂肪酶基因,在大肠杆菌中可高表达并具有生物学活性.     

非对称灭活双亲原生质体融合法选育产果糖基转移酶的米曲霉新菌株的研究

本文以生产果糖基转移酶的米曲霉SBB201(Aspergillus oryzae SBB201)为出发菌株,通过紫外联合氯化锂诱变获得产酶性能与生长性能各自优良的米曲霉菌株UV-A与UV-B,采用非对称灭活法对这两株菌株的原生质体进行紫外以及热灭活双灭活,然后在PEG作为融合剂的介导下进行原生质体双亲融合,筛选出一株果糖基转移酶生产能力有较大提高的米曲霉新菌株,其酶活力达到了172.95 U/g,比出发菌株提高了64.57%。