激光诱变青霉PT95原生质体选育类胡萝卜素高产菌株*

采用激光对青霉PT95菌株的原生质体进行了诱变处理,选育到一株菌核生物量和类胡萝卜素含量均有显著提高的突变菌株L05。与出发菌株相比,L05菌株的菌核生物量提高了98.6％,菌核中的类胡萝卜素含量提高了28.3％,在查氏平板上的类胡萝卜素产率提高了154.0％。所选育的L05菌株经3次传代培养,菌落没有发生扇形变异,菌核生物量和类胡萝卜素含量没有明显改变,说明该菌株具有良好的遗传稳定性。     

外源β-胡萝卜素、光照对青霉PT95菌株菌核分化和类胡萝卜素产率的影响

初步研究了外源β-胡萝卜素和光照对青霉PT95菌株菌核分化和类胡萝卜素产率的影响。结果表明,在培养基中加入外源β-胡萝卜素后,PT95菌株渗出液出现的时间、菌核出现的时间延迟了,但菌核成熟的时间没变。培养基中的外源β胡萝卜素浓度越大,其渗出液、菌核出现的时间越迟。外源β胡萝卜素亦能降低PT95菌株的脂质过氧化水平和菌核中的类胡萝卜素含量。高氧胁迫的光照培养条件有利于PT95菌株的菌核分化和色素在菌核中的积累;与低氧胁迫的黑暗培养条件相比,其菌核生物量和类胡萝卜素产率分别增加了18.7%和101%。以上实验结果表明,若想获得高的菌核生物量和类胡萝卜素产率,应该尽可能在高氧胁迫、无抗氧化剂存在的条件下培养PT95菌株。     

外源β-胡萝卜素、光照对青霉PT95菌株菌核分化和类胡萝卜素产率的影响

初步研究了外源β-胡萝卜素和光照对青霉PT5菌株菌核分化和类胡萝卜素产率的影响。结果表明,在培养基中加入外源β-胡萝卜素后,PT5菌株渗出液出现的时间、菌核出现的时间延迟了,但菌核成熟的时间没变。培养基中的外源β-胡萝卜素浓度越大,其渗出液、菌核出现的时间越迟。外源β-胡萝卜素亦能降低PT5菌株的脂质过氧化水平和菌核中的类胡萝卜素含量。高氧胁迫的光照培养条件有利于PT95菌株的菌核分化和色素在菌核中的积累;与低氧胁迫的黑暗培养条件相比,其菌核生物量和类胡萝卜素产率分别增加了18．7％和101％。以上实验结果表明,若想获得高的菌核生物量和类胡萝卜素产率,应该尽可能在高氧胁迫、无抗氧化剂存在的条件下培养PT5菌株。     

微生物诱导子对青霉PT95菌株菌核生物量和类胡萝卜素产率的影响

菌核是许多丝状真菌形成的一种休眠体.我们从土壤中分离到一株经鉴定属于Penicillium thomii series的PT95青霉菌株,该菌株能在固态培养基上形成大量坚硬的砂粒状的菌核(直径约300μn).PT95菌株的菌核与众不同之处在于可以积累以β-胡萝卜素为主的类胡萝卜素[1].菌核的形成,除了遗传因素外,还受多种因素影响,例如生长环境中的温度、水势( Water potential)、有机物成分等[2-4].Hawker [5]认为对真菌的营养生长( Vegetative growth)有利的物质也对菌核生长有利.我们在以前的研究中也证实了合适的氮、碳源以及植物油的补充有利于提高PT95的菌核生物量[6].然而,菌核生物量的提高,只解决了一半问题.如果在得到大量菌核的同时,菌核中积累的类胡萝卜素含量也提高了,这才能保证PT95菌株类胡萝卜素产率的提高.     

青霉PT95菌株固态发酵产生类胡萝卜素的研究

本文对青霉Penicillium sp. PT95菌株在固态发酵条件下菌核内产生类胡萝卜素进行了初步研究。结果表明,在3种固态发酵培养基中,玉米粉培养基(SMA)比麸皮培养基和棉籽壳培养基更适合于PT95菌株固态发酵产生类胡萝卜素。为了增加菌核干重和提高类胡萝卜素产率,SMA中需要添加氮源、碳源和植物油。在所试的各种氮、碳源中,以硝酸钠和麦芽糖效果最佳。通过正交试验确定了在培养基盐溶液中添加硝酸钠3g/L,麦芽糖10g/L,豆油2.5g/L能使菌核干重由536g/100g提高到970g/100g(干料);类胡萝卜素产率由2149μg/100g提高到5260μg/100g(干料);β-胡萝卜素在类胡萝卜素中的含量由614%提高到71.3%。     

无机盐和碳氦源对青霉PT95类胡萝卜素产率的影响

供试的4种无机盐中,K2HPO4的单因子效应最好;K2HPO4 KCl MgSO4表现出最好的正协同效应。5种碳源都能被PT95菌株利用,麦芽糖和蔗糖是最适碳源。在以酵母膏为氮源的培养基上,PT95菌株的菌核生物量最高;而在以蛋白胨为氮源的培养基上,类胡萝卜素产率最高;铵盐和尿素对菌核形成不利;硝酸钠是最好的无机氮源。培养基中的含氮量保持在0．24～0．48g/L,含碳量保持在5．26～21．05g/L,有利于PT95菌株形成菌核和积累色素;培养基的最适C/N比为25：1。     

青霉PT95菌株菌核内产生类胡萝卜素的研究

从土壤中分离到一株经鉴定属于Penicilliumthomiiseries的菌株PT95,该菌株在6种固体培养基上都能形成大量的橙红色菌核,其中在察氏培养基上形成的菌核量最大,而在马铃薯葡萄糖琼脂培养基上形成的菌核的类胡萝卜素含量最高。培养基初始pH对菌核形成和色素含量无明显影响,但对菌落生长速度有显著影响。光照培养对菌核形成和色素含量无明显影响。薄层色谱分析表明,PT95菌株菌核内产生的类胡萝卜素由两种色素成分组成,其中β-胡萝卜素占总色素量的64．3％。     

红酵母类胡萝卜素形成的生理学研究

从数株红酵母中选出 1株产类胡萝卜素能力较强的红酵母RY 98(生物量、类胡萝卜素含量和产量分别为19.9g/L ,334 .8μg/ g和 6 .7mg/L) ;研究了该菌株产类胡萝卜素的最适营养与环境条件 ,获得了最佳的发酵生理学条件 :葡萄糖 40 g/L ,(NH4 ) 2 SO4 10 g/L ,酵母膏 3g/L ,蕃茄汁 2mL/L ,花生油 0 .5mL/L ,接种量 30mL/L ,初始pH 6 .0和通气量 (培养基装量 ) 4 0mL/ 2 5 0mL。在此初步优化的培养条件下 ,红酵母RY 98经 72h摇瓶发酵其生物量、类胡萝卜素含量和产量分别可达 2 6 .8g/L ,386 .9μg/ g和 10 .4mg/L ,依次比初筛中提高了 34 .7% ,15 .6 %和 5 5 .2 %。     

产类胡萝卜素酵母菌原生质体的制备、再生与诱变

确定了1株产类胡萝卜素红酵母Y-35的原生质体最佳制备条件和再生条件,以及在此基础上的诱变育种,通过实验,初步确定Y-35菌株原生质体形成和再生的适宜条件为;菌龄16h,蜗牛酶浓度1%,30℃处理60min。红酵母Y-35原生原体经紫外线诱变后得到18株诱变菌株,分别测定其生物量,类胡萝卜素含量及产量,获得2株类胡萝卜素产量明显提高的变异菌株RY-10和RY-19。其产量分别比出发菌株提高49%和54%。     

红酵母类胡萝卜素高产菌株的筛选及其发酵生理学条件研究

从数株红酵母中选出了1株产类胡萝卜素能力较强的红酵母RY－98（生物量,类胡萝卜素含量和产量分别为19．9g/L,334.8ug/g和6．7mg/L);研究了该菌株产类胡萝卜素的最适营养与环境条件,获得了最佳的发酵生理学条件;葡萄糖40g/L,(NH4)2SO4 10g/L,酵母膏3g/L,蕃茄汁2mL/L,花生油0.5mL/L,接种量30mLL初始pH6.0和通气量（培养基装置040ml/250mL:,在此初步优化的培养条件下,红酵母RY－98经72小时摇瓶发酵其生物量,类胡萝卜素含量和产量分别可达26．8g/L,386.9ug/g和10．4mg/L,依次比初筛中提高了34．7％,15．6％,和55．2％。     

Laser mutagenesis of protoplasts of Penicillium sp. PT95 for the enhancement of carotenoid yield

Aims: To isolate the protoplasts from Penicillium sp. PT95 and carry out laser mutagenesis to attain high-yield mutant strain for carotenoid production. Methods and Results: The mycelial pellets of PT95 strain were digested with the lytic enzyme for 3 h in order to attain protoplasts. The prepared protoplasts were irradiated using helium neon (He–Ne) laser. Among all regenerated colonies isolated from irradiated protoplasts, five colonies proved to be able to form sclerotia. The five colonies were named as strains L01, L02, L03, L04 and L05, respectively. Whereas, among all regenerated colonies isolated from no-irradiated protoplasts, no colonies were found to form sclerotia. Strains L01, L02, L03, L04 and L05 showed higher carotenoid yield than the original strain in Czapek’s agar medium. Strain L05 gave the highest pigment yield of 381 μg per plate, which was 2·54 times higher than that of original strain. Conclusions: These results suggest that PT95 strain may be mutagenized using laser-irradiation to obtain higher-yield mutant strains for carotenoid production. Significance and Impact of the Study: These data prompted us to consider that several attempts should be made to improve carotenoid production in PT95 by strain selection using classical screening and mutagenesis techniques.     

微生物诱导子对霉素PT95菌株菌核生物量和类胡萝卜素产率的影响

菌核是许多丝状真菌形成的一种休眠体。我们从土壤中分离到一株经鉴定属于Ｐｅｎｉｃｉｌｌｉｕｍｔｈｏｍｉｉｓｅｒｉｅｓ的ＰＴ95青霉菌株 ,该菌株能在固态培养基上形成大量坚硬的砂粒状的菌核 (直径约 30 0 μｍ)。ＰＴ95菌株的菌核与众不同之处在于可以积累以β 胡萝卜素为主的类胡萝卜素[1 ] 。菌核的形成 ,除了遗传因素外 ,还受多种因素影响 ,例如生长环境中的温度、水势 (Ｗａｔｅｒｐｏｔｅｎｔｉａｌ)、有机物成分等[2～ 4] 。Ｈａｗｋｅｒ[5] 认为对真菌的营养生长 (Ｖｅｇｅｔａｔｉｖｅｇｒｏｗｔｈ)有利的物质也对菌核生长有…     

Sclerotia growth and carotenoid production of Penicillium sp PT95 during solid state fermentation of corn meal

Using corn meal as fermentation substrate, the effect of some factors, fermentation time and supplementation of saccharide and nitrogen sources as well as vegetable oil, on the sclerotia growth and carotenoid production of Penicillium sp PT95 during solid state fermentation were studied. When PT95 strain was grown on the amended medium by supplementing of 3g NaNO3, 10g maltose and 2.5g soybean oil per liter of salt solution to basal medium for 20 days, the dry sclerotia weight and carotenoid yield reached 9.70 g and 5260 g / 100 g of substrate, respectively. Without supplementation only 5.36g dry sclerotia and 2149g carotenoid / 100 g of substrate was attained. © Rapid Science Ltd. 1998     

青霉PT95菌固态发酵产生类胡萝卜素的研究

本文对青霉Ｐｅｎｉｃｉｌｌｉｕｍｓｐ．ＰＴ９５菌株在固态发酵条件下菌核内产生类胡萝卜素进行了初步研究。结果表明,在３种固态发酵培养基中,玉米粉培养基（ＳＭＡ）比麸皮２基和棉籽壳培养基更适合于ＰＴ９５菌株固态发酵产生类胡萝卜素。为了增加菌核干重和提高类胡萝卜素产率,ＳＭＡ中需要添加氮源、碳源和植物油。在所度的各种氮、碳源中,以硝酸钠和麦芽糖效果最佳。通过我试验确定了在培养基盐溶液中添加硝酸钠３ｇ／Ｌ     

Sclerotial biomass and carotenoid yield of Penicillium sp. PT95 under oxidative growth conditions and in the presence of antioxidant ascorbic acid

AIMS: To determine the effect of oxidative stress and exogenous ascorbic acid on sclerotial biomass and carotenoid yield of Penicillium sp. PT95. METHODS: In this experiment, high oxidative stress was applied by the inclusion of FeSO(4) in the growth medium and exposure to light. Low oxidative stress was applied by omitting iron from the growth medium and by incubation in the dark. Supplementation of exogenous ascorbic acid (as antioxidant) to the basal medium caused a concentration-dependent delay of sclerotial differentiation (up to 48 h), decrease of sclerotial biomass (up to 40%) and reduction of carotenoid yield (up to 91%). On the contrary, the exogenous ascorbic acid also caused a concentration-dependent decrease of lipid peroxidation in colonies of this fungus. CONCLUSIONS: Under high oxidative stress growth condition, the sclerotial biomass and carotenoid yield of PT95 strain in each plate culture reached 305 mg and 32.94 microg, which were 1.23 and 3.71 times higher, respectively, than those at low oxidative stress growth condition. These data prompted us to consider that in order to attain higher sclerotial biomass and pigment yield, the strain PT95 should be grown under high oxidative stress and in the absence of antioxidants. SIGNIFICANCE AND IMPACT OF THE STUDY: These results suggest that strain PT95 may be used for solid-state fermentation of carotenoid production under high oxidative stress growth conditions.     

Effect of several elicitors on sclerotia biomass and carotenoid yield of Penicillium sp. PT95 during solid-state fermentation of corn meal

Six kinds of heat-released soluble cell-wall fragments (elicitors) were prepared respectively from Neurospora crassa, Monascus purpureus, Sporobolomyces roseus, Rhodotorula rubra, Nocardia corallina N89 and Actinoplanes tuftoflagellus A05. When Penicillium sp. PT95 was grown on corn meal (CM) solid medium containing appropriate amounts of elicitors, both its sclerotia biomass and the amount of carotenoid accumulated in sclerotia were enhanced significantly (P < 0.01). Every one of the elicitors except that fromM. purpureus could also increase significantly the β-carotene fraction of total pigment (P < 0.01). Among elicitors tested, the elicitor (150 μg/g CM) originating from R. rubra gave a maximum value of sclerotia biomass, reaching 15.90 g/100g CM; the elicitor (100 μg/g CM) from M. purpureus gave the highest total carotenoid of 14,446 μg/100 g CM and β-carotene yield of 10,112 μg/100 g CM, which were respectively 2.76 and 2.72 times higher than that of control. Experimental results also showed that the elicitor from M. purpureus could inhibit effectively the occurrence of sectoring during solid-state fermentation of strain PT95. This revised version was published online in July 2006 with corrections to the Cover Date.     

Influence of inocula and grains on sclerotia biomass and carotenoid yield of<Emphasis Type="Italic"> Penicillium</Emphasis> sp. PT95 during solid-state fermentation

Various inocula and grains were evaluated for carotenoid production by solid-state fermentation using Penicillium sp. PT95. Millet medium was more effective in both sclerotia growth and carotenoid production than other grain media. An inoculum in the form of sclerotia yielded higher sclerotia biomass compared to either a spore inoculum or a mycelial pellet inoculum. Adding wheat bran to grain medium favored the formation of sclerotia. However, neither the inoculum type nor addition of wheat bran resulted in a significant change in the carotenoid content of sclerotia. Among grain media supplemented with wheat bran (wheat bran:grain =1:4 w/w, dry basis), a medium consisting of rice and wheat bran gave the highest sclerotia biomass (15.10 g/100 g grain), a medium consisting of buckwheat and wheat bran gave the highest content of carotenoid in sclerotia (0.826 mg/g dry sclerotia), and a medium consisting of millet and wheat bran gave the highest carotenoid yield (11.457 mg/100 g grain).     

Effect of oxidative stress and exogenous beta-carotene on sclerotial differentiation and carotenoid yield of Penicillium sp. PT95

AIMS: To determine the effect of oxidative stress and exogenous beta-carotene on sclerotial differentiation and carotenoid yield of Penicillium sp. PT95. METHODS AND RESULTS: In this experiment, high oxidative stress was applied by inclusion of FeCl(3) (10 micromol l(-1)) in the growth medium and by light exposure. Low oxidative stress was applied by omitting iron from the growth medium and by incubation in the dark. Supplementation of exogenous beta-carotene (as antioxidant) to the basal medium caused a concentration-dependent delay of sclerotial differentiation (up to 72 h), decrease of sclerotial biomass (up to 43%) and reduction of carotenoid yield (up to 92%). On the contrary, the exogenous beta-carotene also caused a concentration-dependent decrease of lipid peroxidation in colonies of this fungus. CONCLUSIONS: Under high oxidative stress growth condition, the sclerotial biomass and carotenoid yield of PT95 strain in each plate culture reached 141 mg and 30.03 microg, which were 1.53 and 3.51 times higher respectively, than that at low oxidative stress growth condition. SIGNIFICANCE AND IMPACT OF THE STUDY: These data prompted us to consider that in order to attain higher sclerotial biomass and pigment yield, the strain PT95 should be grown under high oxidative stress and in the absence of antioxidants.     

Effect of copper-induced oxidative stress on sclerotial differentiation and antioxidant properties of Penicillium thomii PT95 strain

Penicillium thomii PT95 strain was able to form abundant orange, sand-shaped sclerotia in which carotenoids were accumulated. The aim of this work was to determine the effects of copper-induced oxidative stress on the sclerotial differentiation and antioxidant properties of PT95 strain. The results showed that the time of exudates initiation, sclerotial initiation and sclerotial maturation of PT95 strain were advanced in 1–2 days under the copper-induced oxidative stress growth conditions. The analytical results of sclerotial biomass, carotenoids content in sclerotia showed that copper-induced oxidative stress favored the sclerotial differentiation and biosynthesis of carotenoids. Under the copper-induced oxidative stress growth conditions, the total phenolics content and DPPH free radical scavenging activity of sclerotia of this fungus were decreased as compared with the control. However, the oxidative stress induced by a lower amount of CuSO₄ in media could enhance significantly the reducing power of sclerotia.