Biotransformation of p-coumaric acid by Paecilomyces variotii

AIMS: To investigate the biotransformation of p-coumaric acid into p-hydroxybenzoic acid (p-HBA) by Paecilomyces variotii Bainier MTCC 6581. METHODS AND RESULTS: As a result of p-coumaric acid degradation by P. variotii, three phenolic metabolites, p-hydroxybenzaldehyde (p-HBAld), p-HBA and protocatechuic acid were formed. These phenolics were detected using TLC and HPLC. The identity of p-HBA and p-HBAld was further confirmed by mass spectrometry. Various analyses showed that 10.0 mmol l(-1) concentration of p-coumaric acid produced a maximum amount of p-hydroxybenzoic acid, 200 mg l(-1), into the medium at 37 degrees C with high-density cultures. CONCLUSIONS: A catabolic pathway of p-coumaric acid by the fungus P. variotii is suggested for the first time. During the process of p-coumaric acid degradation, p-HBA accumulated in the medium as the major degradation product. SIGNIFICANCE AND IMPACT OF THE STUDY: Microbial degradation of cinnamic acid and hydroxycinnamic acid has continued to be the focus of intensive study. The main goal was to identify the microbial species capable of converting these substances into commercially value-added products such as benzoic acid derivatives or aromatic aldehydes.     

Heat resistance and the effects of continuous pasteurization on the inactivation of Byssochlamys fulva ascospores in clarified apple juice

Aims: To determine thermal resistance, the effect of pasteurization temperature variations (c. 2°C) in a continuous system in the number of decimal reductions (n) of a Byssochlamys strain in clarified apple juice (CAJ). Methods and Results: Thermal destruction kinetics of Byssochlamys fulva IOC 4518 in thermal death tubes were determined at 85°, 90°, 92° and 95°C by using Weibull distribution frequency model. Three processes with different heating and holding temperatures (A: 94°, 92°C; B: 95°, 93°C; C: 96°, 94°C, respectively) were performed in a continuous system. Process time was 30 s. δ (time of first decimal reduction) values were: 42·98, 8·10, 3·62 and 1·81 min. Variable n ranged from 0·16 to >4·78 for process B (equivalent to industrial). Variable n (0·95–2·66 log CFU ml^?1) were obtained in CAJ bottles processed under condition B, while process A resulted in total heat‐resistant mould (HRM) survival and process C in total HRM destruction. Conclusions: This study demonstrates that small variations in temperature during the CAJ pasteurization could result in elimination or survival of HRM due to its nonlogarithmic behaviour. Significance and Impact of the Study: This was the first study to use Weibull frequency method to model inactivation of HRM in fruit juices. Temperature variations could culminate in the presence of HRM in pasteurized juices even when low counts (<10 spores per 100 ml) were present in the raw materials.     

Production, Purification of Exo-Polygalacturonase from Soil Isolate Paecilomyces variotii NFCCI 1769 and Its Application

The aim of the present study was to produce exo-polygalacturonase from potent soil isolate by submerged fermentation and its application for fruit juice treatment. Pectinase producing strains were selectively isolated from pectin industry waste. A selected isolate C2 was found to produce significant amount of exo-polygalacturonase. The isolate was identified as Paecilomyces variotii on the basis of morphological characteristics and 18S rRNA gene sequence analysis. The exo-polygalacturonase produced by the isolate was purified by ammonium sulphate precipitation, size exclusion chromatography and ion exchange chromatography. The purified enzyme had MW of 39.4 kD based on SDS PAGE. Under partially optimized conditions, purified exo-polygalacturonase showed specific activity of 98.49 U/mg protein at pH 6.0 and 30°C. The enzyme was comparatively stable from 10 to 30°C and the activity decreased with increasing temperature. Purified enzyme brought about considerable reduction in viscosity of fruit juice samples.     

玫烟色拟青霉适温性及抗多菌灵特性的种内变异

在15 ℃～35 ℃的温度范围内比较测定了玫烟色拟青霉(Paecilomyces fumosoroseus) 6株野生菌的菌落生长、产孢量及孢子活力.结果显示,3个指标均以25 ℃左右最好,但同一温度下菌株间或同一菌株在不同温度下的差异显著, 菌株Pfr116和Pfr6206具有相对稳定优良的生长、产孢及活孢率性状.对不同浓度多菌灵对6株野生菌的菌落生长和单孢菌落形成的影响测定表明,菌株间存在较大差异.不同多菌灵浓度对单孢菌落形成的抑制率观察值可用逻辑斯蒂模型较好地拟合(r²≥0.90).用所获参数估计的最低禁菌浓度MIC值显示,Pfr4205和Pfr116对多菌灵表现为低抗性（MIC≤20.0 μg·ml^-1）;Pfr153、Pfr612和Pfr2175属于低水平中抗,MIC值仅略大于20 μg·ml^-1;而Pfr6206的MIC值高达93.5 μg·ml^-1,接近MIC>100 μg·ml^-1的高抗标准.因此,Pfr6206为适温性和抗多菌灵特性俱佳的菌株,可作为抗多菌灵和适应不同季节或地区的害虫生防菌剂的候选菌株.     

Detoxification of castor bean residues and the simultaneous production of tannase and phytase by solid-state fermentation using Paecilomyces variotii

In this work, we introduce a biological detoxification method that converts toxic waste from castor beans into animal feed material. This method simultaneously induces the production of tannase and phytase by Paecilomyces variotii; both enzymes have high levels of activity and have the potential to be used in feedstuffs because they decrease overall anti-nutritional factors. The maximum tannase and phytase activities obtained were 2600 and 260 U/g after 48 and 72 h, respectively. SDS-PAGE electrophoresis of the fermented castor cake extracts revealed a reduction in ricin bands during fermentation, and the bands were no longer visible after 48 h. The cytotoxicity of the extracts was evaluated by MTT testing on RAW cells, and a progressive increase in cellular viability was obtained, reaching almost 100% after 72 h of fermentation.     

粉拟青霉类枯草杆菌蛋白酶基因的cDNA克隆及其序列和蛋白质分析

粉拟青霉是一种常见的昆虫病原真菌,被广泛用于生物防治。根据GenBank中已登录的淡紫拟青霉,球孢白僵菌和金龟子绿僵菌的类枯草杆菌蛋白酶基因序列的同源性比较,以它们高度保守的核苷酸序列设计一对引物,采用RT-PCR和3’/5’-RACE相结合的方法,首次从粉拟青霉中克隆出完整的类枯草杆菌蛋白酶cDNA基因。其全序列为2060bp,该序列5’-端和3’-端的非编码序列长度分别为209bp和252bp,开放阅读框为1599bp,编码532个氨基酸,信号肽长度为16个氨基酸。成熟蛋白的氨基酸序列和金龟子绿僵菌小孢变种(CAB63913),玉米赤霉菌(EAA68914),金龟子绿僵菌蚱蜢变种(CAC95047)及柄孢壳(AAC03564)的同源性分别为69%、71%、68%和68%。成熟蛋白具有典型的丝氨酸蛋白酶活性位点,同时有5个半胱氨酸,3个潜在的N-糖基化位点和2个可能的O-糖基化位点。该基因在的密码子第三位碱基的使用上,显示出明显的对C的偏爱。     

Enzymatic analysis and in vitro ovicidal effect of Pochonia chlamydosporia and Paecilomyces lilacinus on Oxyuris equi eggs of horses

The aim of this work was to analyse the enzymatic activity and in vitro ovicidal effect of Pochonia chlamydosporia (VC1 and VC4) and Paecilomyces lilacinus (PL1) on Oxyuris equi eggs of horses. The growth of isolates and their enzymatic production were evaluated on agar media supplemented with gelatin (GA), casein (CA), olive oil (OOA) or starch (SSA). The ovicidal effect was evaluated according to morphological alterations. Following, the P. chlamydosporia crude extract production and proteolytic content was evaluated (VC1 and VC4) in liquid medium at the interval of 15, 30, 45 and 60 min during incubation of P. chlamydosporia and P. lilacinus which grew and showed enzymatic activity on agar media (GA, CA, OOA and SSA). At the 15th day, VC1, VC4 and PL1 showed results on GA, CA, OOA and SSA media, for Type 3 effect of (78, 54, 52 and 68%), (72, 50, 58 and 78%) and (62, 57, 65 and 63%). Pochonia chlamydosporia was able to grow in liquid medium (gelatin) and at Day 5 showed proteolytic activity. The results of the present work suggest that P. chlamydosporia and P. lilacinus can be used in the biological control of O. equi eggs.     

Optimization of physical parameters for exo-biopolymer production in submerged mycelial cultures of two entomopathogenic fungi Paecilomyces japonica and Paecilomyces tenuipes

AIMS: In the present study, two different optimization techniques were used to determine the suitable operating parameters for exo-biopolymer production in submerged mycelial cultures of two entomopathogenic fungi Paecilomyces japonica and Paecilomyces tenuipes. METHODS AND RESULTS: First, the rotating simplex method, a nonstatistical optimization technique, was employed to obtain the best combination of physical parameters (viz. pH, agitation intensity, aeration rate) for maximum exo-biopolymer production by P. japonica in a batch bioreactor. The optimal combination was determined to be a pH of 8.06, an aeration of 3 vvm, without any impeller agitation, producing a 17-time increase in exopolymer production (34.5 g l(-1)) when compared with that achieved in unoptimized flask cultures. Second, the uniform design method, a statistical optimization technique, was employed to determine the best operating parameters for submerged culture of P. tenuipes. The optimal combination for mycelial growth was determined to be a pH of 4.88, an aeration of 2 vvm and an agitation of 350 rpm, while a pH of 4, an aeration of 2 vvm and an agitation of 150 rpm was best for exo-biopolymer production. CONCLUSIONS: The exo-biopolymer production in P. japonica optimized by the rotating simplex method was strikingly improved (max. 34.5 g l(-1)), and the exo-biopolymer production in P. tenuipes optimized by the uniform design method was also significantly increased (max. 3.4 g l(-1)). SIGNIFICANCE AND IMPACT OF THE STUDY: The successful application of these two different optimization techniques in this study implies that these methods are worthy of applying to other fermentation systems for the production of bioactive mycelial biomass and exo-biopolymers in liquid culture of higher fungi.     

Diversity and virulence of soil-dwelling fungi Isaria spp. and Paecilomyces spp. against Solenopsis invicta (Hymenoptera: Formicidae)

To comprehend the diversity and potential control of soil-dwelling fungi, Isaria and Paecilomyces, against the red imported fire ant Solenopsis invicta (Hymenoptera, Formicidae), an investigation was carried out between 2004 and 2008. From 258 soil samples collected from 16 central and southern provinces and cities in China, a total of 171 isolates of the genra Isaria and Paecilomyce were isolated, and the species I. javanicus, P. marquandii and I. fumosoroseus were found more abundant than I. cateniobliquus, P. carneus, P. inflatus and P. lilacinus. Geographic differences of isolating rates were observed as well. Samples from the southern areas had higher fungal isolating rates than those from the central areas. Subsequently, 47 isolates were further tested for pathogencity against the red imported fire ant. All isolates except P115 showed certain pathogenic potential (the mean is 52.3% at 4000 conidiospores/mL) to the ant. I. javanicus was the most effective species with a mean pathogenicity of 80.6%, while pathogenicities of P. marquandii, P. gunni and I. fumosoroseus were 44, 21 and 49%, respectively. Furthermore, the more effective isolates P028 of I. javanicus and P003 of I. fumosoroseus were tested in a virulence experiment. The LD₅₀ values of P028 and P003 against major and miner workers were determined as 412,280 and 854,451 conidiospores/cm², respectively. Meanwhile, the LT₅₀ values at 1000 conidiospores/cm² were 7.1 and 6.6 d in isolate P003 and 6.8 and 6.6 d in isolate P028.     

Heat resistance of MgATPase and contractility in muscle models

1. 1.|During the heating of a synthetic actomyosin suspension, the following sequence of events were observed. First, the rate of superprecipitation decreased; secondly the extent of superprecipitation decreased and finally the MgATPase activity was inhibited. At the same time the dissociating capability of actomyosin decreased in a solution of high ionic strength.

2. 2.|A similar lack of coincidence between the mechanical and the enzymatic activities of actomyosin was observed with an increasing proportion of inactivated myosin occurring in the reconstructed actomyosin complex.

3. 3.|The different heat resistance of contractility and MgATPase activity in muscle models may be caused by inactivated myosin bridges which form in the course of heat treatment so that the dissociating capacity of actomyosin in the presence of ATP is lost.

Molecular and morphological studies of Paecilomyces sinensis reveal a new clade in clavicipitaceous fungi and its new systematic position

The type materials of Paecilomyces sinensis, including herbarium specimen and ex-type strain, were re-examined to clarify its relationships with other species. Morphological observations on the strain grown in various culture media revealed that the fungus was morphologically related to Polycephalomyces, since it produced conidial mass and lanceolate or narrowly lageniform phialides. Six genes, including ITS, nrSSU, nrLSU, tef1, rpb1 and rpb2, were amplified from the type materials and used in phylogenetic analyses to determine the systematic position of the fungus in the framework of clavicipitaceous fungi. The results place P. sinensis with Polycephalomyces formosus, the type species of Polycephalomyces, and Cordyceps ramosopulvinata forming a clade unaffiliated with the known families of clavicipitaceous fungi. Based on both morphological and molecular data, a new combination, Polycephalomyces sinensis, is proposed for Paecilomyces sinensis. The new clade found in this study is designated as Polycephalomyces clade and expands the phylogenetic diversity for clavicipitaceous fungi. The teleomorph–anamorph connection between Berkelella and Polycephalomyces previously conceived cannot be retained as the type species of Polycephalomyces, P. formosus, is closely linked to species of Cordyceps s.l. in the new clade.     

Paecilotoxin production in clinical or terrestrial isolates of Paecilomyces lilacinus strains

The production of paecilotoxin from various isolates of Paecilomyces lilacinus was studied using three different media and high performance liquid chromatography (HPLC). Alkaline medium was found to be suitable for the production of the toxins. Among 20 strains tested, 19 including four clinical isolates were found to produce the toxins. Production patterns of paecilotoxins were very similar in each strain and the main toxins were A and B.     

Comparative efficacy of arbuscular mycorrhizal fungi in combination with bioformulated Paecilomyces lilacinus against Meloidogyne incognita on tomato in two Ultisols of South-eastern Nigeria

Abstract

Greenhouse experiments were conducted to evaluate the efficacy of five arbuscular mycorrhizal fungi (AMF) in combination with bioformulated Paecilomyces lilacinus (PL Gold?) against Meloidogyne incognita on tomato in two Ultisols in Nigeria. Root galling and egg production were higher in Calabar than Nsukka Ultisols. P. lilacinus application did not inhibit tomato root colonisation by any of the AMF. Double application of PL Gold? in combination with Glomus etunicatum caused a significant change in the susceptibility of the tomato cultivar with a gall index (GI) =4.00 to (GI=2.00) in Nsukka Ultisol, while a similar result was obtained with Glomus mosseae, G. deserticola and G. etunicatum in Calabar Ultisols (GI=2.33). Application of both biocontrol agents significantly (P≤0.05) enhanced growth and yield of tomato plants. The highest fresh fruit yield was obtained in Calabar soil when Gi. gigantea was combined with double application of the bionematicide while the same result was obtained with G. deserticola in Nsukka soil.     

Simultaneous and sequential fermentations with yeast and lactic acid bacteria in apple juice

A complex substrate, reconstituted concentrated apple juice, was used for testing the principal processes during yeast and malolactic bacteria fermentations. Interactions between microorganisms were studied based on two controlled inoculation procedures, and at different fermentation temperatures. Temperature had a more important effect on yeast growth than the presence of malolactic bacteria in the medium. Acceleration of the death phase of the bacterial population was detected at increased temperatures. In all cases, malic acid degradation was affected by the fermentation temperature. When experiments were carried out with simultaneous inoculation, acidification of the medium took place at both temperatures tested (15°C and 22°C), that was not observed when the malolactic bacteria were inoculated after completion of alcoholic fermentation by yeasts. Received 4 August 1998/ Accepted in revised form 9 December 1998     

100%枸杞汁对递增负荷运动期间机体HPA轴及血糖调节的影响*

目的: 观察在递增负荷运动期间饮用100%枸杞汁对男大学生血清促肾上腺皮质激素(ACTH)、皮质醇(C)、胰岛素(INS)、胰高血糖素(PG)和血糖(Glu)变化,探讨100%枸杞汁在递增负荷运动期间对机体下丘脑-垂体-肾上腺(HPA)轴应激和血糖调节的作用。方法: 将28名健康男性大学生随机分为对照组(C组,16人)和实验组(E组,12人),所有受试者进行为期32 d、4个阶段的递增负荷运动,运动期间E组每人每天睡前饮用100 ml 100%枸杞汁。在实验前和每阶段结束后次日晨采用血糖仪测试受试者Glu及ELISA法测试血清ACTH、C、INS、PG。结果: ①与运动前相比,递增负荷运动期间两组受试者血清ACTH浓度均在第1、2阶段下降后,第3、4阶段持续升高(P＜0.01),且E组稍高于C组;②递增负荷运动期间受试者血清C呈持续下降趋势,E组第4阶段末显著低于对照组(P＜0.05);③递增负荷运动期间受试者血清INS水平先上升再下降,第4阶段末C组的浓度下降明显(P＜0.05);④实验末期E组血清PG显著降低(P＜0.05);⑤实验后期受试者Glu浓度下降,C组的Glu浓度下降趋势明显(P＜0.05)。结论: 100%枸杞汁可改善递增负荷期间机体HPA轴功能,提高机体对大负荷的应激能力,并调节机体血糖平衡。     

Heat resistance of Cronobacter species (Enterobacter sakazakii) in milk and special feeding formula

Aim:  To determine D - and z -values of Cronobacter species ( Enterobacter sakazakii ) in different reconstituted milk and special feeding formula and the effect of reconstitution of powdered milk and special feeding formula with hot water on the survival of the micro-organism.
Methods and Results:  Five Cronobacter species (four C. sakazakii isolates and C. muytjensii ) were heated in reconstituted milk or feeding formula pre-equilibrated at 52–58°C for various times or mixed with powdered milk or feeding formula prior to reconstitution with water at 60–100°C. The D -values of Cronobacter at 52–58°C were significantly higher in whole milk (22·10–0·68 min) than in low fat (15·87–0·62 min) or skim milk (15·30–0·51 min) and significantly higher in lactose-free formula (19·57–0·66 min) than in soy protein formula (17·22–0·63 min). The z -values of Cronobacter in reconstituted milk or feeding formula ranged from 4·01°C to 4·39°C. Water heated to ≥70°C and added to powdered milk and formula resulted in a > 4 log₁₀ reduction of Cronobacter .
Conclusions:  The heat resistance of Cronobacter should not allow the survival of the pathogen during normal pasteurization treatment. The use of hot water (≥70°C) during reconstitution appears to be an effective means to reduce the risk of Cronobacter in these products.
Significance and Impact of the Study:  This study supports existing data available to regulatory agencies and milk producers that recommended heat treatments are sufficient to substantially reduce risk from Cronobacter which may be present in these products.     

三种来自土壤的有应用前景的拟青霉

从我国新疆和云南土壤中分离到两个拟青霉新记录种,念珠藻状拟青霉(Paecilomyces nostocoides)和雪白拟青霉(P.niveus)。它们的鉴别特征是,前者像念珠藻一样,在同一分生孢子链中具有大小差别十分明显的两类孢子。后者耐高温,菌落白色并有光滑的厚垣孢子。基于rDNA部分序列测序提供的分子证据,将膨大拟青霉大孢变种(P.inflatusvar.major)重新组合为大孢拟青霉(P.major)。     

Influence of culture conditions on production and freeze-drying tolerance of Paecilomyces fumosoroseus blastospores

With the goal of developing a defined medium for the production of desiccation-tolerant blastospores of the bioinsecticidal fungus Paecilomyces fumosoroseus, we evaluated the impact of various media components such as amino acids, carbohydrates, trace metals and vitamins on hyphal growth and sporulation of P. fumosoroseus cultures and on the freeze-drying tolerance of blastospores produced under these conditions. A comparison of 13 amino acids as sole nitrogen sources showed that glutamate, aspartate, glycine and arginine supported biomass accumulations (12–16 mg ml⁻¹) and blastospore yields (6–11 × 10⁸ blastospores ml⁻¹) comparable to our standard production medium which contains casamino acids as the nitrogen source. Using glutamate as the sole nitrogen source, tests with various carbohydrates showed that P. fumosoroseus grew best on glucose (18.8 mg biomass ml⁻¹) but produced similar blastospore concentrations (7.3–11.0 × 10⁸) when grown with glucose, glycerol, fructose or sucrose. P. fumosoroseus cultures grown in media with sodium citrate or galactose as the sole carbohydrate produced lower blastospore concentrations but more-desiccation-tolerant spores. Zinc was the only trace metal tested that was required for optimal growth and sporulation. In a defined medium with glutamate as the nitrogen source, vitamins were unnecessary for P. fumosoroseus growth or sporulation. When blastospores were freeze-dried in the absence of a suspension medium, residual glucose (>2.5% w/v) was required for enhanced spore survival. Thus, a defined medium containing basal salts, glucose, glutamate and zinc can be used to produce optimal concentrations of desiccation-tolerant blastospores of P. fumosoroseus. Received 27 October 1998/ Accepted in revised form 06 May 1999     

中国被毛孢和蝙蝠蛾拟青霉小鼠免疫功能调节作用的比较

目的观察中国被毛孢和蝙蝠蛾拟青霉对小鼠免疫功能的影响。方法分别给予中国被毛孢和蝙蝠蛾拟青霉后,测定观察对小鼠碳粒廓清功能、迟发性变态反应、溶血素抗体、淋巴细胞增殖和NK细胞活性等的影响。结果3．0g／kg中国被毛孢和蝙蝠蛾拟青霉可显著提高小鼠吞噬指数和半数溶血值（P〈0．01）、显著提高T、B淋巴细胞增殖能力（P〈0．01,P〈0．05）、显著提高NK细胞活性（P〈0．05）,0．5～1．5g／kg中国被毛孢都能显著抑制小鼠耳廓肿胀（P〈0．01,P〈0．05）,同时还能抑制脾脏和胸腺增大（P〈0．01,P〈0．05）。结论中国被毛孢和蝙蝠蛾拟青霉均具有免疫调节作用,而中国被毛孢免疫抑制及增强天然免疫系统作用要优于蝙蝠蛾拟青霉,而对适应性免疫系统增强作用蝙蝠蛾拟青霉优于中国被毛孢。     

真菌对唑类抗真菌药物的耐受机制

唑类抗真菌药物广泛用于临床和农业。唑类药物通过与羊毛甾醇14α-去甲基化酶（Erg11p/Cyp51）结合,抑制麦角甾醇合成,同时导致有毒甾醇积累。真菌可快速在转录水平上对唑类药物胁迫作出响应而导致耐药性,尤其是唑类药物外排泵基因和麦角甾醇合成相关基因表达的上调。农业和临床上绝大多数唑类药物耐药菌株的形成都是由麦角甾醇合成基因和唑类药物外排泵表达的变化或是突变所致。一些转录因子（如Pdr1p、Pdr3p、Upc2p、Yap1p、Tac1p、Mrr1p、CCG-8）和信号通路（如cAMP途径、PKC-MAPK途径、HOG MAPK途径、钙调磷酸酶途径）均参与对药物外排泵基因和麦角甾醇合成基因等的调控,影响唑类药物耐药性。针对于这些调控因子设计的抑制剂将有助于提高唑类药物的治疗效果。本文概述了唑类药物的抑菌机制、真菌对唑类药物耐药性形成的原因、真菌对唑类药物适应性响应机理,并对未来此领域的热点和方向进行了展望。