从杨树菇中开发新型食品防腐剂的初步研究

本实验室通过对14株食用真菌的抑菌试验筛选,发现杨树菇具有极强的抑菌作用,且抑菌谱广,可以有效抑制细菌、酵母和部分霉菌。与不同浓度 (0.05％、O.1％、O.2％、O.3％ g/m1) 山梨酸、苯甲酸抑菌能力相比,抑菌效果更为明显。以金黄色葡萄球菌为指示菌研究了其最适发酵条件及有效成分的热稳定性,结果表明：杨树菇的发酵周期为 7—9d,发酵温度 25℃,发酵的起始 pH6.0～7．O,发酵液装量为 80-140ml／500ml 三角瓶,接种量 10％-15％(v／v);发酵液浓缩液经 121℃、30 min 湿热灭菌处理后仍具有很强的抑菌活性。杨树菇是一种具有开发前途的食用真菌。     

一株海洋真菌BY1产伸筋草醇发酵条件的优化

伸筋草醇(Clavatol)是具有多种生物活性的聚酮类三萜醇化合物。本文采用HPLC法分析海洋真菌BY1发酵产物中伸筋草醇的产量,研究不同培养基、海水浓度、培养天数、装液量对BY1菌株产伸筋草醇的影响。结果表明,海洋真菌BY1产伸筋草醇的发酵条件为:接种量1.0 m L种子发酵液,250 m L三角瓶装海水浓度为20%(v/v)的70 m L麦芽汁培养基(MA培养基),28℃,180 r/min,振荡培养9 d。     

浙贝母内生真菌Nectria sp. JZ6分泌抑菌活性代谢产物的初步研究

Nectria sp.JZ6是从浙贝母新鲜鳞茎中首次分离到的一株内生真菌,其发酵液具有抑菌活性。为建立该真菌稳定分泌抑菌活性成分的发酵体系,本研究利用单因素实验和正交实验确定了发酵培养基的配方并初步优化了发酵条件。结果表明,将活化后的菌种接种于改良查氏培养基(8%葡萄糖,0.5%蛋白胨,0.05%KCl,0.1%K2HPO4,0.15%MgSO4,pH6.5)中,28℃、150r/min振荡培养6d后获得的发酵液抑菌活性比优化前提高了26%。该发酵液经100℃水浴30min不失活,在pH1-5时抑菌活性相对稳定,其后逐渐减弱,并在pH9.0及以上时丧失。Nectriasp.JZ6发酵液的乙酸乙酯浸膏抑菌活性最强,对金黄色葡萄球菌、藤黄八叠球菌、枯草芽孢杆菌、绿脓杆菌和大肠杆菌的最低抑菌浓度(MIC)分别为0.625、0.625、1.25、1.25和1.25mg/mL。     

蚀木链霉菌KX6耐热内切葡聚糖酶的产生及酶学性质研究

从堆肥中筛选到一株产耐热内切葡聚糖酶的放线菌菌株,通过形态观察和16S rRNA序列分析,鉴定为蚀木链霉菌(Streptomyces Xylophagus)。实验中对其产酶的液态发酵条件进行了研究,碳源为1%（w/v）羧甲基纤维素钠,氮源为1%（w/v）豆粕粉,250ml三角瓶30 %装液量,接种量为2%,培养基初始pH为8.0,培养温度为40℃,200r/min培养48h后,发酵液中内切葡聚糖酶活达到0.538IU/ml。该酶的最适作用温度和pH分别为50℃和7.0,50℃下酶活保持1 h不变,60℃保温1h,仍有60%的原酶活性,pH为6.0~7.0酶活稳定,该酶属于一种耐热的中性内切葡聚糖酶。     

解淀粉芽孢杆菌LJ1摇瓶发酵条件优化北大核心CSCD

解淀粉芽孢杆菌(Bacillus amyloliquefaciens)LJ1是从天津土壤中分离筛选出的一株具有广谱抑菌效果的生防菌株,该实验旨在明确B.amyloliquefaciens LJ1的抑菌谱并通过优化发酵培养基和发酵条件提升其抑菌效果。通过对峙培养检测菌株LJ1对14种病原真菌的抑制效果;采用单因素试验、正交试验优化发酵培养基和摇瓶发酵条件;最后用孢子萌发法和盆栽试验验证优化后发酵液的抑菌效果。实验结果表明B.amyloliquefaciens LJ1对黄瓜灰霉病菌(Botrytis cinerea)、苹果炭疽病菌(Glomerella cingulata)、苹果腐烂病菌(Valsa ceratosperma)等11种病原真菌有抑制作用;优化后的摇瓶发酵条件为温度30℃、初始p H 5、转速200 r/min、装液量50 m L/250 m L;优化后的发酵培养基为马铃薯200 g,蔗糖10 g,黄豆粉20 g,Mg Cl2·6H2O 1.5 g,蒸馏水1 000 m L。经发酵条件与培养基优化后,B.amyloliquefaciens LJ1发酵液对Botrytis cinerea孢子萌发的抑制率达99.7%,盆栽实验防效达51.08%。     

解淀粉芽孢杆菌LJ1摇瓶发酵条件优化

解淀粉芽孢杆菌(Bacillus amyloliquefaciens)LJ1是从天津土壤中分离筛选出的一株具有广谱抑菌效果的生防菌株,该实验旨在明确B.amyloliquefaciens LJ1的抑菌谱并通过优化发酵培养基和发酵条件提升其抑菌效果。通过对峙培养检测菌株LJ1对14种病原真菌的抑制效果;采用单因素试验、正交试验优化发酵培养基和摇瓶发酵条件;最后用孢子萌发法和盆栽试验验证优化后发酵液的抑菌效果。实验结果表明B.amyloliquefaciens LJ1对黄瓜灰霉病菌(Botrytis cinerea)、苹果炭疽病菌(Glomerella cingulata)、苹果腐烂病菌(Valsa ceratosperma)等11种病原真菌有抑制作用;优化后的摇瓶发酵条件为温度30℃、初始p H 5、转速200 r/min、装液量50 m L/250 m L;优化后的发酵培养基为马铃薯200 g,蔗糖10 g,黄豆粉20 g,Mg Cl2·6H2O 1.5 g,蒸馏水1 000 m L。经发酵条件与培养基优化后,B.amyloliquefaciens LJ1发酵液对Botrytis cinerea孢子萌发的抑制率达99.7%,盆栽实验防效达51.08%。     

重庆低海拔虫草发酵条件及其发酵液抑菌活性研究

从重庆低海拔地区采得一株虫草,分离得到其无性型菌株.以海绵抑菌圈大小为活性指标,考核了无机盐、碳氮比值、发酵温度及培养基初始pH等因素对其发酵液抑菌活性的影响,筛选出抑菌活性物质的发酵培养基为:NaNO3 0.05%,KCl 0.05%,蔗糖3.0%,蚕蛹粉0.05%.摇瓶最佳发酵条件为:pH 6.5,温度25 ℃,转速250 r/min.在此优化条件下,发酵活性在第6 d最高,对枯草芽孢杆菌的抑菌圈为30.4 mm.用海绵抑菌圈法,测定虫草真菌发酵液对不同供试菌的抑菌活性,对真菌酿酒酵母无抑菌作用,对供试的所有革兰氏阴、阳性细菌都有抑菌活性,且对枯草芽孢杆菌的活性最强,其抑菌圈为(26±4) mm.     

利用甘蔗糖蜜酒精发酵液生产腐植酸的菌种鉴定及发酵条件研究

从土壤中筛选出一株适合用甘蔗糖蜜酒精发酵液生产腐植酸的菌株H812。单因素实验和正交实验结果表明,该菌株培养的最适酒精发酵液浓度为16°Bx,最适培养条件为:时间8d、温度34℃、摇床转速200r/min、初始pH7.0、接种量12%和装液量50ml/250ml,其中温度对发酵产品影响显著。在优化的条件下,腐植酸产量为38.12 g/L,较优化前提高了148.34%。对H812菌株进行形态特征分析以及ITS序列分析,推测该菌株为曲霉属真菌。     

梳棉状嗜热真菌发酵产耐热木聚糖酶培养条件的优化

通过多个单因素实验对该菌株发酵产酶的培养条件进行优化,包括碳源、氮源、无机盐、培养基起始pH、培养时间以及培养温度等因素,得出了一个适合该嗜热真菌产酶发酵的条件:麸皮浓度为4%(w/v)、牛肉膏浓度为0.5%(w/v)、NaCl浓度为0.25%(w/v)、K_2HPO_4浓度为0.2%(w/v)、CaCl_2浓度为0.02%(w/v)。培养基初始pH为7.0,装液量为60 mL,发酵温度为55℃,发酵时间为60 h,摇瓶转速为125 r/min。优化之后酶活可达到68.5 U/mL,比未优化之前提高了71.2%。嗜热真菌可以利用麸皮、玉米芯粉等农业废弃物作为能源产木聚糖酶,既可以很好地处理此类农业副产品,又可以获得对人类有利的产物。     

虎奶菇发酵液抑菌成分的理化性质及其分离鉴定

为探明虎奶菇发酵液中的抑菌成分,本文以金黄色葡萄球菌(Staphylococcus aureus)为指示菌,研究了温度、pH、紫外线、蛋白酶K对虎奶菇发酵液抑菌活性的影响;并采用硅胶柱层析及反相中压液相层析追踪分离抑菌活性物质,通过核磁共振并结合相关资料对分离到的单体化合物进行了结构鉴定。结果显示,发酵液抑菌成分对蛋白酶较为稳定,对紫外照射敏感,可在90℃以下、pH1~9的范围内较好的保持其抑菌活性;活性追踪分离得到1个抑菌物质PtrA,经鉴定为2-呋喃甲酸(2-furoic acid)。     

Unusual accumulation of polymorphic microsatellite loci in a specific region of the mitochondrial genome of two mushroom-forming Agrocybe species

The cob/tRNA(Tyr) mitochondrial regions of Agrocybe aegerita and of the related species Agrocybe chaxingu display an unusual clustering of four microsatellite loci constituted by motifs of one to six nucleotides whose number of repeats varied from three to 18. In A. chaxingu, these microsatellite loci are followed by a small region bearing one additional microsatelite and one minisatellite locus constituted by an octanucleotide motif repeated 13-18 times. In A. aegerita, this latter region is deleted. This is the first evidence of such an accumulation of microsatellites in mitochondrial genomes. The analyses of the microsatellite loci in 11 A. aegerita and in four A. chaxingu wild strains have shown extensive intraspecific and interspecific variations in the number of tandem repeats (VNTRs), suggesting that these loci could represent powerful molecular markers for strain fingerprinting. Up to 23 different alleles were present in the 15 Agrocybe studied strains, allowing the definition of 12 different haplotypes.     

一种新杨树菇(Agrocybe aegerita)凝集素的纯化及生化特性

用硫酸铵分级沉淀、离子交换和分子筛等方法 ,从食用菌杨树菇子实体中分离纯化了一种凝集素 ,称作为AAVP(Agrocybeaegeritaantiviralprotein) .经SDS PAGE测定其亚基的相对分子质量为15 8kD ,凝胶过滤分析分子量为 32kD .IEF PAGE计算其等电点为 3 8.AAVP不含糖 ,是一种N端焦谷氨酰环化封闭的蛋白质 ,经N端去封闭后测得N端氨基酸序列为QGVNIYNIVAGA ,用胰蛋白酶消化后得到一大片段 ,测定的氨基酸序列为PDGPWLVEK .AAVP可以凝集供试的 12种动物血和3种血型人血的血红细胞 ,但对各种血红细胞凝集滴度不同 .糖抑制实验表明 ,在供试的 18种单糖和 3种糖蛋白中 ,只有猪胃粘蛋白强烈抑制AAVP的凝血活性 .AAVP具有较好的热稳定性 ,能够忍受极端的酸碱条件 .AAVP的凝血活性不受Ca2 + 、Mg2 + 、Zn2 + 等二价阳离子的影响 .抗肿瘤活性检测表明 ,AAVP对胃癌细胞株SGC 790 1,MGC 80 3,BGC82 3及人急性白血病细胞株HL 6 0有明显的抑制作用 .AAVP对小鼠腹腔注射的半致死剂量为 15 85mg kg .     

Cyclooxygenase inhibitory and antioxidant compounds from the fruiting body of an edible mushroom, Agrocybe aegerita.

In the search for bioactive natural products from edible mushrooms, we have investigated the fruiting body of Agrocybe aegerita. The methanol extract of this mushroom yielded a fatty acid fraction (FAF), along with palmitic acid (1), ergosterol (2), 5,8-epidioxy-ergosta-6,22-dien-3beta-ol (3), mannitol (4) and trehalose (5). The composition of FAF was confirmed by GC-MS and by comparison to the retention values of authentic samples of palmitic, stearic, oleic and linoleic acids. The structures of 1-5 were established using spectroscopic methods. FAF and compounds 1-3 showed cyclooxygenase (COX) enzyme inhibitory and antioxidant activities. The inhibition values of liposome peroxidation by FAF, compounds 1 and 2 at 100 microg/ml were 75, 45, and 43%, respectively. The inhibition values of COX-I enzyme by FAF and 1-3 at 100 microg/ml were 80, 39, 19, and 57%, respectively. Similarly, COX-II enzyme activity was reduced by FAF and 1-3 at 100 microg/ml with values of 88, 45, 28, and 22%, respectively. Compounds 1, 3 and fatty acids were isolated here for the first time from the fruiting body of A. aegerita.     

A lectin with mycelia differentiation and antiphytovirus activities from the edible mushroom Agrocybe aegerita

A lectin named AAL has been purified from the fruiting bodies of the edible mushroom Agrocybe aegerita. AAL consisted of two identical subunits of 15.8 kDa, its pI was about 3.8 determined by isoelectric focusing, and no carbohydrate was discerned. Being treated by pyrogultamate aminopeptidase, the blocked N-terminus of AAL was sequenced as QGVNIYNI. AAL agglutinated human and animal erythrocytes regardless of blood type or animal species. Its hemagglutinating activity was unaffected by acid or alkali treatment and demetalization or addition of divalent metals Mg(2+), Ca(2+) and Zn(2+). AAL was toxic to mice: its LD50 was 15.85 mg per kilogram body weight by intraperitoneal injection. In this study, two novel activities of AAL were proved. It showed inhibition activity to infection of tobacco mosaic virus on Nicotiana glutinosa. The result of IEF suggested that AAL attached to TMV particles. Mycelia differentiation promotion was the other interesting activity. AAL promoted the differentiation of fruit body primordia from the mycelia of Agrocybe aegerita and Auricularia polytricha. AAL antiserum was prepared and immunologically cross-reactived with several proteins from five other kinds of mushrooms. These results suggested that AAL probably was a representative of a large protein family, which plays important physiological roles in mushroom.     

杨树菇凝集素AAVP具有抗病毒和促进菌丝分化功能

杨树菇凝集素 (AAVP)是一种新的真菌凝集素 .用半叶法检测证明 ,AAVP具有抑制烟草花叶病毒 (TMV)侵染的活性 .等电聚焦法证实了AAVP可以与TMV的外壳蛋白结合 .用滤纸圆片法检测 ,AAVP对 3种植物病原真菌没有抑制作用 .AAVP滴加在菌丝的表面 ,显著地促进了杨树菇和毛木耳的菌丝分化 ,促进子实体的形成 .Western印迹表明 ,AAVP存在于菌丝、菌柄和菌盖等组织中 ;假猴头 ,香菇 ,草菇 ,杏孢菇 ,灵芝等大型真菌的子实体中存在着多种与AAVP的抗血清有交叉反应的蛋白质 .大多数大型真菌中可能存在着与AAVP具有相似血清学特征的蛋白质家族 ,在防御反应及菌丝分化等多种生理过程中发挥重要的作用     

培养条件对头孢霉脂肪酸链长和ω-3脱饱和的影响*

为了获得高产量的长链ω-3多不饱和脂肪酸,用十八碳脂肪酸和十六碳脂肪酸的比值考察碳链延长,用α-亚麻酸和亚油酸的比值考察ω-3脱饱和;探讨了八种因子对脂肪酸链长和ω-3脱饱和的影响。有利于碳链延长的条件为：麦芽糖10g/L、(NH4)2SO4 3g/L、起始pH为4.0、500mL三角瓶装50mL培养基、接种20%（V/V）、20℃培养6d。有利于ω-3脂肪酸生成的条件为：蔗糖30g/L、NH4Cl 3g/L、培养基起始pH为4.0、500mL三角瓶装50mL培养基、接种20%（V/V）、10℃培养10d。     

培养条件对头孢霉脂肪酸链长和ω-3脱饱和的影响

为了获得高产量的长链ω-3多不饱和脂肪酸,用十八碳脂肪酸和十六碳脂肪酸的比值考察碳链延长,用α-亚麻酸和亚油酸的比值考察ω-3脱饱和;探讨了八种因子对脂肪酸链长和ω-3脱饱和的影响。有利于碳链延长的条件为：麦芽糖10g/L、(NH4)2SO4 3g/L、起始pH为4.0、500mL三角瓶装50mL培养基、接种20%（V/V）、20℃培养6d。有利于ω-3脂肪酸生成的条件为：蔗糖30g/L、NH4Cl 3g/L、培养基起始pH为4.0、500mL三角瓶装50mL培养基、接种20%（V/V）、10℃培养10d。     

Sequence of the URA1 gene encoding dihydroorotate dehydrogenase from the basidiomycete fungus Agrocybe aegerita.

The URA1 gene encoding dihydroorotate dehydrogenase (DHOdehase) from the edible basidiomycete, Agrocybe aegerita, has been cloned by complementation of the Escherichia coli pyrD mutation. The nucleotide sequence of a 1531-bp genomic fragment carrying URA1 revealed two uninterrupted open reading frames (ORFs) separated by 61 bp. The larger ORF can encode a 328-amino acid (aa) DHOdehase that has 53% homology with the corresponding protein from E. coli. Comparison with other DHOdehase aa sequences showed essentially conservation of the cofactor-binding site of flavoproteins.     

Pleurotus and Agrocybe hemolysins,new proteins hypothetically involved in fungal fruiting

Novel hemolytic proteins, ostreolysin and aegerolysin, were purified from the fruiting bodies of the edible mushrooms Pleurotus ostreatus and Agrocybe aegerita. Both ostreolysin and aegerolysin have a molecular weight of about 16 kDa, have low isoelectric points of 5.0 and 4.85, are thermolabile, and hemolytic to bovine erythrocytes at nanomolar concentrations. Their activity is impaired by micromolar Hg(2+) but not by membrane lipids and serum low-density lipoproteins (LDL). The sequence of respectively 50 and 10 N-terminal amino acid residues of ostreolysin and aegerolysin has been determined and found to be highly identical with a cDNA-derived amino acid sequence of putative Aa-Pri1 protein from the mushroom A. aegerita, Asp-hemolysin from Aspergillus fumigatus, and two bacterial hemolysin-like proteins expressed during sporulation. We found that ostreolysin is expressed during formation of primordia and fruiting bodies, which is in accord with previous finding that the Aa-Pri1 gene is specifically expressed during fruiting initiation. It is suggestive that the isolated hemolysins play an important role in initial phase of fungal fruiting.