Corncob-induced endo-1,4-beta-d-xylanase of Aspergillus oryzae MTCC 5154: production and characterization of xylobiose from glucuronoxylan

Eight different fungi were cultivated in a peptone-yeast extract medium containing 1% oat spelt xylan (OSX) to evaluate endo-1,4-beta-xylanase secretion for xylooligosaccharide (XOS) production. Aspergillus oryzae MTCC 5154, Aspergillus flavus , Aspergillus niger , and Aspergillus ochraceus showed significant titers of endoxylanases, which were further used for the production of XOS from birch wood xylan (BWX). A. oryzae produced 89.5 +/- 1.13% XOS in the hydrolysate at 24 h of reaction. The effect of OSX, BWX, and raw corncob on the induction of endoxylanase in A. oryzae was studied, and the xylanase activity was maximum at 96 h of cultivation in 3% corncob containing medium. XOS produced at 36 h of reaction was 5.87 +/- 0.53 mg/mL (12 +/- 2% xylose, 48 +/- 2.43% xylobiose, and 40 +/- 3.6% higher oligomers) from 1% BWX . HPLC/refractive index detection and ESI/MS analysis of fractions obtained by GPC corresponded to neutral and 4- O-methyl-alpha- d-glucuronic acid substituted acidic oligosaccharides. The major fraction, beta- d-xylopyranosyl-(1-->4)- d-xylanopyranose was characterized using (13)C NMR.     

不同粮仓中稻谷优势霉菌的鉴定及差异性分析

为了研究稻谷中优势霉菌的种类、数量以及变化趋势,本试验以湖南省3个不同地区稻谷为研究对象,通过初步判断各粮仓优势霉菌,结合分子生物学方法对其ITS 序列进行分析,并对稻谷霉菌进行带菌量测定。结果表明,3个粮仓中,金牛仓稻谷中优势菌株分别为根霉、米曲霉、毛霉、黑曲霉、烟曲霉、黄曲霉、白曲霉、桔青霉;金山仓稻谷中优势菌株分别为根霉、米曲霉、毛霉、黑曲霉、黄曲霉、白曲霉、桔青霉;银光仓稻谷中优势菌株分别为根霉、米曲霉、毛霉、黑曲霉、黄曲霉。3个粮仓中根霉数量分布均表现为上层>下层>中层,米曲霉为中层>上层>下层,毛霉、黑曲霉、黄曲霉的分布为上层>中层>下层,同一霉菌在同一粮仓不同粮层间数量存在显著差异(P<0.05);随着储藏时间的延长,米曲霉、黑曲霉、黄曲霉数量逐渐减少,根霉、毛霉数量增加,同一霉菌在不同储藏时间霉菌数量存在差异显著(P<0.05)。本研究结果为稻谷安全储藏与霉菌防控提供了一定的理论依据。     

Evidence for formation of heterooligosaccharides by Lactobacillus sanfranciscensis during growth in wheat sourdough

Lactobacillus sanfranciscensis is a key organism of the lactic microflora in traditional and industrial sourdough fermentations. In this paper we provide evidence for the formation of heterooligosaccharides (HeOS) by L. sanfranciscensis during growth in sourdough. To identify the HeOS based on HPAEC-PAD analysis, HeOS standards were synthesized by enzymatic reactions with L. sanfranciscensis levansucrase in a chemically defined system in the presence of raffinose, maltotriose, maltose, xylose, or arabinose as acceptor carbohydrates. The oligosaccharides known to originate from the corresponding acceptor reactions, 1(F)-beta-fructosylraffinose, 1(F)-beta-fructofuranosylmaltotriose, erlose (1(F)-beta-fructofuranosylmaltose), xylsucrose, 1(F)-beta-fructosylxylsucrose, and arabsucrose, were identified by HPAEC-PAD. Evidence for the formation of further tri-, tetra-, and pentasaccharides was provided. Wheat doughs with sucrose were fermented with L. sanfranciscesis TMW 1.392 or the isogenic, levansucrase-negative strain TMW 1.392Deltalev, and the analysis of dough extracts or invertase-treated dough extracts provided evidence for the formation of arabsucrose and erlose in sourdough in addition to 1-kestose and nystose.     

Kinetics of hydrolysis of fructooligosaccharides in mineral-buffered aqueous solutions: influence of pH and temperature

High-performance anion exchange chromatography coupled with a pulsed amperometric detection system (HPAEC-PAD) was used to evaluate the extent of chemical hydrolysis of three fructooligosaccharides (FOS) including 1-kestose (beta-D-Fru-(2-->1)(2)-alpha-D-glucopyranoside, GF2), nystose (beta-D-Fru-(2-->1)(3)-alpha-D-glucopyranoside, GF3), and fructofuranosylnystose (beta-D-Fru-(2-->1)(4)-alpha-D-glucopyranoside, GF4). A kinetic study was carried out at 80, 90, 100, 110, and 120 degrees C in aqueous solutions buffered at pH values of 4.0, 7.0, and 9.0. Under each experimental condition, the determination of the respective amounts of reactants and hydrolysis products showed that FOS hydrolysis obeyed pseudo-first-order kinetics as the extent of hydrolysis, which decreased at increasing pH values, increased with temperature. The three oligomers were found to be degraded mainly under acidic conditions, and at the highest temperature value (120 degrees C), a quick and complete acid degradation of each FOS was observed. Using the Arrhenius equation, rate constants, half-life values, and activation energies were calculated and compared with those obtained from sucrose under the same experimental conditions. It appeared that the hydrolysis of FOS took place much more easily at acidic pH than at neutral or basic pH values.     

水稻白叶枯病黄单胞菌rpfF基因的定位及其在致病调控中的作用

检测到水稻白叶枯病菌能产生一种“跨菌调控因子”，在固体平板上，这种因子由白叶枯病菌分泌到细胞外后可以扩散并进入甘蓝黑腐病菌，调控甘蓝黑腐病菌致病因子的表达．本研究从水稻白叶枯病菌中克隆鉴定了一个与这种“跨菌”调控因子合成有关的基因ｒｐｆＦ，通过转座子诱变和标记置换（ｍａｒｋｅｒｅｘｃｈａｎｇｅ）技术，构建了ｒＰｆＦ突变体，突变体丧失了产生这种“跨菌”调控因子的能力，在水稻植株上的致病能力明显减弱，ＤＮＡ序列分析得知ｒｐｆＦ基因编码的产物和大肠杆菌的３－酮脂酰－ＣＯＡ硫解酶（３－ｋｅｔｏａｃｙｌ－ＣＯＡｔｈｉｏｌａｓｅ）同源。     

Methyl jasmonate stimulates aflatoxin B1 biosynthesis by Aspergillus parasiticus.

Aflatoxin B1 (AFB1) is a highly toxic and carcinogenic metabolite produced by certain Aspergillus species on agricultural commodities. One factor promoting the production of aflatoxin is the presence of high levels of fatty acid hydroperoxides often found in plant material under stress. Jasmonic acid (JA) and its methyl ester (MeJA) are derived from linolenic acid, and their biosyntheses involve the production of lipid hydroperoxides. Exposure of aflatoxigenic mold to jasmonates is likely because the mold attacks plant material and possibly initiates the production of jasmonates. In this study the effect of MeJA on the growth of Aspergillus parasiticus and AFB1 biosynthesis is reported. MeJA, at a final concentration of 10(-4) M in yeast extract sucrose medium, did not have any apparent effect on mycelial growth during the 16 days of observation but did increase significantly the levels of AFB1 after the seventh day of growth. After the ninth day, AFB1 production was decreased in contrast to the control cultures, where the production was constantly increasing. AFB1 determination was performed by immunoaffinity and HPLC after derivatization to AFB2a.     

Occurrence of naphtho-gamma-pyrones- and ochratoxin A-producing fungi in French grapes and characterization of new naphtho-gamma-pyrone polyketide (aurasperone G) isolated from Aspergillus niger C-433

A survey on the occurrence on grape of fungi species in 2001 and their capability to produce ochratoxin A (OTA) and naphtho-gamma-pyrones (NGPs) was conducted in different vineyards from several French viticulture regions. The total numbers of fungal isolates, from setting to harvest, were 732. The Aspergillus genus was essentially represented by section Nigri (98.53%) and it was predominant (74.72%) when compared to Penicillium (25.27%). Approximately one third (30.46%) of the fungal isolates were OTA producers, and 94.17% belong to black aspergilli; Aspergillus carbonarius was the main OTA producer. Moreover, 8.33% of isolates (belong to A. carbonarius and A. niger) were NGP producers. However, none of the Penicillium spp. or other Aspergillus spp. isolates can produces NGP derivatives under the conditions used. No other study on NGPs production by fungi isolated from grapes has been reported. In the second part, a novel NGP, named aurasperone G (1), was isolated from the fermentation broth of the culture extracts of Aspergillus niger C-433, strain producer of OTA, along with the known compound aurasperone F (2). The chemical structure of the new polyketide was proposed based on complete (1)H and partial (13)C, COSY, HMQC, 1D NOE NMR spectra as well as UV and MS spectra. This new NGP was not reported before in nature or prepared synthetically.     

黑曲霉和米曲霉发酵改善豆渣口感

豆渣作为豆制品生产的副产品,富含营养。为了解决豆渣颗粒大,口感差,难以直接食用的问题,该文对利用黑曲霉和米曲霉发酵豆渣降低其粒度分布进而改善其口感、增加其可食性进行了研究。结果表明:利用黑曲霉和米曲霉在28℃,相对湿度为95%的条件下发酵,能使渣感减弱,吞咽变易,口感明显改善;对发酵10d后豆渣的外观形态、显微镜观察、粒度分布进行考察,均一致表现为发酵后豆渣颗粒显著变小;黑曲霉发酵豆渣对渣感的降低效果好于米曲霉发酵豆渣和未发酵豆渣;发酵使豆渣颗粒变小是口感改善的主要原因;口感改善的根本原因是发酵豆渣过程中所产生的纤维素酶和半纤维素酶降解了豆渣中的纤维素和半纤维素,导致豆渣颗粒变小的缘故。该研究对豆渣的综合利用提供了新途径。     

氯嘧磺隆高效降解真菌F8的分离和鉴定

从田间多年施用氯嘧磺隆的土壤中,驯化分离得到一株能够以氯嘧磺隆为唯一碳源和能源生长的真菌F8,F8在麦芽汁液体培养基中发酵96h,对10mgL-1氯嘧磺隆的降解率为93.07%。F8明显促进土壤中氯嘧磺隆的降解,20μgkg-1氯嘧磺隆的降解半衰期由对照的46.52d缩短为15.03d。F8在麦芽汁平板培养基上形成的菌落为乳白色,圆形,干燥,绒毛状或粉状,中心突起;经电镜观察,细胞长宽比大于2,形成发达的假菌丝,连接成树枝状;糖类发酵试验表明:F8能发酵葡萄糖、蔗糖、麦芽糖、半乳糖,不能发酵淀粉、乳糖;同化碳源试验表明:F8能利用葡萄糖、蔗糖、麦芽糖,不能利用淀粉、乙酸钠;同化氮源试验表明:F8不能利用硝酸钾,能利用硫酸铵,尿素;能分解脂肪,能胨化牛奶,不能液化明胶,不能同化乙醇,不能产生淀粉化合物,不能产酸,不能产酯。根据其形态特征,生理生化特性、18SrRNA序列分析,初步鉴定F8为酿酒酵母(Saccharomyces cerevisiae)。     

Enrichment of cheeses manufactured from cow's and sheep's milk blends with sheep-like species-related alkylphenols

Enhancement of concentrations of species-related sheep-like alkylphenols, p- and m-cresols and 3- and 4-ethylphenols, in experimental Manchego-type cheeses manufactured from cow's and sheep's milk blends (80:20) by using arylsulfatases was investigated. A food-grade arylsulfatase from Aspergillus oryzae (ATCC 20719) was produced using a stimulatory medium, and crude dried cells were used as the enzyme source. Exogenous arylsulfatases from Helix pomatia and A. oryzae were added to cheese curd, and the amounts of species-related alkylphenols were measured. Arylsulfatase from H. pomatia released limited amounts of alkylphenols in the cheese only when used at a high level. Arylsulfatase from A. oryzae released substantial amounts of alkylphenols during 2 months of ripening. The concentrations of alkylphenols in A. oryzae arylsulfatase-treated cheese were comparable to the previously reported levels present in aged Manchego-type cheeses manufactured from pure sheep's milk.     

Comparative study of the effects of solid-state fermentation with three filamentous fungi on the total phenolics content (TPC), flavonoids, and antioxidant activities of subfractions from oats (Avena sativa L.)

The aim of present work was to investigate the effect of solid-state fermentation with filamentous fungi (Aspergillus oryzae var. effuses, Aspergillus oryzae, and Aspergillus niger) on total phenolics content (TPC), flavonoids, and antioxidant activities of four subfractions of oat, namely, n-hexane, ethyl acetate (EA), n-butanol, and water, and compare them to their corresponding subfractions of unfermented oat. The TPC and total flavonoids increased dramatically, especially in EA subfractions (p < 0.05). The levels of antioxidant activity of subfractions were also significantly enhanced (p < 0.05). The highest antioxidant activities were also found in the EA subfractions. The polyphenols in EA were analyzed by high-performance liquid chromatography at 280 nm. Most polyphenols were increased remarkably, especially ferulic and caffeic acids. There was a clear correlation between the TPC and antioxidant activity. In conclusion, fungi fermentation is a potential bioprocess for increasing the TPC, flavonoids, and antioxidant activities of oat-based food.     

不同培养基对赭曲霉生长及产毒能力的影响

赭曲霉广泛分布在粮食和饲料中,其次级代谢产物赭曲霉毒素A(OTA)具有强毒性和高稳定性。由于同一产毒菌株在不同培养基上产毒能力不同,为筛选得到赭曲霉生长、产毒最优的培养基,选取多种固体培养基、液体培养基和天然培养基接种赭曲霉,对比其生长和产毒的差异性;并通过高效液相色谱法(HPLC)对比5种常用提取方法的回收率。结果表明,赭曲霉在不同培养基上产毒差异较大,天然培养基中产毒最高的是玉米和小麦培养基,OTA产量均在250μg·g~(-1)以上;固体培养基中产毒最高的是DG18培养基,OTA达到107.93 ng·mm~(-2);赭曲霉在液体培养基中的产毒十分微弱且只有在YES液体培养基中产毒,OTA产量仅为15.39 ppb。HPLC检测发现,5种常用的OTA提取方法中,甲醇直接提取法的回收效果最好,回收率达到106.64%;氯仿萃取法次之,回收率为87.28%。本研究结果为OTA检测、赭曲霉生长和产毒机制的探究提供了理论依据。     

酸性β-甘露聚糖酶高产菌株的诱变育种

摘要：以一株实验室保藏的酸性β-甘露聚糖酶产生菌黑曲霉（Aspergillus niger）LW-1作为原始出发菌株,首先经自然分离筛选出一株产酶较稳定的N-9作为诱变出发菌株,再经真空微波和甲基磺酸乙酯（EMS）逐级诱变处理,采用基础发酵培养基固态发酵筛选以及斜面传代培养,获得了一株高产、稳产酸性β-甘露聚糖酶的E-30菌株。其产β-甘露聚糖酶活性达36 675 U/g,是原始出发菌株（17 048 U/g）的2.15倍。E-30菌株三角瓶麸曲种子保藏两个月,产酶活性稳定。     

水稻白叶枯病黄单胞菌编码顺乌头酸酶的rpfA基因的鉴定

通过生化功能分析，证实了水稻白叶枯病黄单胞菌野生型菌株Ｔ３０００产生两种顺乌头酸酶（分别命名为ＸｏｏＡｃａｎＡ和ＸｏｏＡｃａｎＢ）。通过三亲本接合的方法，将获得的含水稻白叶枯病黄单胞菌ｒｐｆ基因簇的重组质粒ｐＧＸＮ３０００导入甘蓝黑腐病黄单胞菌ｒｐｆＡ基因突变体８４７６中。经生化功能分析证实，８４７６／ｐＧＸＮ３０００中含有水稻白叶枯病黄单胞菌的顺乌头酸酶ＸｏｏＡｃａｎＡ；同时还证实了ＰＧＸＮ３０００能互补８４７６，恢复其合成胞外酶和胞外多糖的能力及致病性．这表明在ＰＧＸＮ３０００中含有一个完整的水稻白叶枯病黄单胞菌的顺乌头酸酶基因，该基因具有类似甘蓝黑腐病黄单胞菌ｒｐｆＡ基因的功能．因此将该基因命名为水稻白叶枯病黄单胞菌ｒｐｆＡ基因。     

一株苯酚降解菌的筛选、鉴定及其降解特性

本研究采用逐量分批驯化的方法,从造纸废水中分离得到一株能够以苯酚为唯一碳源生长的苯酚降解菌株F5-1。经形态观察、生理生化特性鉴定及16S rDNA序列分析,将该菌株鉴定为克雷伯菌（Klebsiella sp.）。该菌株能够在7h时完全降解初始浓度为100mg/L的苯酚,降解苯酚主要发生在生长对数期;在pH5.0～9.0,NaCl浓度0～80g/L,温度20～40℃范围内,菌株F5-1均可有效降解初始浓度为100～1200mg/L的苯酚;能够耐受的最大苯酚浓度为1500mg/L。本研究结果表明,F5-1菌株对处理环境条件复杂的含酚废水具有潜在的应用前景。     

水稻秸秆降解复合菌系的筛选构建及其田间应用效果

  【目的】  筛选并构建适宜原位还田水稻秸秆快速腐解的高效多功能复合菌系,以提高秸秆原位还田的腐解速率。  【方法】  秸秆原位腐解菌株从水稻田带有腐烂秸秆的表层土壤中筛选分离,分别采用DNS法、摇瓶培养观察法和失重法测定了腐解菌株的羧甲基纤维素酶活性、滤纸崩解能力及水稻秸秆降解能力,对降解效果较好的菌株进行了16S rRNA或18S rRNA鉴定。选取无拮抗作用的菌株构建复合菌系,并在实验室条件下测定各复合菌系的滤纸酶活性和秸秆降解率。挑选降解效果较好的复合菌系进行田间试验,分析了秸秆降解率、土壤有机质及速效养分含量,调查了水稻产量。  【结果】  从土壤中共分离到秸秆降解率均高于19.8%的菌株有6个,包括B2、B5、B6、F1、F5、F12,经鉴定分别为贝莱斯芽孢杆菌(Bacillus velezensis,B2)、耐盐芽孢杆菌(Bacillus halotolerans,B5)、枯草芽孢杆菌(Bacillus subtilis,B6)、米曲霉(Aspergillus oryzae,F1)、黑曲霉(Aspergillus niger,F5)、长枝木霉(Trichoderma longibrachiatum,F12),其中F12 (长枝木霉)的秸秆降解率最高(29.1%)。以该6个菌株构建了7组复合菌系,复合菌系B2+F12、F1+F5+F12、B2+F5+F12的秸秆降解效果较好,在液体培养基中,对秸秆的降解率分别达到32.9%、29.8%、40.3%。利用这3组复合菌系进行田间试验,结果表明,复合菌系B2+F5+F12表现最好,与未施菌剂相比,原位还田的水稻秸秆降解率提高了37.1个百分点,土壤有机质、碱解氮、速效磷、速效钾含量分别增加了2.1 g/kg、1.9 mg/kg、0.6 mg/kg、1.7 mg/kg,水稻产量提高了6.3% (P<0.05)。  【结论】  施用复合菌系B2+F5+F12不仅具有良好的水稻秸秆降解能力,同时能增加土壤有机质、碱解氮、速效磷、速效钾含量,并促进作物产量的提高,具有较好的应用潜力。     

Disease control effect of strevertenes produced by Streptomyces psammoticus against tomato fusarium wilt

During screening of microorganisms producing antifungal metabolites, Streptomyces psammoticus strain KP1404 was isolated. The culture extract of this strain showed potent disease control efficacy against Fusarium wilt on tomato plants. The antifungal metabolites ST-1 and ST-2 were isolated from the culture extract using a variety of chromatographic procedures. On the basis of MS and NMR spectrometric analysis, the structures of the antifungal active compounds ST-1 and ST-2 were determined to be the polyene antibiotics strevertene A and strevertene B, respectively. In vitro, strevertenes A and B showed inhibitory effects against the mycelial growth of Alternaria mali , Aspergillus oryzae , Cylindrocarpon destructans , Colletotrichum orbiculare , Fusarium oxysporum f.sp. lycopersici, and Sclerotinia sclerotiorum , even at concentrations of 4-16 μg/mL. Fusarium wilt development on tomato plants was strongly retarded by treatment with 1 μg/mL of these strevertenes. The disease control efficacies of strevertenes on Fusarium wilt were as remarkable as that of benomyl.     

Changes in free amino acid and sugar levels of dried figs during aflatoxin B1 production by Aspergillus flavus and Aspergillus parasiticus

An aqueous slurry of gamma-irradiated sterilized dried figs was inoculated with toxigenic strains of Aspergillus flavus and Aspergillus parasiticus. During incubation at 28 degrees C, pH, fructose, glucose, and free amino acids were determined by high-performance liquid chromatography and liquid chromatography (LC)/mass spectrometry, respectively, over 13 time points (1-20 days). At the same 13 time points using a LC/time-of-flight mass spectrometry screening method, aflatoxin B 1 and other secondary metabolites were simultaneously monitored. During the course of incubation, the pH significantly decreased and aflatoxin B 1 formation correlated with a reduction in proline content for both fungi. Of the 22 free amino acids that were monitored, only proline and cystine were found to be critical in supporting aflatoxin production. Levels of fructose and glucose steadily declined during incubation, until glucose was almost exhausted after 21 days. These time-course experiments confirmed the need for carbon and nitrogen sources for aflatoxin production in dried figs, and the favorable composition of figs as a fungal growth medium.     

蒸汽爆破预处理和微生物发酵对玉米秸秆降解率的影响

为了提高玉米秸秆的利用效率,首先对玉米秸秆进行蒸汽爆破预处理（压力2.5 Mpa,维压200 s）,然后再进行米曲霉发酵,研究物理和生物学处理对秸秆成分及相关酶活变化的影响。结果表明,蒸汽爆破使秸秆中纤维素、半纤维素和木质素的降解率分别达到8.47%、50.45% 和36.65% （p＜0.05）。爆破预处理的秸秆再经米曲霉发酵6 d后,秸秆中纤维素和半纤维素的降解率分别为27.89%和64.80% （p＜0.05）,发酵秸秆中的滤纸酶、羧甲基纤维素酶、淀粉酶和蛋白酶活力分别达到335.10、1138.92、1954.20和201.99 U/g。爆破预处理后进行米曲霉发酵,对于提高玉米秸秆的降解率具有非常重要的意义。     

Production of trichothecenes and other secondary metabolites by Fusarium culmorum and Fusarium equiseti on common laboratory media and a soil organic matter agar: an ecological interpretation

Fusarium culmorum and F. equiseti were characterized with regard to production of trichothecenes and other secondary metabolites. Results following growth on laboratory media are interpreted with the aim of increasing the understanding of fungal metabolism in the field environment. While trichothecene production was detected for 94 of 102 F. culmorum isolates, only 8 of 57 F. equiseti isolates were positive. Profiles of secondary metabolites were compared by following growth on yeast extract sucrose agar (YES), potato sucrose agar (PSA), and an agar medium, prepared from soil organic matter (SOM), which was included to simulate growth conditions in soil. SOM supported the production of chrysogine by F. culmorum. The two species utilized the media differently. F. culmorumproduced zearalenone (ZEA) on YES, whereas some F. equiseti isolates produced ZEA on PSA. Other F. equiseti isolates produced equisetin. These differences may reflect that F. culmorum depends on a pathogenic life style while F. equiseti has a more saprotrophic mode of existence.