Recovery of ganoderic acids from <Emphasis Type="Italic">Ganoderma lucidum</Emphasis> mycelia by macroporous adsorption resins

In this work, the performance and adsorption characteristics of macroporous resins for the recovery and enrichment of ganoderic acid (GA)-Mk and GA-T from Ganoderma lucidum mycelia were systematically evaluated. ADS-8 resin displayed the best adsorption and desorption capacities among the tested resins based on batch experiments. The interaction between solute and ADS-8 resin at different temperatures was described in terms of Langmuir and Freundlich isotherms, and the equilibrium experimental data were well fitted to the two isotherms. Thermodynamic analysis indicated the exothermic and spontaneous nature of the adsorption process. The adsorption capacity of ADS-8 resin was found to depend strongly on the pH value of the initial solution. Dynamic adsorption and desorption tests were performed on an ADS-8 resin-packed column to obtain optimal parameters for recovering GA-Mk and GAT from G. lucidum extract. Under optimized conditions, a laboratory scale-up preparation of GA-Mk and GA-T was carried out. The contents of GA-Mk and GA-T were increased from 45 to 22 mg/g in the crude extract to 352 and 141 mg/g in the final product with recovery yields of 90.1 and 72.2%, respectively. These results demonstrated that ADS-8 resin chromatography could act as a useful approach for obtaining ganoderic acids from G. lucidum mycelia.     

Enhanced recovery of four antitumor ganoderic acids from Ganoderma lucidum mycelia by a novel process of simultaneous extraction and hydrolysis

Ganoderic acids (GAs) Mk, T, S and R exhibit promising anti-tumor effect, but they are difficult to purify from Ganoderma lucidum mycelia due to the presence of numerous analogs. In this work, a novel and efficient extraction/hydrolysis method was developed for the recovery of these four GAs from the mycelia of G. lucidum. By using a 50% aqueous ethanol solution containing 50 mmol/l HCl as extractant, extraction of GAs from mycelia and conversion of analogs impurities into the products of interest could be achieved in one step. This one-pot extraction/hydrolysis process increased the yield of GA-Mk, -T, -S and -R to 242%, 389%, 189% and 420%, respectively, compared to a raw sample without hydrolysis. Simultaneous purification of these four GAs was readily achieved in a single RP-HPLC run due to the conversion of analog impurities into corresponding desired GAs, and the purity and recovery of these four GAs were over 97% and 90%, respectively. The results demonstrated that the simultaneous extraction and hydrolysis process is simple and efficient and thus can act as a useful approach for enhanced recovery of those four GAs from G. lucidum mycelia.     

Optimization of physiological factors for direct saccharification of cassava starch to glucose by Rhizopus oligosporus 145f

Direct saccharification of 2.64% cassava starch by Rhizopus oligosporus 145F was attempted under various cultural conditions. Maximum glucose yield of 18.0 g/L culture filtrate was obtained with an initial pH 3.8, 2% (v/v) inoculum of R. oligosporus spores, and an incubation temperature of 45 degrees C in shake flask cultures for 48 h. This concomitantly produced 2.7 g mycelia/100g cassava starch containing 20.2% true protein. The production of glucose and mycelia was accomplished with 92.8% starch saccharification having 67.9% starch to glucose conversion efficiency.     

Ganoderic acid T from Ganoderma lucidum mycelia induces mitochondria mediated apoptosis in lung cancer cells

Ganoderma lucidum is a well-known traditional Chinese medicinal herb containing many bioactive compounds. Ganoderic acid T (GA-T), which is a lanostane triterpenoid purified from methanol extract of G. lucidum mycelia, was found to exert cytotoxicity on various human carcinoma cell lines in a dose-dependent manner, while it was less toxic to normal human cell lines. Animal experiments in vivo also showed that GA-T suppressed the growth of human solid tumor in athymic mice. It markedly inhibited the proliferation of a highly metastatic lung cancer cell line (95-D) by apoptosis induction and cell cycle arrest at G(1) phase. Moreover, reduction of mitochondria membrane potential (Delta psi(m)) and release of cytochrome c were observed during the induced apoptosis. Our data further indicate that the expression of proteins p53 and Bax in 95-D cells was increased in a time-dependent manner, whereas the expression of Bcl-2 was not significantly changed; thus the ratio of Bcl-2/Bax was decreased. The results show that the apoptosis induction of GA-T was mediated by mitochondrial dysfunctions. Furthermore, stimulation of the activity of caspase-3 but not caspase-8 was observed during apoptosis. The experiments using inhibitors of caspases (Z-VAD-FMK, Z-DEVD-FMK and Z-IETD-FMK) confirmed that caspase-3 was involved in the apoptosis. All our findings demonstrate that GA-T induced apoptosis of metastatic lung tumor cells through intrinsic pathway related to mitochondrial dysfunction and p53 expression, and it may be a potentially useful chemotherapeutic agent.     

灵芝悬浮培养中添加微颗粒Talc对灵芝酸产生的影响

灵芝酸是灵芝中重要的药理活性物质,其低产量限制了它的广泛应用和深入研究,高效提高液体发酵中灵芝酸的含量十分必要。以CGMCC 5.65为材料,在悬浮培养条件下,研究添加微颗粒Talc对灵芝酸产生的影响。结果表明,微颗粒Talc添加显著减小了灵芝细胞粒径,对照组为(3.33±0.16)mm,15g/L微颗粒Talc添加组为(2.04±0.12)mm。灵芝细胞中单体灵芝酸和总灵芝酸的含量在微颗粒Talc添加条件下也显著提高。15g/L微颗粒Talc添加组的总灵芝酸含量达到(1.51±0.02)mg/100mg细胞干重,GA-Mk、GA-T和GA-Me的含量最高为(6.02±0.29)、(5.08±0.14)和(1.71±0.09)μg/100mg细胞干重,分别是对照的1.6、4、1.9和1.4倍。另外,15g/L微颗粒Talc添加条件下鲨烯和羊毛甾醇的最大积累量分别为(3.69±0.23)和(34.86±6.41)μg/100mg细胞干重,是对照的2.6和4.2倍;灵芝酸生物合成途径关键基因fps和cyp-5150l8的表达量最高为对照的2.35和1.53倍。     

Heterologous protein secretion by Aspergillus niger growing in submerged culture as dispersed or aggregated mycelia

The secreted production of a heterologous enzyme, hen egg-white lysozyme, by Aspergillus niger was studied in shake flasks containing media of different initial viscosities. Raising the viscosity of the medium by addition of polyvinylpyrrolidone (PVP) brought about a transition in the form of growth from aggregated mycelia (pellets) to dispersed mycelia. The specific yield of lysozyme in cultures containing an initial concentration of 5% (w/v) starch was 8 mg lysozyme/g dry weight. Addition of 2% (w/v) PVP to the medium resulted in a specific yield of 14 mg lysozyme/g dry weight.     

适合飞虱虫疠霉菌丝生产的液体培养基组分及发酵条件

蚜虫、飞虱、叶蝉等是威胁农业生产的刺吸式口器害虫,长期以来依赖化学农药防治,严重影响农产品的安全性和农业生态环境.探讨这类害虫的生物防治途径,减轻农产品化学农药的残留污染,一直受到国内外学者的关注.虫霉(En-tomophthorales)的许多种类具有优异的诱病杀虫能力,尤其通过主动弹射孢子而在害虫种群中高强度流行的特征,可在短期内迅速压低虫口密度,是极其重要的杀虫微生物资源[1～3].     

Effects of Zn supplementation on the growth,amino acid composition,polysaccharide yields and anti-tumour activity of <Emphasis Type="Italic">Agaricus brasiliensis</Emphasis>

The effects of Zn supplementation on the growth, amino acid composition, polysaccharide yields and anti-tumour activity of Agaricus brasiliensis were studied. An initial Zn concentration within the range of 0–300 mg/l had a significant effect on the cell growth and Zn biosorption. At an initial Zn concentration of 300 mg/l, a maximal extracellular polysaccharide (EPS) yield of 5.08 ± 0.25 g/l was obtained, as well as a maximal intracellular polysaccharide (IPS) of 12.25 ± 0.31 mg/g DW. Amino acid analysis results showed that the total amino acid contents of mycelia and culture filtrate decreased from 1090.08 ± 0.76 (233.62 ± 0.06) to 1077.40 ± 0.77 mg/100 g DW (229.52 ± 0.05 mg/l), respectively, while the total essential amino acid contents of mycelia and culture filtrate markedly increased from 429.51 ± 0.86 (58.84 ± 0.05) to 476.9 ± 0.85 mg/100 g DW (59.99 ± 0.04 mg/l), respectively. The anti-tumour activity of Zn-enriched mycelial powder against sarcoma 180 in mice showed that the tumour inhibition ratio was 61.5% and was enhanced markedly as compared to normal mycelial powder of 30.8. The fundamental information obtained in this study will be useful for efficient production of Zn-enriched foods or drugs.     

Decomposition of myceliar matrix and extraction of chitosan from Gongronella butleri USDB 0201 and Absidia coerulea ATCC 14076

Free chitosan, 2 g/100g mycelia from Gongronella butleri and 6.5 g/100g mycelia from Absidia coerulea were isolated by 1M NaOH at 45 degrees C for 13 h and 0.35 M acetic acid at 95 degrees C for 5 h. Both myceliar matrixes did not break down under these conditions. However, myceliar matrix could be decomposed by treatment with 11 M NaOH at 45 degrees C for 13 h and 0.35 M acetic acid at 95 degrees C for 5 h and then extracted the total chitosan, 8-9 g/100g mycelia from both fungi. According to these results, G. butleri has higher amount of complexed chitosan and A. coerulea has higher amount in free chitosan.     

Synthetic activity enhancement of membrane-bound lipase from <Emphasis Type="Italic">Rhizopus chinensis</Emphasis> by pretreatment with isooctane

The cell-bound lipase from Rhizopus chinensis CCTCC M201021 with high catalysis ability for ester synthesis was located as a membrane-bound lipase by the treatments of Yatalase™ firstly. In order to improve its synthetic activity in non-aqueous phase, the pretreatments of this enzyme with various organic solvents were investigated. The pretreatment with isooctane improved evidently the lipase synthetic activity, resulting in about 139% in relative synthetic activity and 115% in activity recovery. The morphological changes of mycelia caused by organic solvent pretreatments could influence the exposure of the membrane-bound enzyme from mycelia and the exhibition of the lipase activity. The pretreatment conditions with isooctane and acetone were further investigated, and the optimum effect was obtained by the isooctane pretreatment at 4°C for 1 h, resulting in 156% in relative synthetic activity and 126% in activity recovery. When the pretreated lipases were employed as catalysts for the esterification production of ethyl hexanoate in heptane, higher initial reaction rate and higher final molar conversion were obtained using the lipase pretreated with isooctane, compared with the untreated lyophilized one. This result suggested that the pretreatment of the membrane-bound lipase with isooctane could be an effective method to substitute the lyophilization for preparing biocatalysts used in non-aqueous phase reactions.     

Comparative study on chemical pretreatment methods for improving enzymatic digestibility of crofton weed stem

In order to utilize and control the invasive weed, crofton weed (Eupatorium adenophorum Spreng), a potential pathway was proposed by using it as a feedstock for production of fermentable sugars. Three chemical pretreatment methods were used for improving enzymatic saccharification of the weed stem. Mild H2SO4 pretreatment could obtain a relatively high yield of sugars in the pretreatment (32.89%, based on initial holocellulose), however, it led to only a slight enhancement of enzymatic digestibility. NaOH pretreatment could obtain a higher enzymatic conversion ratio of cellulose compared with H2SO4 pretreatment. Peracetic acid (PAA) pretreatment seemed to be the most effective for improving enzymatic saccharification of the weed stem in the three chemical pretreatment methods under the same conditions. The conversion ratio of cellulose in the sample pretreated by PAA under the "optimal" condition was increased to 50% by cellulase loading of 80 FPU/g cellulose for 72 h incubation. A number of empirical quadratic models were successfully developed according to the experimental data to predict the yield of sugar and degree of delignification.     

Microbial production of methane from municipal refuse

A heat and caustic pretreatment process has been tested to determine the increase in gas yield that can be obtained from fermentation of organic municipal refuse. A treatment temperature of 130°C and a NaOH concentration of 3 g/100 g of dry solids resulted in the highest gas yield for the conditions tested. The probable increase in gas yield was 20% for the best conditions tested. The treatment procedure also substantially increased the rates of gas production. A high conversion efficiency is possible at much shorter reactor retention times with pretreatment than without pretreatment.     

玉米芯发酵法生物制氢

在批式培养试验中, 以牛粪堆肥为天然产氢菌源, 玉米芯为底物, 通过厌氧发酵生产氢气。系统考察了底物预处理条件、初始pH值和底物浓度对玉米芯产氢能力的影响。在初始pH 8.0, 1.0%盐酸预处理底物30 min, 底物浓度10 g/L的最佳产氢条件下, 玉米芯最大产氢能力〔每克TVS（总挥发性固体物）产氢量〕和最大产氢速率（每克TVS每小时产氢量）分别为107.9 mL /g、4.20 mL/g·h-1。玉米芯经酸预处理后半纤维素含量由42.2%下降至3.0%, 而酸预处理的玉米芯产氢前后纤维素、半纤维素和木质素含量只有少量变化。产氢菌主要用酸预处理产生的可溶性糖产氢, 故底物的酸预处理对玉米芯的发酵产氢非常重要。用傅里叶变换红外光谱(FTIR)分析显示酸预处理和产氢过程中玉米芯的特征峰发生变化, 酸预处理过程降解了底物纤维素的无定形区和半纤维素, 产氢微生物对纤维素的结晶区有破坏作用。     

玉米芯发酵法生物制氢

在批式培养试验中, 以牛粪堆肥为天然产氢菌源, 玉米芯为底物, 通过厌氧发酵生产氢气。系统考察了底物预处理条件、初始pH值和底物浓度对玉米芯产氢能力的影响。在初始pH 8.0, 1.0%盐酸预处理底物30 min, 底物浓度10 g/L的最佳产氢条件下, 玉米芯最大产氢能力〔每克TVS（总挥发性固体物）产氢量〕和最大产氢速率（每克TVS每小时产氢量）分别为107.9 mL /g、4.20 mL/g·h-1。玉米芯经酸预处理后半纤维素含量由42.2%下降至3.0%, 而酸预处理的玉米芯产氢前后纤维素、半纤维素和木质素含量只有少量变化。产氢菌主要用酸预处理产生的可溶性糖产氢, 故底物的酸预处理对玉米芯的发酵产氢非常重要。用傅里叶变换红外光谱(FTIR)分析显示酸预处理和产氢过程中玉米芯的特征峰发生变化, 酸预处理过程降解了底物纤维素的无定形区和半纤维素, 产氢微生物对纤维素的结晶区有破坏作用。     

Adsorption of Lithocholic Acid to Fusarium equiseti M41 as an Essential Process in Its Conversion to Ursodeoxycholic Acid

Fusarium equiseti M41 converts lithocholic acid to ursodeoxycholic acid. Adsorption of lithocholic acid particles to mycelia of F. equiseti M41 is essential in the conversion of lithocholic acid to ursodeoxycholic acid. Production of ursodeoxycholic acid was negligible when particles of lithocholic acid were absent. As the concentration of lithocholic acid particles increased, both the amount of mycelium-bound lithocholic acid and the production of ursodeoxycholic acid increased hyperbolically (K_1/2 = 1.9 g/liter and K_mapparent = 1.9 g/liter. A fluorescent lithocholic acid derivative was used to confirm that insoluble particles of lithocholic acid attached to the surface of the mycelia. The hydrophobic nature of this binding was estimated from the close relationship observed between the hydrophobicity of bile acids and their binding capacity to the mycelia. By repeated washing with 30% dimethyl sulfoxide, two binding modes of lithocholic acid were distinguished, i.e., surface binding (59% of bound lithocholic acid) and tight binding (41% of bound lithocholic acid). From the amount of tightly bound lithocholic acid, the intracellular concentration of lithocholic acid was calculated to be 1,433-fold higher than its saturating concentration in the reaction mixture, thus promoting effective conversion to ursodeoxycholic acid in the mycelia. Several lines of evidence indicated that glycoproteins of the cell wall participated in the binding of lithocholic acid.     

响应面法优化富硒荷叶离褶伞菌丝体胞内粗多糖发酵条件

为了优化富硒荷叶离褶伞菌丝体胞内粗多糖发酵条件,在单因素试验基础上,考察灭菌条件、培养时间、硒添加量和硒添加时间对富硒荷叶离褶伞菌丝体胞内粗多糖的影响,并通过Box-Benhnken实验设计和响应面分析法确定最优发酵条件。结果表明,在121℃、20min条件下对培养基和Na₂SeO₃溶液分别灭菌后混合,在发酵至第129h,发酵液中添加4µg/mL Na₂SeO₃,继续发酵至第244h,发酵产生的富硒荷叶离褶伞菌丝体胞内粗多糖含量为349.8mg/100mL。     

提高蛋白激发子产量的培养基及发酵条件的优化

为了进一步提高极细链格孢菌产蛋白激发子的产量,通过单因子和多因子试验与分析,筛选优化了适于极细链格孢菌产生蛋白激发子的培养基和培养条件,并检测了发酵过程中pH、还原糖、氨基氮和菌丝量变化以及与蛋白激发子产量的关系。结果表明,土豆淀粉和黄豆粉对蛋白激发子产量影响最大,其次是蛋白胨和无机盐。优化的发酵培养基主要成分（g／L）：碳源I 15、葡萄糖5、玉米淀粉5、土豆淀粉20、谷氨酸10、氮源I5、黄豆粉10、硫酸铵5。确定了优化的培养条件,调整培养基起始pH为7．0～7．5,将18h菌龄的种子培养液按10％接种量接种到装液量为75mL的500mL摇瓶中,在温度（28±1）℃、摇床转速180r／min下培养可获得理想的蛋白产量。在优化的培养基和培养条件下,发酵12～48h该菌进入对数生长期,48h进入稳定生长期,60h菌丝扣蛋白激发子产量达最高。蛋白产量与菌体生物量呈正相关,当还原糖、总糖量消耗到最低水平时,菌丝产量和蛋白激发子产量达最高。优化的培养基菌丝干重收率迭3．9g／100mL,蛋白激发子产量达到5．17g／L,比普通的土豆液体培养基提高近4倍。     

Morphological diversity ofMortierella alpina: Effect of consumed carbon to nitrogen ratio in flask culture

The influence of the consumed carbon to nitrogen (C/N) ratio on mycelial morphology was investigated in cultures ofMortierella alpina using shake flasks. The consumed C/N ratio was varied from 5 to 32 under the condition that the total initial amount of the carbon and nitrogen sources was 50 g/L. The whole mycelia and filamentous mycelia exhibited no relationship with the consumed C/N ratio below a consumed C/N ratio of 20 in the presence of either excess carbon or excess nitrogen. However, when the consumed C/N ratio increased higher than 20, the mycelial sizes increased in proportion to the consumed C/N ratio. However, the area ratio of filamentous mycelia to total mycelia was found to be independent of the consumed C/N ratio, and remained constant at 0.82. In the case of a fixed consumed C/N ratio of 20, the whole mycelia and filamentous mycelia increased in proportion to the degree of the medium strength, yet the area ratio of filamentous mycelia to total mycelia remained unchanged at 0.76. Accordingly, these results show that fungal morphology and mycelial size are both affected by the ratio of carbon to nitrogen. The findings of the current study will be helpful in obtaining the efficient production of useful bioproducts from fungal cultures.