Modeling growth,substrate consumption and product formation of <Emphasis Type="Italic">Penicillium nalgiovense</Emphasis> grown on meat simulation medium in submerged batch culture

Penicillium nalgiovense is the most widely used starter mold for cured and fermented meat products. The development of a biomass film on the surface of these products prevents a large degree undesirable growth of various fungal contaminants and contributes to the ripening process with production of metabolites. This work presents an attempt to model the growth of P. nalgiovense and to relate it to substrate consumption and product release. Because of the extremely complex nature of the meat product fermentation, submerged culture was employed in a bioreactor system that enabled on-line monitoring, using a meat simulation medium, which contained peptones and lactate as carbon, nitrogen and energy sources. The unstructured model presented is based on a partial association of substrate assimilation and product formation with growth. Experimentally derived values for peptones and lactate were compared with model-derived values and their proportions corresponding to growth associated parts, used for biosynthesis, and non-growth associated parts, used for maintenance. The model was applied for the products ammonia, carbon dioxide and protons. Both peptones and lactate were used mainly for biosynthesis (85 and 80% of the total amounts provided, respectively). Assimilation of lactate and ammonia formation from amino acid metabolism resulted in a proton exchange, which was mainly growth associated. The contribution of the growth associated mechanism to the total proton exchange was estimated to be 75% while the contribution of the non-growth associated mechanism increased during the growth phase and reached a maximum of 25%. For carbon dioxide production, the contribution of a maintenance mechanism was evident at 40 h, while production was growth-associated and remained such even at the end of fermentation at 168 h when growth rate was very low. The partially growth associated model showed good agreement with the experimental data and allows accurate determination of the proportions of substrates or products related to biosynthesis and cell maintenance.     

Microbial transformation of baicalin and baicalein

As a result of microbial transformation of baicalin and baicalein the products of 4′-hydroxylation of the B ring, O-methylation at C-6, and both O-methylation at C-6 and hydroxylation at C-4′ were obtained. Transformations of baicalin were accompanied by the reaction of hydrolysis.     

Penicamedine A,a Highly Oxygenated Hexacyclic Indole Alkaloid from Penicillium camemberti

A highly oxygenated hexacyclic indole alkaloid, penicamedine A ( 1 ), bearing a rare furan ring, was isolated from the culture broth of Penicillium camemberti, together with two known analogs, iso‐α‐cyclopiazonic acid ( 2 ) and cyclopiazonic acid ( 3 ). The structure of 1 was elucidated by comprehensive spectroscopic analyses including NMR and HR‐ESI‐MS. Its absolute configuration was further confirmed unambiguously by single‐crystal X‐ray diffraction analysis. Compound 1 was evaluated for anti‐HIV activity with p24 assays and tested for cytotoxic activities against five human cancer cell lines, including HL‐60, SMMC‐7721, A‐549, MCF‐7, SW480, and the immortalized non‐cancerous human pulmonary epithelial cell line BEAS‐2B by MTS method.     

海洋真菌菌核青霉FS50固体发酵代谢产物

采用硅胶柱、反相硅胶柱、凝胶柱、薄层制备和HPLC等色谱技术对1株分离自南海沉积物的海洋真菌菌核青霉Penicillium sclerotiorum FS50的大米发酵提取物进行分离纯化,从中分离得到7个化合物。通过波谱数据分析,化合物1-7分别被鉴定为:6,8‐dihydroxyisocoumarin‐3‐butyl formate(1),6,8‐dihydroxyisocoumarin‐3‐carboxylic acid(2),6‐methoxy‐8‐hydroxyisocoumarin‐3‐carboxylic acid(3),(+)‐sclerotiorin(4),4‐methyl‐5,6‐dihydropyren‐2‐one(5),5‐羟甲基糠醛(6),胡萝卜甙(7)。化合物1为新化合物,化合物2,3,6为首次从青霉属中分离得到。     

Natural mortality of immature stages of Bactrocera oleae (Diptera: Tephritidae) in traditional olive groves from north-eastern Portugal

From 2006 to 2008, we studied the natural mortality of olive fly, Bactrocera oleae (Rossi), eggs and larvae as collected in fruit on the tree, in two to five ‘traditional’ olive groves of Trás-os-Montes (north-eastern Portugal), per year. We also studied the fate of 2044 puparia that were buried in the soil from November to May for two seasons, by using exclusion cages to estimate predation. Mortality of eggs inside the fruit was estimated as between 5.4±2.4 and 16.6±6.1%, and as 10.7±2.1 and 100.0% for young larvae. Similarly, the estimated mortality of mature larvae was between 0 and 66.7±33.3%. The highest mortality levels for young larvae could reach 100% during August, and 47.4% during the first fortnight of December, coincident with high temperatures in summer or low temperatures at the end of autumn and early winter. Mortality (winter disappearance) of puparia was estimated at up to 98.5% of the population. Predation of eggs was low, at 0.6±0.3 to 6.3±4.0% in 2006, 0 to 4.1±1.1% in 2007, and 0 to 3.6±3.6% in 2008. Parasitism was insignificant during the study, and only one parasitoid was identified, the eulophid Pnigalio agraules (Walker). We also surveyed entomopathogenic fungi associated with the insect in 12 olive groves, and evaluated three of the latter against larvae, puparia, and adult B. oleae in the laboratory. Of the 15 fungal species identified from immature stages, three were considered to be entomopathogenic (i.e. Cordyceps bassiana, Penicillium corylophilum and Mucor hiemalis). We observed up to 20.0±3.2% and 94.0±2.5% mortality of larvae and puparia, respectively when treated with P. corylophilum and up to 32.5±11.1% of adults when treated with M. hiemalis.     

来源于青霉XMZ-9两个低温脂肪酶的基因克隆、原核表达与性质测定

低温脂肪酶在低温条件下仍具有较高活性,在食品添加剂、洗涤添加剂及有机合成等产业具有非常独特的应用前景。从低温菌株中分离低温脂肪酶基因是开发新的低温脂肪酶的有效手段。首先利用油脂同化平板与三丁酸甘油酯-维多利亚蓝平板从冰川土样中筛选分离获得一株具有较高脂肪酶活性的真菌,18S rDNA鉴定其属于青霉属,命名为Penicillium sp.XMZ-9。根据真菌脂肪酶多序列比对获得的保守区,设计简并引物,利用降落PCR与染色体步移的方法从Penicillium sp.XMZ-9中克隆到2个完整的脂肪酶基因,分别记为LipA与LipB。LipA全长1 014 bp,无内含子,编码337个氨基酸。而LipB全长1 232 bp,cDNA长1 122 bp,含有2个内含子,编码373个氨基酸。将两基因的cDNA序列克隆到pET30a(+)载体上,转化大肠杆菌Escherichiacoli BL21(DE3)。经低温诱导表达后,LipA大部分表达为包涵体,包涵体经复性后具有脂肪酶活性,并表现出低温适应性;LipB则大部分表达为可溶性蛋白,Ni-亲和层析柱纯化后,其亦具有低温脂肪酶活性。青霉菌株XMZ-9的获得与低温脂肪酶的克隆表达研究,为研究低温菌株与低温酶的适冷机制提供了宝贵的资源,也为进一步开发利用低温脂肪酶奠定了基础。     

桔青霉摇瓶发酵生产核酸酶P1的动力学研究

研究了桔青霉发酵生产核酸酶P1的发酵动力学特性：以Logistic方程和Luedeking—Piret方程为基础,进行最优参数估计和非线性拟合,得到了描述整个发酵过程中的茵体生长、产物合成和基质消耗的动力学模型。对实验数据与模型预测值进行比较,发现模型预测值与实验数据能较好地拟合,基本上反映了桔青霉发酵过程的动力学特征,为以后进一步研究和预测核酸酶P1发酵过程奠定了理论基础。     

Fungi and ochratoxin A detected in healthy grapes for wine production

AIMS: The mycoflora of healthy grapes (i.e. without visible symptoms of rot) for wine production in Portuguese wine-making regions was assessed and its potential for ochratoxin A (OTA) production evaluated. The OTA content of grapes was also determined. METHODS AND RESULTS: A total of 386 fungal strains were isolated by plating methods. The most frequent genera found in grapes were non-ochratoxigenic species: Cladosporium (28%), Penicillium (24%), Botrytis (13%) and Aspergillus (9%). Two OTA-producing strains were isolated, belonging to the species Aspergillus carbonarius and Aspergillus ochraceus. OTA was detected in three of four grape samples, up to 116 ng l(-1). CONCLUSIONS: OTA is being produced in healthy berries by Aspergillus species, namely A. carbonarius, at levels below the maximum recommended limit of 2,000 ng l(-1) in wine. SIGNIFICANCE AND IMPACT OF THE STUDY: The OTA concentration detected in healthy Portuguese grapes does not represent a risk to wine regarding the legal limit established.     

Effect of conditions and protectants on the survival of Penicillium bilaiae during storage

Penicillium bilaiae is a fungal microorganism used to promote soil-bound phosphorous uptake in several crop species such as wheat, canola, and pulse crops. In this study, an air-dried granular starch-based form of the inoculant was studied for improved longevity. Experiments were performed to determine optimal water activity, packaging atmosphere, and the effects of several protective chemicals. Viable spore fractions were measured at 1, 3, and 6 months. Longevity of the microorganism was found to suffer at intermediate values of water activity and with the addition of glycerol. However, skim milk powder, sucrose, MSG, and proprietary PBX3000 were found to improve survival, with a maximum survival fraction of 15% at 6 months using the best formulation. Cell concentration was not found to influence shelf-life. High viability loss during storage in a nitrogen environment resulted in less than 1% survival.     

Identification of <Emphasis Type="Italic">Penicillium marneffei</Emphasis> in Paraffin-Embedded Tissue Using Nested PCR

Penicillium marneffei is one of the unique thermally dimorphic fungi in Penicillium species that causes a disseminated, progressive and life threatening infection in immunocompromised patients. The diagnosis of Penicilliosis marneffei depends on culture that may delay the treatment due to the time-consuming process. In the present study, we evaluated the specificity and sensitivity of nested PCR to identify Penicillium marneffei from paraffin-embedded tissue. Two sets of oligonucleotide primers were derived from the sequence of 18S rRNA of Penicillium marneffei. The outer primers (RRF1 and RRH1) were specific to fungi. The inner primers (Pm1 and Pm2) were specific to Penicillium marneffei. The specific fragment of approximately 400 bp was amplified from all paraffin-embedded tissues from 14 patients with Penicilliosis marneffei and 10 bamboo rats. The detectable DNA concentration of single PCR and nested PCR were 14 pg/μl and 14 fg/μl, respectively. Further studies are required in order to use nested PCR for early diagnosis of the disease.