重组大肠杆菌利用木糖发酵产高纯度L-乳酸

对重组大肠杆菌JH16利用木糖产高纯度的三一乳酸进行研究。通过无氧管驯化EscherwhiacdiJH12菌株得到E．coliJH16,驯化后的菌株茵体浓度提高了31％,乙酸积累减少了43％;在摇瓶中考察不同Mg2＋浓度对EcoliJHl6产三一乳酸的影响,确定最适Mg2＋质量浓度为0．25g/L;EcoEJH16以60g/L木糖为C源,在7L全自动发酵罐中添加0．25g／LMg2＋,乳酸积累量提高了18％,达38．18g/L,乳酸纯度高达95％;E．coliJH16在30g／L木糖和30g／L葡萄糖混合C源中,优先利用葡萄糖,当葡萄糖质量浓度低于1．56g/L后,菌体开始利用木糖进行乳酸发酵,最终得到39g／L乳酸。     

外加肌醇对嗜鞣管囊酵母发酵的酒精产量及其耐酒精能力的影响

嗜鞣管囊酵母(Pachysolen tannophilus)是可以同时发酵葡萄糖和木糖为酒精的菌种,在其生长和发酵培养基中分别添加不同浓度((0～200mg/L)的肌醇以及不同起始浓度的酒精,以考察外加肌醇对嗜鞣管囊酵母生长、产酒精能力和耐酒精能力的影响.结果 表明,添加肌醇前后,嗜鞣管囊酵母的生物量及发酵的酒精产量均有所增加.外加肌醇对嗜鞣管囊酵母生长有轻微的刺激作用,酵母生长最适肌醇浓度为150mg/L;而对酵母生长的耐酒精能力却有明显的影响, 并且,菌种在YEPD培养基中的耐酒精能力高于在YEPX培养基中的耐酒精能力.经实验测定,肌醇对嗜鞣管囊酵母产酒精能力及发酵的耐酒精能力均有显著的影响.发酵培养基中未添加起始浓度的酒精时,菌种发酵的最适肌醇浓度为100mg/L,此时生成的酒精产量为45.20g/L.当分别添加起始酒精浓度为10%和12%时,随着肌醇浓度的增加,菌种发酵生成的酒精浓度均呈上升趋势;肌醇浓度为200mg/L时,两种起始酒精浓度下,酒精的净生成量均达到最大,分别为17.18g/L和16.68g/L.     

响应面法优化褐藻胶降解菌Cobetia sp.20发酵培养基

为了提高褐藻胶降解菌株Cobetia sp.20产褐藻胶裂解酶的能力,利用响应面法优化其发酵产褐藻胶裂解酶的培养基。首先利用单因素法分别对发酵培养基中的不同碳源、碳源添加量、不同氮源、氮源添加量以及氯化钠添加量、磷酸二氢钾添加量、硫酸镁添加量和pH进行探究,研究各因素对产酶的影响。在单因素实验的基础上,通过Plackett-Burman试验确定Cobetia sp.20发酵培养基中影响产酶的主要因素。通过响应面试验建立回归方程。研究结果表明,Cobetia sp.20最优发酵培养基配方为褐藻胶15.00 g/L、硫酸铵7.50 g/L、氯化钠15.00 g/L、硫酸镁0.50 g/L、磷酸二氢钾5.30 g/L、硫酸亚铁0.01 g/L、pH值7.58。优化后酶活为142.79 U/mL,比优化前提高了26.36%。褐藻胶裂解酶活的提高,为褐藻胶裂解酶的工业化生产提供了参考。     

L-鸟氨酸的混合菌种发酵研究

谷氨酸棒杆菌可发酵葡萄糖产L-鸟氨酸。通过连续筛选与驯化筛选到了产量较高的生产菌株CG1006。以GC/MS对发酵液成分的定性分析及鸟氨酸的产量为依据,优化了培养条件,产量从最初的14.346g/L提高到40.416g/L。初步考察了酵母菌CG1006-1对发酵液成分及产量的影响。结果表明混合菌种发酵可纯化发酵液成分。     

产酸丙酸杆菌耐酸菌株的选育及其应用

为了解除微生物发酵生产丙酸过程中代谢产物(丙酸)对菌体生长的抑制作用,以实验室保藏的产酸丙酸杆菌(耐30g/L丙酸)为出发菌株P-0,通过丙酸压力筛选获得了一株耐10g/L丙酸的产酸性能良好的菌株P-10,降低了发酵过程中丙酸对菌体生长的抑制作用。菌株P-10做摇瓶发酵,发酵周期168h,丙酸浓度为49.66g/L,产酸速率为0.30g/(L·h),较出发菌株P-0提高了53.04%;7L发酵罐实验表明,菌株P-10发酵周期168h,丙酸浓度为55.63g/L,产酸速率0.33g/(L·h)。同时对菌株P-10做二次接种实验,结果表明,84h为二次接种最适时间段,且84h进行二次接种时,丙酸浓度提高了17.77%,二次接种实验不但有利于有机酸的积累,而且可以提高菌株的产酸能力和耐酸能力;经过选育的菌株P-10具有优良的产酸稳定性,有利于菌种的工业化生产和应用,同时对后续的发酵分离耦合具有重要意义。     

产酸丙酸杆菌耐酸菌株的选育及其应用

为了解除微生物发酵生产丙酸过程中代谢产物(丙酸)对菌体生长的抑制作用,以实验室保藏的产酸丙酸杆菌(耐30g/L丙酸)为出发菌株P-0,通过丙酸压力筛选获得了一株耐10g/L丙酸的产酸性能良好的菌株P-10,降低了发酵过程中丙酸对菌体生长的抑制作用。菌株P-10做摇瓶发酵,发酵周期168h,丙酸浓度为49.66g/L,产酸速率为0.30g/(L·h),较出发菌株P-0提高了53.04%;7L发酵罐实验表明,菌株P-10发酵周期168h,丙酸浓度为55.63g/L,产酸速率0.33g/(L·h)。同时对菌株P-10做二次接种实验,结果表明,84h为二次接种最适时间段,且84h进行二次接种时,丙酸浓度提高了17.77%,二次接种实验不但有利于有机酸的积累,而且可以提高菌株的产酸能力和耐酸能力;经过选育的菌株P-10具有优良的产酸稳定性,有利于菌种的工业化生产和应用,同时对后续的发酵分离耦合具有重要意义。     

紫外诱变驯化提高酿酒酵母木糖发酵的抑制物耐受性

利用驯化和紫外处理结合驯化的手段对一株木糖发酵工业菌株的抑制物耐受性进行提升。在反复批次培养过程中不断提高含9种抑制物的混合抑制物浓度,使细胞的生长和发酵逐渐适应高浓度抑制物环境,分离突变菌株并进行评价。紫外处理结合驯化比直接驯化能更有效的提升细胞对高浓度抑制物的耐受能力;通过突变菌株分离和筛选,获得3株抑制物耐受能力高于出发菌株的突变菌株,它们在抑制物浓度100%MI(甲酸1 g/L,乙酸3.5 g/L,乙酰丙酸1.5 g/L,糠醛1.5 g/L,5-羟甲基糠醛1.5g/L,丁香醛0.1 g/L,香草醛0.1 g/L,松柏醛0.025 g/L,肉桂酸0.025 g/L)条件下的木糖消耗率比出发菌株高出11.3%-23.2%。紫外诱变处理结合驯化过程可以有效提高酿酒酵母对混合抑制物的耐受性。     

耐盐性毒死蜱降解菌HY-1 的产酶培养基及发酵条件优化

为了明确生化处理和微生物降解的关系,通过增加耐盐菌的比例可以提高农药废水生化处理效果。从农药厂废水中分离到1株耐盐性毒死蜱降解菌——蜡状芽孢杆菌(Bacillus cereus HY-1),以从该菌中提取到的降解酶比活力为指标,进行产酶培养基和发酵条件的优化研究。通过单一因素试验和正交试验,对细菌HY-1的产酸培养基和发酵条件进行了优化。运用SPSS软件进行结果分析,所获优化培养基配方为:葡萄糖6.0 g/L,胰蛋白胨2.2 g/L,K2HPO4 2.0 g/L,KH2PO4 0.2 g/L,MgSO4.7H2O 0.1 g/L,NaCl 0.1 g/L和微量元素溶液2 mL/L。得到菌株发酵培养的最佳优化条件为:种子液培养时间为16 h,发酵培养时间为18 h,接种量为1%(V/V),发酵培养基初始pH值为7.0。氯化钠浓度为0?30 g/L时降解酶比活力不受影响,这是已报道的耐盐性最强的一株毒死蜱降解菌。     

耐高糖米根霉菌株的生理特性

高糖发酵是提高产物浓度的常用方法,但天然菌株不能耐受高的糖浓度。为了解析高糖环境对菌株生理代谢的影响,以驯化获得的耐高糖菌株和原始菌株为对象,研究不同糖浓度对二者生理特性的影响。结果发现:与原始菌株相比,耐高糖菌株细胞膜不饱和脂肪酸的含量、胞内三磷酸腺苷(ATP)含量较高,且表现出更强的侧系呼吸强度。在此基础上,尝试通过调节碳氮比(C/N)或添加甘氨酸的方式补足原始菌株的代谢不足。结果表明:在150 g/L糖质量浓度条件下,调节C/N为500时,原始菌株富马酸产量可由37.4 g/L增至40.2 g/L。而添加0.5g/L甘氨酸,原始菌株富马酸产量可以增至43.8 g/L,提高了17.1%。     

种子培养基成分对裂殖壶菌发酵产DHA的影响

考察种子培养基中谷氨酸钠质量浓度和NaCl质量浓度（盐度）对Schizochytrium sp. HX-308菌种质量及发酵性能的影响。结果表明：40 g/L谷氨酸钠条件下培养菌种,发酵后DHA质量分数达到（53.25±0.48）%,而其油产量、油脂质量分数和DHA产量分别为（24.9±0.4）g/L、（45.2±0.6）%、（8.6±0.35）g/L; 利用不含谷氨酸钠的种子培养基发酵,其油产量、油脂质量分数和DHA产量分别为（30.1±0.8）g/L、（54.3±0.5）%、（12.1±0.5）g/L,比含40 g/L谷氨酸钠时分别提高了20.88%、20.13%和40.70%,为菌种驯化提供了新的思路和方向。此外,低盐度培养条件有利于菌种生产性能的发挥,6.25 g/L NaCl条件下发酵后油质量分数、DHA产量及DHA质量分数最高,分别为（62.1±0.8）%、（10.0±0.3）g/L、（48.97±0.42）%。     

Identification and analysis of the metabolic functions of a high-salt-tolerant halophilic aromatic yeast <Emphasis Type="Italic">Candida etchellsii</Emphasis> for soy sauce production

Salt-tolerant yeasts are very important for the flavor formation in soy sauce fermentation production. A halophilic aromatic yeast was isolated on the basis of the molecular biological and metabolic functions from soy sauce. The ITS nucleotide sequence alignment method was used to identify the strain as Candida etchellsii by subjecting the sequence to NCBI-BLAST in comparison with that of the C. etchellsii strain Miso 0208 (a typical high-salt-tolerant halophilic aromatic yeast strain). Organic acids, amino acids and volatile flavor compounds were produced by the yeast strain which were analyzed by HPLC and SPME-GC/MS methods. Tartaric acid (0.979 ± 0.040 g/l), formic acid (0.636 ± 0.030 g/l), lactic acid (2.80 ± 0.10 g/l), α-alkone glutaric acid (0.132 ± 0.015 g/l), citric acid (2.59 ± 0.10 g/l) and succinic acid (3.03 ± 0.20 g/l) were detected at 72 h of fermentation, respectively. Free and acid hydrolyzed amino acids at levels of 3.7355 ± 0.0027 and 11.5604 ± 0.0037 g/l, respectively, 4-ethyl guaiacols as well as other volatile flavor compounds were also detected.     

Bioconversion of ferulic acid to 4-vinylguaiacol and 4-ethylguaiacol and of 4-vinylguaiacol to 4-ethylguaiacol by halotolerant yeasts belonging to the genus Candida

In order to examine the genesis of the characteristic flavors of soy sauce and miso, seven novel halotolerant yeast strains of two types, which showed convertibility of ferulic acid (FA) to 4-vinylguaiacol (4-VG) and to 4-ethylguaiacol (4-EG), were isolated from miso-koji and miso pastes. Two of these strains were identified as Candida guilliermondii (anamorph of Pichia guilliermondii), and Candida fermentati (anamorph of Pichia caribbica), based on sequence analyses of a partial 26S ribosomal RNA gene and the region of internal transcribed spacers 1 and 2, and the 5.8S ribosomal RNA gene. Moreover, we also found three Candida etchellsii strains which showed convertibility of FA to 4-VG, but not to 4-EG, and two atypical strains of Candida versatilis which showed no convertibility of FA to 4-VG, but did show convertibility of 4-VG to 4-EG from soy sauce mashes. The bioconversion pathway from FA to 4-EG via 4-VG in halotolerant yeasts and bacteria is discussed.     

Identification and typing of miso and soy sauce fermentation yeasts, Candida etchellsii and C. versatilis, based on sequence analyses of the D1D2 domain of the 26S ribosomal RNA gene, and the region of internal transcribed spacer 1, 5.8S ribosomal RNA gene and internal transcribed spacer 2

We analyzed sequences of the D1D2 domain of the 26S ribosomal RNA gene (26S rDNA sequence), and the region of internal transcribed spacer 1, 5.8S ribosomal RNA gene and internal transcribed spacer 2 (ITS sequence) of the miso and soy sauce fermentation yeasts, Candida etchellsii and Candida versatilis, in order to evaluate the usefulness of this sequence analysis for identification and typing of these two species. In the 26S rDNA sequence method, the numbers of base substitutions among C. etchellsii strains were up to 2 in 482 bp (99.6% similarity), and they were divided into three types (types A, B, and C). Those of C. versatilis strains were also up to 2 in 521 bp (99.6% similarity) and they were divided into three types (types 1, 2, and 3). In the ITS sequence method, those of C. etchellsii strains were zero in 433 bp (type a, 100% similarity). Those of C. versatilis were 5 in 409 bp (98.8% similarity), divided into 4 types (types I, II, III and IV). It was found that molecular methods based on the sequences of the 26S rDNA D1D2 domain and the ITS region were rapid and precise compared with the physiological method for the identification and typing of these two species.     

以玉米淀粉糖渣为原料制备米曲发酵酱油

以玉米淀粉糖渣为原料制备米曲,米曲中的中性蛋白酶活力能够达到4000 U/g(干基),糖化酶比活力可达到120 U/g左右,满足酿造酱油所需要求。用糖渣米曲发酵酱油,氨基态氮和总氮质量浓度分别能够达到7.1 g/L和11.9 g/L,还原糖质量浓度为13.2 g/L,各项理化指标均可达到国家二级酱油标准。     

一株高效利用木糖的酵母菌的分离及鉴定

从256个自然试样中筛选到1株高效转化D-木糖为木糖醇的酵母菌株441-28—1。初始木糖质量浓度为90g/L的条件下,24h内的木糖利用效率为3．0g／（L·h）。通过高效液相分析,菌株441-28—1的主要代谢产物为木糖醇。在初始木糖质量浓度为65g/L的条件下,摇瓶分批发酵,木糖醇生成速率达1．1g／（L·h）,木糖醇转化率为70％。经过形态、生理生化特征测定,以及ITS序列分析（GenBank的登记号为EU121523）,将441-28—1菌株鉴定为热带假丝酵母（Candida tropicalis）。Candida tropicalis（热带假丝酵母）已保存于中国高校工业微生物资源数据平台,保藏编号CICIM Y0092。     

EXPLORING THE OPTIMUM CONDITIONS FOR MAXIMIZING THE MICROBIAL GROWTH OF Candida intermedia BY RESPONSE SURFACE METHODOLOGY

Exploring optimum and cost-efficient medium composition for microbial growth of Candida intermedia Y–1981 yeast culture growing on whey was studied by applying a multistep response surface methodology. In the first step, Plackett–Burman (PB) design was utilized to determine the most significant fermentation medium factors on microbial growth. The medium temperature, sodium chloride and lactose concentrations were determined as the most important factors. Subsequently, the optimum combinations of the selected factors were explored by steepest ascent (SA) and central composite design (CCD). The optimum values for lactose and sodium chloride concentrations and medium temperature were found to be 18.4 g/L, 0.161 g/L, and 32.4°C, respectively. Experiments carried out at the optimum conditions revealed a maximum specific growth rate of 0.090 1/hr; 42% of total lactose removal was achieved in 24 h of fermentation time. The obtained results were finally verified with batch reactor experiments carried out under the optimum conditions evaluated.     

Bioconversion of Ferulic Acid to 4-Vinylguaiacol and 4-Ethylguaiacol and of 4-Vinylguaiacol to 4-Ethylguaiacol by Halotolerant Yeasts Belonging to the Genus Candida

In order to examine the genesis of the characteristic flavors of soy sauce and miso, seven novel halotolerant yeast strains of two types, which showed convertibility of ferulic acid (FA) to 4-vinylguaiacol (4-VG) and to 4-ethylguaiacol (4-EG), were isolated from miso-koji and miso pastes. Two of these strains were identified as Candida guilliermondii (anamorph of Pichia guilliermondii), and Candida fermentati (anamorph of Pichia caribbica), based on sequence analyses of a partial 26S ribosomal RNA gene and the region of internal transcribed spacers 1 and 2, and the 5.8S ribosomal RNA gene. Moreover, we also found three Candida etchellsii strains which showed convertibility of FA to 4-VG, but not to 4-EG, and two atypical strains of Candida versatilis which showed no convertibility of FA to 4-VG, but did show convertibility of 4-VG to 4-EG from soy sauce mashes. The bioconversion pathway from FA to 4-EG via 4-VG in halotolerant yeasts and bacteria is discussed.     

A high salt tolerant neutral protease from Aspergillus Oryzae: Purification, characterization and kinetic properties

A neutral high salt tolerant protease from Aspergillus oryzae CICIM F0899 which could be used for soy sauce production and other relevant applications under high-salt conditions was purified to homogeneity through ammonium sulfate precipitation, ion-exchange chromatography and gel filtration chromatography with overall recovery of 2%. Its molecular weight was estimated to be 50 kDa by SDS-PAGE. The optimum pH and temperature for activity of the extracellular protease of A. oryzae CICIM F0899 were shown to be between 7.0–9.0, and 50°C, respectively. The protease behaved high salt tolerance in 18% NaCl and retained 72% of initial activity after 14 days, indicating the high stability. The enzyme activity was inhibited by metal ions such as Al³⁺ and Ag⁺, and slightly activated by Mn²⁺ and Cu²⁺. A kinetic model incorporating the Debye-Hückel limiting law was proposed for A. oryzae CICIM F0899 protease hydrolysis of casein at ionic strength NaCl from 0.10 to 3.18 M. It was found that, with the higher ionic strength, the Michaelis constant K _m of the protease monotonically increased while the turnover number k _cat decreased in accordance with first order kinetic model. The high-salt tolerant protease has been demonstrated to be promising for the soy sauce production process.     

Investigation of soy sauce treated with nitrite in the chromosomal aberration test in vitro and the micronucleus test in vivo

Soy sauce pretreated with 2300 ppm nitrite caused no more aberrations than did untreated soy sauce in the chromosomal aberration test in vitro using a Chinese hamster fibroblast cell line with or without S9 mixture. The aberration induction by soy sauce is likely to be caused by the 17% sodium chloride it contains. Soy sauce with or without pretreatment with 2300 ppm nitrite was orally given to ICR mice at a dose of 14 ml/kg body weight once or 6 ml/kg body weight/day for 5 consecutive days. This oral administration did not induce any significant increase in micronuclei in the micronucleus test in vivo.     

Candida potacharoeniae sp. nov. and Candida spenceri sp. nov., two novel galactose-containing ascomycetous anamorphic yeast species isolated in Thailand

Fifteen strains of anamorphic yeasts isolated from various natural substrates collected in various places in Thailand were found to represent two novel species of anamorphic yeast genus Candida based on the sequence analysis of the D1/D2 domain of the large subunit rRNA genes, chemotaxonomic and conventional properties used for the classification of yeasts. These strains are located in the clade including Candida etchellsii and Candida magnoliae. Fourteen strains represented by ST-490(T) (BCC 15176(T)=NBRC 106439(T)= CBS 11674(T)) are closely related to Candida sorbosivorans in the D1/D2 sequences but 11 nucleotides (2.4%) were substituted. The remaining strain, ST-594(T) (=BCC 15278(T)=NBRC 106446(T)=CBS 11673(T)) showed a close relationship to Candida geochares but 21 nucleotides (4.7%) were substituted. Apparently, these strains represent two novel Candida species of the Starmerella clade. The two species are described as Candida potacharoeniae sp. nov. and Candida spenceri sp. nov. in the present paper. Like the most species of this clade, the two species contain galactose in the cells in addition to glucose and mannose and have high mol% G + C of 54.4-55.9 and 54.9, respectively.