两株耐碱性木聚糖酶产生菌的筛选及产酶条件研究

本文主要研究了从造纸厂碱性土壤中筛选得到的，能够产生耐碱木聚糖酶的两株放线菌X24-14和X15-17。通过16 S rRNA基因序列分析并结合菌株的形态特征以及生理生化特性，初步认为菌株X15-17为拟诺卡氏菌属（Nocardiopsis）的一个潜在新种；菌株X24-14为纤维化纤维菌（Cellulosimicrobium cellulans）。 在此基础上探索了菌株X24-14和菌株X15-17所产木聚糖酶的基本酶学性质。研究发现，两株菌所产的木聚糖酶的耐碱性均较强： 1）菌株X24-14所产的木聚糖酶，在pH 4.2～9.4的范围内能维持较高的活力，pH 9.4条件下，仍能保持80％的酶活力；2）菌株X15-17所产的木聚糖酶在pH 4.0～9.0的范围内能维持较高的活力，pH 9.0条件下，仍能保持80％的酶活力；3）两株菌所产的木聚糖酶均具有较好的pH稳定性，在pH 2.0～11.0范围内稳定，pH 11.0、4 ℃条件下处理24 h仍具有75％的活力。 本文还重点研究了菌株X24-14在不同培养基成分及不同培养条件下的产酶情况，确定了其适宜的产酶条件。结果显示，菌株X24-14的最适碳源为麸皮；最适氮源为蛋白胨；最适产酶pH为pH 8.5。菌株X24-14适宜的产酶条件为：麸皮60 g/L，蛋白胨10 g/L，K2HPO4 7.0 g/L，pH 8.5，接种量为5％，37 ℃，200 r/min发酵培养108 h。 Two strains of actinomycetes, X24-14 and X15-17, which produced alkali-tolerant xylanase were screened from the soil samples collected from a pulp mill in china. Based on the morphological, physiochemical characteristics and 16S rRNA sequence, X24-14 was priminarily identified as cellulosimicrobium cellulans ; X15-17 was priminarily identified as a new species of Nocardiopsis. The investigation examined the enzyme activities which produced by X24-14 and X15-17 under different pH and different temperatures. The results showed that : 1）The xylanase from X24-14 had characteristic of alkali-tolerance: It remains 80% relative activity at pH ranges between pH 4.2 and pH 9.4 under 50℃. 2)The xylanase from X15-17 also showed characteristic of alkali-tolerance, it remains 80% relative activity at pH ranges between pH 4.0and pH 9.0 under 50℃. 3）The xylanase from the two strains showed alkali-stable characteristics. They were stable at pH ranges between pH 2.0 and pH 11.0, showing 75% of its maximal activity remaining under 24 hours of treatment at 4℃. We also studied the effect of different growth conditions: carbon source, nitrogen sources, inoculum size, and initial pH on the production of xylanase of strain X24-14. The results showed that :The optimal carbon source was wheat bran; The optima nitrogen source was peptone; The maximum xylanase activity was achieved in the medium containing 60 g/L wheat bran, 10 g/L peptone, 7 g/L K2HPO4, inoculum size 5% and pH 8.5, under 37℃ in 108 h.

Lovastatin production by Aspergillus terreus using lignocellulose biomass in large scale packed bed reactor

The effect of superficial air velocity on lovastatin production by Aspergillus terreus PL10 using wheat bran and wheat straw was investigated in a 7 l and a 1200 l packed bed reactor. Mass transfer and reaction limitations on bioconversion in the 1200 l reactor was studied based on a central composite design of experiments constructed using the superficial air velocity and solid substrate composition as variables and lovastatin production as response.
The surface response prediction showed a maximum lovastatin production of 1.86 mg g-1 dry substrate on day 5 of the bioconversion process when the reactor was operated using 0.19 vvm airflow rate (23.37 cm min-1 superficial air velocity) and 54% substrate composition (wC). Lovastatin production did not increase significantly with superficial air velocity in the 7 l reactor. Variation in temperature and exit CO2 composition was recorded, and the Damköhler number was calculated for lovastatin production at these two scales. The results showed that in larger reactors mass transfer limitation controlled bioconversion while in smaller reactors bioconversion was controlled by reaction rate limitations. In addition, mass transfer limitations in larger reactors reduced the rate of metabolic heat removal, resulting in hot spots within the substrate bed.

Extracellular beta-Glucosidase production by marine Aspergillus sydowii BTMFS 55

The beta-glucosidase enzyme purified from the marine fungus, Aspergillus sydowii BTMFS 55 showed a good yield of enzyme production under solid state fermentation. The statistical optimization of the media components revealed that moisture content, concentration of peptone and inoculum are the major parameters which supported the maximal enzyme production. The purified enzyme showed low pH activity and stability, glucose tolerance and activation by ethanol. It could produce ethanol from wheat bran and rice straw by simultaneous saccharification and fermentation with yeast.The glucosidase purified from Aspergillus sydowii BTMFS 55 shows great potential for several biotechnological applications such as the production of bio-ethanol from agricultural biomass and improvement in the aromatic character of wines and fruit juices through the hydrolysis of flavour glucosidic precursors. There is immense scope for the application of this marine fungus in the biofuel production besides in other industries provided further studies are pursued in exploiting this enzyme and the organism particularly scale up studies with respect to application. There is also ample scope for cloning of the gene encoding beta-glucosidase in domesticated hosts such as Pichia pastoris or S. cerevisiae that can produce ethanol directly from cellulosic biomass.

Chitinase production by.marine fungi

In this thesis an attempt is made to explore the potential of marine fungi for the production of chitinolytic enzymes and to recognize the ability to hydrolyse native chitin through submerged as well as solid substrate fermentation culture conditions, using wheat bran and shellfish processing waste such as ‘prawn waste’ as solid substrates. Attempt was made to isolate a potential chitinase producing fungus from marine environment and to develop an ideal bioprocess for the production ofchitolytic enzymes.Present study indicate scope for utilization of B. bassiana for industrial production of chitinase using prawn waste as solid substrate employing solid substrate fermentation.

Banana waste as substrate for a-amylase production by Bacillus subtilis (CBTK 106) under solid-state fermentation

Bacillus subtilis CBTK 106, isolated from banana wastes, produced high titres of a-amylase when banana fruit stalk was used as substrate in a solid-state fermentation system. The e¤ects of initial moisture content, particle size, cooking time and temperature, pH, incubation temperature, additional nutrients, inoculum size and incubation period on the production of a- amylase were characterised. A maximum yield of 5 345 000 U mg~1 min~1 was recorded when pretreated banana fruit stalk (autoclaved at 121 ¡C for 60 min) was used as substrate with 70% initial moisture content, 400 lm particle size, an initial pH of 7.0, a temperature of 35 ¡C, and additional nutrients (ammonium sulphate/sodium nitrate at 1.0%, beef extract/peptone at 0.5%, glucose/sucrose/starch/maltose at 0.1% and potassium chloride/sodium chloride at 1.0%) in the medium, with an inoculum-to-substrate ratio of 10% (v/w) for 24 h. The enzyme yield was 2.65-fold higher with banana fruit stalk medium compared to wheat bran

Impact of process parameters on chitinase production by an alkalophilic marine Beau6eria bassiana in solid state fermentation

A chitinolytic fungus, Beau6eria bassiana was isolated from marine sediment and significant process parameters influencing chitinase production in solid state fermentation using wheat bran were optimised. The organism was strongly alkalophilic and produced maximum chitinase at pH 9·20. The NaCl and colloidal chitin requirements varied with the type of moistening medium used. Vegetative (mycelial) inoculum was more suitable than conidial inoculum for obtaining maximal enzyme yield. The addition of phosphate and yeast extract resulted in enhancement of chitinase yield. After optimisation, the maximum enzyme yield was 246·6 units g 1 initial dry substrate (U gIDS 1). This is the first report of the production of chitinase from a marine fungus.

Production, purification and partial characterization of a novel protease from marine Engyodontium album BTMFS10 under solid state fermentation

Engyodontium album isolated from marine sediment produced protease, which was active at pH 11. Process parameters influencing the production of alkaline protease by marine E. album was optimized. Particle size of <425 mm, 60% initial moisture content and incubation at 25 8C for 120 h were optimal for protease production under solid state fermentation (SSF) using wheat bran. The organism has two optimal pH (5 and 10) for maximal enzyme production. Sucrose as carbon source, ammonium hydrogen carbonate as additional inorganic nitrogen source and amino acid leucine enhanced enzyme production during SSF. The protease was purified and partially characterized. A 16-fold purified enzyme was obtained after ammonium sulphate precipitation and ion-exchange chromatography. Molecular weight of the purified enzyme protein was recorded approximately 38 kDa by SDS-PAGE. The enzyme showed maximum activity at pH 11 and 60 8C. Activity at high temperature and high alkaline pH suggests suitability of the enzyme for its application in detergent industry

Short Communication: Polystyrene-an inert carrier for L·glutaminase production by marine Vibrio costico/a under selld-state fermentation

Polystyrene beads, impregnated with mineral salts/glutamine medium as inert support, were used to produce L-glutaminase from Vibrio costicola by solid-state fermentation. Maximum enzyme yield, 88 U/g substrate, was after 36 h. Glucose at 10 g/kg enhanced the enzyme yield by 66%. The support system allowed glutaminase to be recovered with higher specific activity and lower viscosity than when a wheat-bran system was used

Extracellular β-glucosidase Production by a Marine Aspergillus sydowii BTMFS 55 under Solid State Fermentation Using Statistical Experimental Design

A potential fungal strain producing extracellular β-glucosidase enzyme was isolated from sea water and identified as ^ëéÉêJ Öáääìë=ëóÇçïáá BTMFS 55 by a molecular approach based on 28S rDNA sequence homology which showed 93% identity with already reported sequences of ^ëéÉêÖáääìë=ëóÇçïáá in the GenBank. A sequential optimization strategy was used to enhance the production of β-glucosidase under solid state fermentation (SSF) with wheat bran (WB) as the growth medium. The two-level Plackett-Burman (PB) design was implemented to screen medium components that influence β-glucosidase production and among the 11 variables, moisture content, inoculums, and peptone were identified as the most significant factors for β-glucosidase production. The enzyme was purified by (NH4)2SO4 precipitation followed by ion exchange chromatography on DEAE sepharose. The enzyme was a monomeric protein with a molecular weight of ~95 kDa as determined by SDS-PAGE. It was optimally active at pH 5.0 and 50°C. It showed high affinity towards éNPG and enzyme has a hã and sã~ñ of 0.67 mM and 83.3 U/mL, respectively. The enzyme was tolerant to glucose inhibition with a há of 17 mM. Low concentration of alcohols (10%), especially ethanol, could activate the enzyme. A considerable level of ethanol could produce from wheat bran and rice straw after 48 and 24 h, respectively, with the help of p~ÅÅÜ~êçãóÅÉë=ÅÉêÉîáëá~É in presence of cellulase and the purified β-glucosidase of ^ëéÉêÖáääìë=ëóÇçïáá BTMFS 55.

Effects of Different Feeding Regimes on the Digestibility and Faecal Excretion of Nitrogen, Soluble Carbohydrates and Fibre Fractions in Water Buffaloes kept under Subtropical Conditions

In the course of the ‘Livestock Revolution’, extension and intensification of, among others, ruminant livestock production systems are current phenomena, with all their positive and negative side effects. Manure, one of the inevitable secondary products of livestock rearing, is a valuable source of plant nutrients and its skillful recycling to the soil-plant interface is essential for soil fertility, nutrient - and especially phosphorus - uses efficiency and the preservation or re-establishment of environmentally sustainable farming systems, for which organic farming systems are exemplarily. Against this background, the PhD research project presented here, which was embedded in the DFG-funded Research Training Group 1397 ‘Regulation of soil organic matter and nutrient turnover in organic agriculture ’ investigated possibilities to manipulate the diets of water buffalo (Bubalus bubalis L.) so as to produce manure of desired quality for organic vegetable production, without affecting the productivity of the animals used. Consisting of two major parts, the first study (chapter 2) tested the effects of diets differing in their ratios of carbon (C) to nitrogen (N) and of structural to non-structural carbohydrates on the quality of buffalo manure under subtropical conditions in Sohar, Sultanate of Oman. To this end, two trials were conducted with twelve water buffalo heifers each, using a full Latin Square design. One control and four tests diets were examined during three subsequent 7 day experimental periods preceded each by 21 days adaptation. Diets consisted of varying proportions of Rhodes grass hay, soybean meal, wheat bran, maize, dates, and a commercial concentrate to achieve a (1) high C/N and high NDF (neutral detergent fibre)/SC (soluble carbohydrate) ratio (HH), (2) low C/N and low NDF/SC ratio (LL); (3) high C/N and low NDF/SC ratio (HL) and (4) low C/N and high NDF/SC (LH) ratio. Effects of these diets, which were offered at 1.45 times maintenance requirements of metabolizable energy, and of individual diet characteristics, respectively, on the amount and quality of faeces excreted were determined and statistically analysed. The faeces produced from diets HH and LL were further tested in a companion PhD study (Mr. K. Siegfried) concerning their nutrient release in field experiments with radish and cabbage. The second study (chapter 3) focused on the effects of the above-described experimental diets on the rate of passage of feed particles through the gastrointestinal tract of four randomly chosen animals per treatment. To this end, an oral pulse dose of 683 mg fibre particles per kg live weight marked with Ytterbium (Yb; 14.5 mg Yb g-1 organic matter) was dosed at the start of the 7 day experimental period which followed 21 days of adaptation. During the first two days a sample for Yb determination was kept from each faecal excretion, during days 3 – 7 faecal samples were kept from the first morning and the first evening defecation only. Particle passage was modelled using a one-compartment age-dependent Gamma-2 model. In both studies individual feed intake and faecal excretion were quantified throughout the experimental periods and representative samples of feeds and faeces were subjected to proximate analysis following standard protocols. In the first study the organic matter (OM) intake and excretion of LL and LH buffaloes were significantly lower than of HH and HL animals, respectively. Digestibility of N was highest in LH (88%) and lowest in HH (74%). While NDF digestibility was also highest in LH (85%) it was lowest in LL (78%). Faecal N concentration was positively correlated (P≤0.001) with N intake, and was significantly higher in faeces excreted by LL than by HH animals. Concentrations of fibre and starch in faecal OM were positively affected by the respective dietary concentrations, with NDF being highest in HH (77%) and lowest in LL (63%). The faecal C/N ratio was positively related (P≤0.001) to NDF intake; C/N ratios were 12 and 7 for HH and LL (P≤0.001), while values for HL and LH were 11.5 and 10.6 (P>0.05). The results from the second study showed that dietary N concentration was positively affecting faecal N concentration (P≤0.001), while there was a negative correlation with the faecal concentration of NDF (P≤0.05) and the faecal ratios of NDF/N and C/N (P≤0.001). Particle passage through the mixing compartment was lower (P≤0.05) for HL (0.033 h-1) than for LL (0.043 h-1) animals, while values of 0.034 h-1 and 0.038 h-1 were obtained for groups LH and HH. At 55.4 h, total tract mean retention time was significantly (P≤0.05) lower in group LL that in all other groups where these values varied between 71 h (HH) and 79 h (HL); this was probably due to the high dietary N concentration of diet LL which was negatively correlated with time of first marker appearance in faeces (r= 0.84, P≤0.001), while the dietary C concentration was negatively correlated with particle passage through the mixing compartment (r= 0.57, P≤0.05). The results suggest that manure quality of river buffalo heifers can be considerably influenced by diet composition. Despite the reportedly high fibre digestion capacity of buffalo, digestive processes did not suppress the expression of diet characteristics in the faeces. This is important when aiming at producing a specific manure quality for fertilization purposes in (organic) crop cultivation. Although there was a strong correlation between the ingestion and the faecal excretion of nitrogen, the correlation between diet and faecal C/N ratio was weak. To impact on manure mineralization, the dietary NDF and N concentrations seem to be the key control points, but modulating effects are achieved by the inclusion of starch into the diet. Within the boundaries defined by the animals’ metabolic and (re)productive requirements for energy and nutrients, diet formulation may thus take into account the abiotically and biotically determined manure turnover processes in the soil and the nutrient requirements of the crops to which the manure is applied, so as to increase nutrient use efficiency along the continuum of the feed, the animal, the soil and the crop in (organic) farming systems.

The effect of carrier substrate, dose rate and time of application on biocontrol efficacy of fungal antagonists against Armillaria root rot of strawberry plants.

In a glasshouse experiment using potted strawberry plants (cv. Cambridge Favourite) as hosts, the effect of selected fungal antagonists grown on 25 or 50 g of mushroom compost containing autoclaved mycelia of Agaricus bisporus, or wheat bran was evaluated against Armillaria mellea. Another glasshouse experiment tested the effect of application time of the antagonists in relation to inoculations with the pathogen. A significant interaction was found between the antagonists, substrates and dose rates. All the plants treated with Chaetomium olivaceum isolate Co on 50 g wheat bran survived until the end of the experiment which lasted 482 days, while none of them survived when this antagonist was added to the roots of the plants on 25 g wheat bran or 25 or 50 g mushroom compost. Dactylium dendroides isolate SP had a similar effect, although with a lower host survival rate of 33.3%. Trichoderma hamatum isolate Tham 1 and T. harzianum isolate Th23 protected 33.3% of the plants when added on 50 g and none when added on 25 g of either substrate, while 66.7% of the plants treated with T. harzianum isolate Th2 on 25 g, or T viride isolate TO on 50 g wheat bran, survived. Application of the antagonists on mushroom compost initially resulted in development of more leaves and healthier plants, but this effect was not sustained. Eventually, plants treated with the antagonists on wheat bran had significantly more leaves and higher health scores. The plants treated with isolate Th2 and inoculated with Armillaria at the same time had a survival rate of 66.7% for the duration of the experiment (475 days), while none of them survived that long when the antagonist and pathogen were applied with an interval of 85 days in either sequence. C. olivaceum isolate Co showed a protective effect only, as 66.7% of the plants survived when they were treated with the antagonist 85 days before inoculation with the pathogen, while none of them survived when the antagonist and pathogen were applied together or the infection preceded protection.

Cultural techniques for improvement in biocontrol potential of fungal antagonists for biological control of Armillaria root rot of strawberry plants under glasshouse conditions

Several in vitro and in vivo experiments were conducted to develop an effective technique for culturing potential fungal antagonists (isolates of Trichoderma harzianum, Dactylium dendroides, Chaetomium olivaceum and one unidentified fungus) selected for activity against Armillaria mellea. The antagonists were inoculated onto (1) live spawn of the oyster mu shroom (Pleurotus ostreatus), (2) extra-moistened or sucrose-enriched mushroom composts containing living or autoclaved mycelia of P. ostreatus or Agaricus bisporus (button mushroom), (3) pasteurized compost with or without A. bisporus mycelium, wheat bran, wheat germ and (4) spent mushroom composts with living mycelia of A. bisporus, P. ostreatus or Lentinus edodes (the Shiitake mushroom). In one experiment, a representative antagonist (isolate Th2 of T. harzianum) was grown together with the A. bisporus mycelium, while in another one, the antagonist was first grown on wheat germ or wheat bran and then on mushroom compost with living mycelium of A. bisporus. Some of the carrier substrates were then added to the roots of potted strawberry plants in the glasshouse to evaluate their effectiveness against the disease. The antagonists failed to grow on the spawn of P. ostreatus even after reinoculations and prolonged incubation. Providing extra moisture or sucrose enrichment also did not improve the growth of Th2 on mushroom composts in the presence of living mycelia of A. bisporus or P. ostreatus. The antagonist, however, grew rapidly and extensively on mushroom compost with autoclaved mycelia, and also on wheat germ and wheat bran. Colonization of the substrates by the antagonist was positively correlated with its effectiveness in the glasshouse studies. Whereas only 33.3% of the inoculated control plants survived in one experiment monitored for 560 days, 100% survival was achieved when Th2 was applied on wheat germ or wheat bran. Growth of the antagonist alone on pasteurized or sterilized compost (without A. bisporus mycelia) and simultaneous growth of the antagonist and mushroom on pasteurized compost did not improve survival over the inoculated controls, but growth over mushroom compost with the living mycelium resulted in 50% survival rate. C. olivaceum isolate Co was the most effective, resulting in overall survival rate of 83.3% compared with only 8.3% for the inoculated and 100% for the uninoculated (healthy) controls. This antagonist gave the highest survival rate of 100% on spent mushroom compost with L. edodes. T harzianum isolate Th23, with 75% survival rate, was the most effective on spent mushroom compost with P. ostreatus, while D. dendroides isolate SP resulted in equal survival rates of 50% on all the three mushroom composts.

Modest doses of beta-glucan do not reduce concentrations of potentially atherogenic lipoproteins

BACKGROUND: In 1997, the US Food and Drug Administration passed a unique ruling that allowed oat bran to be registered as the first cholesterol-reducing food at a dosage of 3 g beta-glucan/d. OBJECTIVE: The effects of a low dose of oat bran in the background diet only were investigated in volunteers with mild-to-moderate hyperlipidemia. DESIGN: The study was a double-blind, placebo-controlled, randomized, parallel study. Sixty-two healthy men (n = 31) and women (n = 31) were randomly allocated to consume either 20 g oat bran concentrate (OBC; containing 3 g beta-glucan) or 20 g wheat bran (control) daily for 8 wk. Fasting blood samples were collected at weeks -1, 0, 4, 8, and 12. A subgroup (n = 17) was studied postprandially after consumption of 2 meals (containing no OBC or wheat bran) at baseline and after supplementation. Fasting plasma samples were analyzed for total cholesterol, HDL cholesterol, triacylglycerol, glucose, and insulin. LDL cholesterol was measured by using the Friedewald formula. The postprandial samples were anlayzed for triacylglycerol, glucose, and insulin. RESULTS: No significant difference was observed in fasting plasma cholesterol, LDL cholesterol, glucose, or insulin between the OBC and wheat-bran groups. HDL-cholesterol concentrations fell significantly from weeks 0 to 8 in the OBC group (P = 0.05). There was a significant increase in fasting glucose concentrations after both OBC (P = 0.03) and wheat-bran (P = 0.02) consumption. No significant difference was found between the OBC and wheat-bran groups in any of the postprandial variables measured. CONCLUSIONS: A low dosage of beta-glucan (3 g/d) did not significantly reduce total cholesterol or LDL cholesterol in volunteers with plasma cholesterol concentrations representative of a middle-aged UK population.

Consumption of a Flaxseed-Rich Food Is Not More Effective Than a Placebo in Alleviating the Climacteric Symptoms of Postmenopausal Women

Our objective in this work was to test the effects of daily intake of bread produced with partially defatted ground flaxseed on the climacteric symptoms and endometrial thickness of postmenopausal women. A double-blind, placebo-controlled, randomized clinical trial was performed with 38 women who had been postmenopausal for 1-10 y and consumed 2 slices of bread containing 25 g of flaxseed (46 mg lignans) or wheat bran (<1 mg lignans; control) every day for 12 consecutive weeks. The outcome variables were the daily number of hot flashes, the Kupperman Menopausal Index (KMI), and endometrial thickness. The plasma lipid profile (total cholesterol and HDL, LDL, and VLDL cholesterol fractions and triglycerides) and the hormones estradiol, follicle-stimulating hormone, thyroid-stimulating hormone, and free thyroxine also were measured. Food intake was evaluated by means of 2 24-hrecalls, before and after the treatment. Twenty patients in the study group and 18 in the control group completed the study. The general characteristics did not differ between the 2 groups at the start of the study. Both had significant, but similar, reductions in hot flashes and KMI after 3 mo of treatment. Moreover, endometrial thickness was not affected in either group. Our findings clearly show that although flaxseed is safe, its consumption at this level (46 mg lignans/d) is no more effective than placebo for reducing hot flashes and KMI. J. Nutr. 140: 293-297, 2010.

Optimization of laccase production from Trametes versicolor by solid fermentation

The regulation of culture conditions, especially the optimization of substrate constituents, is crucial for laccase production by solid fermentation. To develop an inexpensive optimized substrate formulation to produce high-activity laccase, a uniform design formulation experiment was devised. The solid fermentation of Trametes versicolor was performed with natural aeration, natural substrate pH (about 6.5), environmental humidity of 60% and two different temperature stages (at 37 °C for 3 days, and then at 30 °C for the next 17 days). From the experiment, a regression equation for laccase activity, in the form of a second-degree polynomial model, was constructed using multivariate regression analysis and solved with unconstrained optimization programming. The optimized substrate formulation for laccase production was then calculated. Tween 80 was found to have a negative effect on laccase production in solid fermentation; the optimized solid substrate formulation was 10.8% glucose, 27.7% wheat bran, 9.0% (NH4)2SO4, and 52.5% water. In a scaled-up verification of solid fermentation at a 10 kg scale, laccase activity from T. versicolor in the optimized substrate formulation reached 110.9 IU/g of dry mass.