pH对灵芝液态发酵代谢物及抗氧化活性的影响

已有研究报道灵芝栽培生长的最适pH在中性偏酸环境,在碱性范围的生长及代谢情况鲜见报道。本研究主要探究广泛pH对灵芝液态发酵代谢物及其抗氧化活性的影响。采用摇瓶液态培养后分析代谢物中灵芝三萜、胞内外多糖、菌丝体蛋白及抗氧化活性等指标,系统比较灵芝菌丝体在pH值2-11的生长和代谢情况。研究结果表明,灵芝菌丝体生长、合成灵芝三萜、胞内多糖、30E胞外多糖、菌丝体蛋白和菌丝体水解氨基酸的最适pH值分别为10、3、2、7、2和2。对应结果分别为17.13 g/L、33.86 mg/g、72.73 mg/g、7.86 g/L、71.42 mg/g和107.10 mg/g。比对照分别提高28.5%、77.3%、22.4%、96.5%、97.1%和70.8%。胞内多糖组分1和组分2最高分子量均在初始pH 4,分别为1.016×10⁸ g/mol和9.280×10⁴ g/mol,胞外多糖组分1最高分子量在初始pH 10,为4.946×10⁶ g/mol;对菌丝体的总抗氧化能力、1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2- picrylhydrazyl,DPPH)自由基清除能力、羟自由基清除能力分析结果表明最佳的初始pH分别为3、7、9。本研究为液态发酵方式下灵芝生长及其代谢物定向调控发酵的工艺优化提供参考依据,同时发现灵芝菌丝体中优质蛋白及抗氧化活性可在功能性食品和化妆品领域推广应用。     

绒毛栓孔菌液体培养过程中胞外酶活性的研究

本研究以绒毛栓孔菌为材料,采用液体培养的方法分析其在发酵过程中胞外酶的活性变化,并对其菌丝体生物量和发酵液pH值进行了测定。结果表明:胞外酶活性与菌丝体生长状况密切相关。菌丝体生物量增长呈"S"型,6～8d增长最快,第12天达到最大值,在此过程中漆酶、锰过氧化物酶、淀粉酶、羧甲基纤维素酶、果胶酶和蛋白酶活性均出现高峰。酶活性的变化表明,在液体培养过程中绒毛栓孔菌首先分解木质素,其次利用淀粉和纤维素作为碳源,蛋白质作为氮源。若要获得最大菌丝体生物量,缩短培养时间,就必须在培养过程中保证碳氮源的均衡供给。本试验说明不同的酶其分泌高峰期可以作为判断菌丝体营养利用情况和培养周期的依据,以此获取最大菌丝体生物量,为工业生产利用奠定基础。     

通气量对灵芝菌丝体液态深层发酵合成灵芝三萜的影响

采用5 L搅拌式发酵罐研究灵芝菌丝体液态深层发酵合成灵芝三萜的扩大培养条件,考察了不同条件下的通气量对灵芝菌丝体生长、比生长速率、灵芝三萜合成、比合成速率、还原糖消耗、还原糖比消耗速率、铵根离子消耗以及铵根离子比消耗速率的影响。研究结果表明,通气量能够显著影响菌丝体的生长以及灵芝三萜的合成,当通气量为8 L/min时,菌丝体合成灵芝三萜得率和生产强度最高,分别为0.204 g/L和0.00131 g/(L·h),最大菌丝体得率达到8.77 g/L。发酵过程中菌丝体最大比生长速率为0.0704 1/h,灵芝三萜最大比合成速率为0.0256 1/h,还原糖和铵根离子最大比消耗速率分别为0.562 1/h和0.0171 1/h。     

灵芝液体发酵条件的优化研究

通过单因素和正交试验,优化灵芝5760深层培养的营养需求与环境条件对灵芝菌丝体生物量及次生代谢产物发酵液多糖的作用。结果表明,适宜灵芝液体菌种培养和液体发酵的培养基配方为麦麸粉5%,黄豆饼粉3%,KH2PO40.10%,MgSO40.09%;适宜的环境条件为初始pH5.5,装液量32%,生物量最大时为第8～9d;适宜接种的种龄为6d,接种量为10%;发酵周期6d,搅拌转速150r/min,消沫剂添加量可为0.20%。从硒对灵芝5760菌丝体生长及深层发酵生物量的影响,灵芝5760富硒培养适宜浓度为100μg/mL;从加硒时间对生物量的作用,在菌龄适宜时期加入即第1d。     

金针菇液态发酵培养基的筛选

研究不同碳源、氮源及无机盐对金针菇菌丝体产量的影响,采用正交设计法进行金针菇液态发酵培养基配方筛选和优化,确定了最佳培养基配方为大米粉3%、酵母粉1%、KH2PO40.1%、MgSO4.7H2O 0.05%。同时利用此配方培养测定了发酵过程pH、还原糖和氨基氮含量的变化,结果表明:发酵过程中pH变化很小,培养末期略有上升;还原糖含量呈先增后降的变化,氨基氮含量呈上升趋势。     

芦竹内生真菌F0238细胞生长及其代谢调控*

对芦竹内生真菌F0238的细胞生长和代谢产曲酸量进行了代谢调控。结果表明,F0238生长及产曲酸的营养和环境条件为:PDA培养基,8%淀粉为碳源,0.2%蛋白胨为N源,发酵温度28℃,初始pH值为6.5,发酵时间5d/(120h),装液量80mL/500mL三角瓶。在摇瓶试验的基础上,对该菌发酵过程作了初步放大试验(10L全自动发酵罐),得到F0238发酵过程的动态曲线。动态曲线反映了在一个发酵周期内,发酵液的pH值、DO值及残糖的降低趋势和生物量与抗菌产物量的上升趋势。     

产糖化酶黑曲霉的固定化研究

采用多孔聚酯材料作为固定化载体,考察并比较了载体吸附固定化黑曲霉菌丝细胞的条件,当菌丝体细胞与载体预培养的条件为pH值5.0、孢子浓度为105个/ml、固液比为1/75时,有利于菌丝体的生长、吸附固定及发酵产酶.在产糖化酶的发酵过程中,与游离菌丝体细胞相比,发酵过程持续产酶时间有一定程度的延长,产糖化酶活力始终高于游离菌丝体.     

芦竹内生真菌F0238细胞生长及其代谢调控

对芦竹内生真菌F0238的细胞生长和代谢产曲酸量进行了代谢调控。结果表明,F0238生长及产曲酸的营养和环境条件为：PDA培养基,8％淀粉为碳源,0．2％蛋白胨为N源,发酵温度28℃,初始pH为6．5,发酵时间5d／（120h）,装液量80mL/500mL三角瓶。在摇瓶试验的基础上,对该菌发酵过程作了初步放大试验（10L全自动发酵罐）,得到F0238发酵过程的动态曲线。动态曲线反映了在一个发酵周期内,发酵液的pH值、DO值及残糖的降低趋势和生物量与抗菌产物量的上升趋势。     

假蜜环菌液体深层发酵条件的研究

对该真菌菌丝体的液体深层培养条件进行了探讨为进一步工业化发酵提供参考数据。方法：在基本培养基的基础上分别改变碳源、氮源、无机元素和C／N比对该菌的液体深层发酵情况,包括测量各培养条件下耗糖情况、绘制生长曲线,观察菌体的生长形态等等。结果：假蜜环菌的菌丝产量在第6d可达到最大值,其后会逐渐降低,并伴有溶菌的现象发生。在培养初期加入微量钴元素有利于刺激糖代谢提高菌体对糖的利用率,其作用主要是缩短延滞期。进入指数生长期后连续补加碳源有利于菌体迅速生长。优化后培养基组成为：葡萄糖l％和蔗糖1％为碳源,以酵母浸膏l％为氮源,KH2PO4 0．1％,MgSO4 0．05％Z,元素钴适量。液体深层发酵培养6d后菌丝生长状态已达到最佳,在此条件下搅拌发酵培养可收获20mg/ml(干重)的菌丝体。     

海洋真菌Asteromyces sp.TM76木聚糖酶生产初步研究

研究了海洋真菌Asteromyces sp．TM76的生长动力学和发酵产酶情况。在生长培养基中,TM76菌的最大比生长速率为1．875（1／d）,倍增时间为8．87h;TM76发酵过程中所产木聚糖酶有两个生产高峰,第7d,有最大木聚糖酶活力470．5U／mL。发酵起始pH为6．5时对产酶最佳。酶学性质研究表明该菌所产木聚糖酶的最适反应pH和温度分别为6．5和55℃,在不同温度保温1h,测定酶的半失活温度为63℃。     

钙对金针菇生长发育的影响

研究了CaCO3粉末对金针菇(Flammulina velutipes)生长发育的影响及子实体的富钙作用。结果表明,钙能显著刺激金针菇菌丝体和子实体的生长,缩短出菇时间,提高产量和子实体含钙量。这些生理生化变化与钙浓度密切相关,低质量分数(0~5%)和高质量分数(5.5~10%)的CaCO3粉末造成了相似的变化。初步分析认为,这可能与金针菇细胞内存在两种不同的钙信号转导途径有关。     

Purification and characterization of a fibrinolytic protease from a culture supernatant of Flammulina velutipes mycelia

In this study we purified a fibrinolytic enzyme from the culture supernatant of Flammulina velutipes mycelia by ion exchange and gel filtration chromatographies, it was designated as F. velutipes protease (FVP-I). This purification protocol resulted in 18.52-fold purification of the enzyme at a final yield of 0.69%. The molecular mass of the purified enzyme was estimated to be 37 kDa by SDS-PAGE, fibrin-zymography and size exclusion by FPLC. This protease effectively hydrolyzed fibrin, preferentially digesting alpha-chain over beta-and gamma-gamma chain. Optimal protease activity was found to occur at a pH of 6.0 and a temperature of 20 to 30 degrees C. The protease activity was inhibited by Cu2+, Fe2+ and Fe3+ ions, but was found to be enhanced by Mn2+ and Mg2+ ions. Furthermore, FVP-I activity was potently inhibited by EDTA and EGTA, and it was found to exhibit a higher specificity for chromogenic substrate S-2586 for chymotrypsin, indicating that the enzyme is a chymotrypsin-like metalloprotease. The first 20 amino acid residues of the N-terminal sequence of FVP-I were LTYRVIPITKQAVTEGTELL. They had a high degree of homology with hypothetical protein CC1G_11771, GeneBank Accession no. EAU86463.     

金针菇富锌条件及锌结合形态的研究

对金针菇深层发酵富锌培养条件进行了研究,并对Zn的生物有机化程度进行了初步探讨。结果表明,金针菇富锌的深层培养较好的Zn源为ZnAc_2,初始pH值为6.5,加入0.1％柠檬酸和0.4％的CMC-Na利于富锌和提高生物量。金针菇富锌过程中,93％以上的ZnAc_2以有机锌态存在,其中50％以上结合为Zn-蛋白,34％与糖及脂肪类物质结合。     

An agglutinin with mitogenic and antiproliferative activities from the mushroom Flammulina velutipes

A hemagglutinin with a molecular mass of 12 kDa was isolated from the fruiting bodies of the mushroom Flammulina velutipes. Its molecular mass is similar to that of the fungal immunomodulatory protein isolated from F. velutipes (FIP-fve) with ice-cold 5% acetic acid and 50 mM 2-mercaptoethanol as extraction medium and to that of the larger 12 kDa subunit of F. velutipes lectin isolated with phosphate buffer as extraction medium. Its hemagglutinating activity cannot be inhibited by a variety of carbohydrates tested. The activity is stable between pH 4 and pH 11. Loss in activity occurred when the temperature is raised to 60 C and 70 C. Activity is indiscernible at and above 80 C. Its N-terminal sequence shows differences from that of FIP-fve. F. velutipes hemagglutinin stimulates [3H-methyl] thymidine uptake by mouse splenocytes. It inhibits proliferation of leukemia L1210 cells with an IC50 of 13 microM.     

两种精油熏蒸处理对金针菇贮藏品质的影响

为了开发高效的食用菌绿色保鲜剂,以新鲜的白色金针菇为供试材料,以花椒精油和丁香精油为供试熏蒸剂,分别在常温（25±1）℃和低温（4±1）℃条件下开展了适用于金针菇保鲜的精油种类和浓度的筛选试验,并对金针菇贮藏期内的感官评价、失重率、呼吸强度、褐变度、多酚氧化酶（PPO）活性、苯丙氨酸解氨酶（PAL）活性、丙二醛（MDA）以及总酚含量进行了测定。结果显示,常温（25±1）℃条件下用0.1 mL·kg-1花椒精油和0.5 mL·kg-1丁香精油保鲜效果优于其他处理,且有统计学意义（P<0.05）,0.1 mL·kg-1花椒精油和0.5 mL·kg-1丁香精油处理组感官评分分别高于对照组23.4%和27.8%,二者均能够抑制金针菇褐变、减轻腐败变质,且有统计学意义（P<0.05）;在低温（4±1）℃贮藏试验中发现,0.1 mL·kg-1花椒精油和0.5 mL·kg-1丁香精油均能有效抑制呼吸强度和PPO活性的升高（P<0.05）,其呼吸高峰较对照组分别降低了28.3%和39.6%;贮藏15 d后,精油处理组PPO活性较对照组分别降低了8.2%和16.6%;精油处理有效降低了MDA含量的产生,保持着较高的总酚含量、减轻腐烂褐变的程度。第15天时,对照组MDA含量为1.75 μmol·g-1,而花椒精油和丁香精油处理组MDA含量分别比对照组低0.15、0.40 μmol·g-1,丁香精油处理组显著低于花椒精油处理组和对照组（P<0.05）。研究结果表明,0.1 mL·kg-1花椒精油和0.5 mL·kg-1丁香精油均对金针菇采后贮藏保鲜效果显著,其中,0.5 mL·kg-1丁香精油的保鲜效果最明显,在15d的贮藏期内,金针菇依然保持着良好的品质,而对照组已经轻微褐变,部分开始腐烂。研究结果为花椒精油和丁香精油应用于金针菇采后贮藏保鲜提供了理论依据。     

金针菇担孢子核相及遗传属性的研究

以3个不同的金针菇菌株为材料,研究了其担孢子的核相及遗传属性。荧光染色观察显示,担孢子核相以双核为主,双核孢子、单核孢子和无核孢子分别占80.2%、7.5%和12.3%。源于单孢分离物的菌丝为有隔膜、无锁状联合的多核菌丝。在交配试验中,源于不同菌株单孢分离物的菌丝原生质体的配对形成具锁状联合的菌落,而源于同一单孢分离物的菌丝原生质体的配对则形成无锁状联合的菌落,暗示担孢子中的两个核具有相同的交配型。RAPD分析显示,源于同一单孢分离物的菌丝原生质体为10个随机引物所扩增的图谱彼此完全相同,印证了担孢子中的双核是同质的。此外,观察表明,一个担子上着生有4个担孢子。因此,金针菇是一种具4个含同质双核担孢子的四极性蕈菌。     

金针菇褐化病毒(FvBV)脱毒方法

【目的】评价5种不同脱毒方法对金针菇(Flammulina velutipes)菌株的脱毒效果,筛选出脱毒率高和脱毒后金针菇菌株菌丝生长速度、生物量、漆酶活力等性状改善明显的脱毒方法。【方法】以栽培金针菇菌株F-4889为研究材料,从菌丝体中提取大小约2.0 kb的病毒dsRNA,经RT-PCR鉴定该病毒为金针菇褐化病毒(FvBV)。采用菌丝尖端分离、原基组织分离、原生质体单核化、有性生殖和核迁移5种脱毒方法对金针菇菌株进行脱毒处理,利用dsRNA技术和RT-PCR检测脱毒效果。【结果】菌丝尖端分离脱毒后得到1株脱毒菌株;原基组织分离法未能脱毒;原生质体单核化脱毒法得到3株脱毒单核菌株和2株原单杂交脱毒菌株;有性生殖脱毒法获得脱毒孢子单核菌株23株和单孢杂交脱毒菌株8株;核迁移脱毒后得到5株核迁移脱毒菌株。脱毒率依次为25.0%、0、7.5%、57.5%和100%。脱毒菌株的菌丝生长速度、生物量、漆酶活力等均优于出发菌株、菌丝尖端和原基组织分离菌株。【结论】这5种方法中原生质体单核化、有性生殖和核迁移脱毒法脱毒效果较佳,均能有效脱除FvBV,脱毒率高,脱毒后菌株菌丝生长速度、生物量、漆酶活力等均明显提高。     

Anti-complementary activity of endopolymers produced from submerged mycelial culture of higher fungi with particular reference to Lentinus edodes

The anti-complementary activity (immuno-stimulating activity) was tested for the endo-polymer (intra-cellular polymer) produced from submerged mycelial cultures of 21 types of higher fungi. Anti-complementary activity of endo-polymer from Lentinus edodes showed the highest (85%) followed by Flammulina velutipes (77%) and Phellinus linteus (71%). © Rapid Science Ltd. 1998     

The first characterized asparaginase from a basidiomycete, Flammulina velutipes

Flammulina velutipes enjoys high popularity as an edible mushroom in Asian cuisines. Investigating the secretion of peptidases in nutrient media enriched with gluten, an enzyme was noticed that catalyzed the deamidation of L-asparagine and L-glutamine. The enzyme was purified to electrophoretic homogeneity by foaming and SEC. PAGE analysis revealed a protein of about 85 kDa with 13 kDa subunits indicating a hexameric protein. Degenerated primers were deduced from peptide fragments and the complete coding sequence of 372 bp was determined. The gene of Flammulina velutipes asparaginase (FvNase) over expressed in E. coli achieved an L-asparagine-hydrolyzing activity of 16 U/mL in crude extract, which was five times higher than its L-glutamine-hydrolyzing ability. The enzyme showed a pH-optimum at pH 7, remarkable tolerance towards elevated temperature and sodium chloride concentration in both the native and recombinant form, and no significant homology to any conserved domains of published asparaginases or glutaminases.     

Host feeding,damage and control of the mushroom pest,Brennandania lambi (Acari: Pygmephoroidea) in China

The pygmephoroid mite, Brennandania lambi (Krczal), is the most serious mite pest of mushroom production in Shanghai. China. This mite could not develop and reproduce on the mycelia of Lentinus edodes, Flammulina velutipes, Pleurotus ostreatus or Pleutotus sajor-caju, but 12–20% of the females survived 15 to 31 days on these hosts, whereas all mites died after 8 days in absence of food. When fed the mycelia of Auricularia polytricha, B. lambi adults could become gravid and reproduce but active larvae failed to develop to the next stage. B. lambi developed and reproduced on the mycelia of Auricularia auricula, Hericium erinaceus, Tremella fuciformis and Agaricus bisporus. Mobile stages of B. lambi damage A. bisporus by feeding on its mycelia. Field surveys of mite damage during 1983–1989 in the suburbs of Shanghai revealed that contaminated spawn was the major source of the mite problem and losses of the crop averaged between 10% and 20%. Experiments on the effects of temperature on mite survival indicate that exposure of mites at 50° C for 1 hour or-10°C for 24 hours killed all stages of B. lambi. Thus, heating treatment during well-executed phase II composting and freezing of mushroom spawn at-10° C for 24 hours should eliminate or at least reduce the damage to mushrooms by this pest.