Effect of nitrogen source on mycelial morphology and arachidonic acid production in cultures of Mortierella alpina

The effects of nitrogen source on arachidonic acid (AA) production and morphological changes during the culture of Mortierella alpina were investigated using an image analysis system. When yeast extract, gluten meal, or corn steep liquor was used, a circular pellet morphology was obtained. However, when Pharmamedia, fish meal, or soybean meal was used, M. alpina formed radial filamentous mycelia. The radial filamentous area in the case of soybean meal was 75% of the total mycelial area. In a jar fermentor culture, M. alpina morphology varied with the cultivation period: (i) at 0-6 h culture, the inoculated pellet-like mycelia were adapted to the new environment, (ii) at 6 h-1 d culture, filamentous mycelia grew exponentially which yielded a feather-like morphology, (iii) at 1-2 d culture, the filamentous mycelia became disentangled as a result of the mechanical agitation; consequently, the proportion of filamentous mycelia was increased, (iv) at 2-4 d culture, mycelia showed stationary growth, but the AA concentration increased rapidly, and (v) at 4-6 d culture, hyphae grew thick radially with the AA concentration continuing to increase gradually. In the case of the cultures with feather-like morphology obtained using soybean meal, the AA yield was 0.14 g/g dry cell weight, which was two times higher than that in cultures grown using yeast extract. These results suggest that the feather-like morphology of culture of M. alpina is suitable for AA production.     

Transformation of oil-producing fungus, Mortierella alpina 1S-4, using Zeocin, and application to arachidonic acid production

The arachidonic acid-producing fungus Mortierella alpina 1S-4, an industrial strain, was endowed with Zeocin resistance by integration of the Zeocin-resistance gene at the rDNA locus of genomic DNA. Plasmid DNA was introduced into spores by microprojectile bombardment. Twenty mg/ml Zeocin completely inhibited the germination of M. alpina 1S-4 spores, and decreased the growth rate of fungal filaments to some extent. It was suggested that preincubation period and temperature had a great influence on transformation efficiency. Four out of 26 isolated transformants were selected. Molecular analysis of these stable transformants showed that the plasmid DNA was integrated into the rDNA locus of the genomic DNA. We expect that this system will be applied for useful oil production by gene manipulation of M. alpina 1S-4 and its derivative mutants. On the basis of the fundamental transformation system, we also tried to overexpress a homologous polyunsaturated fatty acid elongase gene, which has been reported to be included in the rate-limiting step for arachidonic acid production, thereby leading to increased arachidonic acid production.     

肉毒碱脂酰转移酶基因沉默对高山被孢霉脂质积累的影响

高山被孢霉是一种工业化生产多不饱和脂肪酸的产油真菌。通过分析高山被孢霉脂肪酸氧化途径中相关基因的转录水平,发现肉毒碱脂酰转移酶(carnitine acyl-transferase,CAT)基因与高山被孢霉脂肪酸氧化紧密相关,进一步利用RNA干扰技术实现在高山被孢霉中CAT基因的沉默,分析发现,在高山被孢霉重组菌中,CAT活性下调了33.3%,总脂肪酸积累量相比野生型菌株提高38.1%,表明通过抑制β氧化途径可以提高高山被孢霉的脂质积累量,这为推动高山被孢霉在脂质生物合成工业化方面的应用奠定了理论基础。     

被孢霉菌菌丝体在贮存过程中油脂的变化

报导了花生四烯酸生产菌被孢霉菌（Ｍｏｒｔｉｅｒｅｌａｓｐ．Ｍ１０）菌丝体在贮存过程中油脂的变化。研究结果表明，不同培养时间的菌丝体（３～７ｄ）在室温下贮存１５ｄ，菌丝体中总脂含量由１８％～３０％上升至３６％～４１％；菌丝体中ＡＡ含量由１．１％～２．６％上升至２．６％～３．７％。但菌丝体油脂中ＡＡ含量在贮存过程中根据不同培养时间显示出不同变化规律。不同贮存温度（５～２５℃）对菌丝体中ＡＡ含量没有显著影响。在ｐＨ为８．０时贮存更有利于菌丝体中花生四烯酸含量的提高。     

Effect of Culture Variables on Mycelial Arachidonic acid Production by Mortierella alpina

Effect of culture conditions on biomass, lipid, and arachidonic acid production was investigated in the oleaginous fungus Mortierella alpina CBS 528.72 under shake flask conditions. Several factors have been found to affect the biomass buildup and lipogenesis in this fungus, complicated by the fact that different strains demonstrate varying optimization conditions. Growth, lipid accumulation, and arachidonic acid production in the strain investigated were influenced by media, pH, temperature, carbon source, nitrogen source, etc. The results indicated that the most effective medium for growth and arachidonic acid production was glucose yeast extract medium. The optimum pH and temperature were found to be 6.5 and 28°C, respectively. On the same weight basis, glucose was the most efficient carbon source for biomass and lipid production in this fungal strain which yielded 6.8 g/L dry biomass and 40.2% (w/w) total lipid after 7 days of cultivation. Maximum arachidonic acid (ARA) production of 40.41% achieved in rhamnose-containing media was not concomitant with higher biomass and lipid yields. Efficacy of organic carbon sources, viz, yeast extract and peptone over inorganic sources like sodium nitrate, ammonium sulfate, ammonium chloride, etc, was established in the present study. M. alpina CBS 528.72 grown in peptone acquired the highest lipid content (42.0% (w/w)). However, the ARA content (28.74%) proved to be significantly less than that grown in yeast extract (35.28%). Furthermore, it was found that the biomass and ARA production declined drastically in a medium with vegetable oils as the sole carbon source but triggered the lipogenic pathway leading to higher accumulation of total lipids. Under the ideal conditions mentioned above, the maximum biomass, total lipid, and arachidonic acid production were 6.8 g/L, 41.6%, and 35.28% total fatty acid, respectively, in shake flask system.     

利用被孢霉发酵生产花生四烯酸工艺配方优化研究

利用高山被孢霉Mortierella alpina I49-N18发酵生产花生四烯酸。优化筛选出最适于高山被孢霉斜面培养、种子摇瓶培养以及摇瓶发酵培养的配方,稳定的培养基配方为后续发酵生产提供保障。通过培养基配方单因子试验分别对碳源、氮源、无机盐、氨基酸、植物油等原料的合适添加量进行初步确定,然后设计正交实验进行验证。考察初始葡萄糖浓度在高山被孢霉发酵生产花生四烯酸过程中对脂肪酸组分的影响,分析脂肪酸主要组分在发酵过程中的变化趋势。考察结果显示发酵培养基中补充添加800 1000mg/kg磷酸盐,添加600 1000mg/kg谷氨酸钠盐、添加0.04%0.12%植物油可以有效提高发酵生产花生四烯酸的产量。通过发酵工艺放大,在200m3发酵罐规模的发酵生产中得到应用,发酵产量得到大幅度提升,培养7d发酵生产花生四烯酸(ARA)的产量达到10g/L以上。     

碳氮源及碳氮比对BAA中温α-淀粉酶发酵的影响研究

研究了BAA中温α-淀粉酶生产过程中,碳、氮源种类及碳氮比对发酵产酶的影响,结果表明,采用以40%玉米粉和60%玉米淀粉作碳源,以标准棉籽饼粉为氮源,碳氮源质量比为14：5,添加万分之四的进口专用发酵消泡剂,同时采用补料工艺,在最佳发酵工艺条件下,发酵酶活力突破900U/mL,平均832U/mL,较优化前的600U/mL平均提高38%。     

响应面法优化深黄被孢霉发酵生产花生四烯酸的培养基条件

为了降低深黄被孢霉YZ-124生产花生四烯酸的成本,研究了不同添加量的玉米黄浆水对发酵的影响,与葡萄糖培养基相比,在发酵培养基中添加一定量的玉米黄浆水对发酵产量无显著影响.在单因素实验的基础上,利用Design Expert设计了响应面实验,研究了葡萄糖浓度、不同添加量的玉米黄浆水和初始pH对花生四烯酸产量的影响.结果表明,最佳的培养基条件是葡萄糖浓度为90g/L、添加体积分数为25％的玉米黄浆水、初始pH6时,花生四烯酸(ARA)产量达到最大,为3.11g/L.     

Effect of carbon source on the mycelial growth and exo-biopolymer production by submerged culture of Paecilomyces japonica

A significant difference was observed in the production pattern of exo-biopolymer and broth rheology during submerged culture of Paecilomyces japonica when two efficient carbon sources (maltose and sucrose) were employed. The maximum concentration of biopolymers in maltose medium (30 g/l) was higher than that in sucrose medium (25 g/l), whereas maximum mycelial concentration displayed an inverse result (i.e., 25 g/l in sucrose medium and 20 g/l in maltose medium). The broth rheology and morphology of P. japonica were also markedly different between the two culture media.     

Maximizing the production of γ-linolenic acid in Mortierella ramanniana var. ramanniana as a function of pH,temperature and carbon source,nitrogen source,metal ions and oil supplementation

A locally isolated (Perryvale, Alta., Canada) fungal genus, Mortierella ramanniana var. ramanniana, was evaluated as a potential industrial producer of γ-linolenic acid (GLA). Six growth variables (pH, temperature, carbon source, nitrogen source, and metal ions and oil supplementation) were systematically manipulated. The results indicated that the GLA production for this particular strain, could be maximized by using a basal growth medium consisting of 5% dextrose and 1% yeast extract, supplemented with 5 mg/L Mn²⁺ with incubation at 20 °C. The lipid yield under optimum conditions was 54.2% of the total dry biomass and consisted of 84.3% unsaturated fatty acids. The yield of GLA per gram biomass represented 13.3% of the total lipid content, slightly higher than that of the typical evening primrose oil GLA yield. The study establishes this variety as an effective intermediate fungal source of GLA.     

微生物发酵生产花生四烯酸的研究进展

花生四烯酸是二十碳多不饱和脂肪酸,在有机体中发挥着独特的生物学功能,因此在食品、医药、化妆品等方面有着巨大的应用价值,利用微生物发酵生产花生四烯酸的研究也引起了人们的广泛关注.综述了花生四烯酸的结构、理化特性、代谢、生理功能、应用以及发酵研究的概况.     

Effect of the addition of pulse ingredients to milk on acid production by probiotic and yoghurt starter cultures

Pulses contain carbohydrates, proteins, minerals and vitamins which are essential requirements in the human diet and which could also serve as growth nutrients for probiotic and yogurt starter cultures. In this study, milk supplementation with pulse ingredients is examined as a means to increase the nutritional properties of yogurt and probiotic type beverages. The acid production rate of two yogurt starters (A and B) and two probiotic cultures (Lactobacillus rhamnosus and Lactobacillus acidophilus) was followed in milk supplemented with the following soy and pulse ingredients: pea protein, chickpea flour, lentil flour, pea fibre, soy protein concentrate and soy flour. The pulse ingredients had no negative effect on the acidification trends of the fermented milks. On the contrary, with yogurt culture B, pea fibre, pea protein and lentil flour significantly enhanced the acidification rate. All ingredients used for supplementation improved the acidification rate of probiotic cultures, and the highest effects were obtained with lentil and soy flour. Lentil flour had the lowest pH after 12 h which was significantly lower than the product enriched with the same quantity of skim milk powder. The effect of ingredient supplementation on the microbial composition (ratio of cocci to bacilli) of the yoghurt products was also examined. The ratio of cocci to bacilli was between 1.8 and 2.5 for all supplemented yogurt samples obtained with culture A, and these variations were not judged to be statistically significant (p < 0.05). With yogurt products obtained from culture B, however, there was a higher proportional level of lactobacilli in all supplemented samples, as compared to the milk control; the enhanced growth of the lactobacilli was particularly noted when lentil flour was added to milk.     

不同碳源对阿魏菇菌丝生长量及多糖产量的影响

阿魏菇是一种极具发展前景的食药两用大型真菌,实验用液体摇床培养法与平板培养法培养阿魏菇,对其在不同碳源培养基中的胞外多糖产量及菌丝生长情况进行了研究。结果表明,蔗糖为阿魏菇生长利用的最佳碳源,液体培养基中菌球长势好,胞外多糖产量高,平板培养菌丝生长速度快。葡萄糖作碳源培养前期胞外多糖量增加快,但胞外多糖产量和菌丝量不如蔗糖作碳源时高。以麦芽糖作碳源液体培养菌球生长不完整。阿魏菇对乳糖利用情况最差,胞外多糖产量及菌丝量均为最低。     

高山被孢霉GDP-L-岩藻糖合成途径中GDP-甘露糖-4,6-脱水酶的克隆、表达和功能鉴定

GDP-L-岩藻糖是糖复合物生物合成和糖类代谢的重要中间产物,作为岩藻糖转移酶的供体参与岩藻糖基化反应,具有重要的生理功能。高山被孢霉是重要的产油真菌,是唯一在分子水平上证明可合成GDP-L-岩藻糖的真菌。GDP-L-岩藻糖从头合成途径中的GDP-甘露糖-4,6-脱水酶(GMD)能催化GDP-D-甘露糖合成GDP-4-酮基-6-脱氧-D-甘露糖。对高山被孢霉中的GMD基因进行克隆、表达和功能鉴定,可进一步阐明GDP-L-岩藻糖体内代谢的分子生物机制。首先对GMD序列进行分析,并以p ET28a+质粒为骨架构建了GMD的表达载体,然后转化至大肠杆菌BL21中进行诱导表达。进一步利用Ni金属螯合层析纯化目的蛋白,采用液相色谱-质谱法分析酶反应产物,表明纯化蛋白具有GMD活性。最后对高山被孢霉进行发酵培养,发现GDP-L-岩藻糖产量在氮源耗尽后较高,可达0.10 mg/g。同时GMD的转录水平在氮源耗尽后发生了明显的上调,表明GMD在氮源耗尽后对高山被孢霉体内GDP-L-岩藻糖的合成具有重要作用。这为进一步利用高山被孢霉发酵生产GDP-L-岩藻糖和酶法转化生产GDP-L-岩藻糖奠定了理论基础。     

花生四烯酸的生理学功能及利用真菌生产的研究进展

花生四烯酸是人体必需脂肪酸,具有极高的保健价值,利用真菌发酵生产花生四烯酸已成为国内外研究的热点.本文对花生四烯酸的生理功能以及利用真菌生产的研究进行概括.     

Improving fruit juice origin assignment by combined carbon and nitrogen isotope ratio determination in pulps

The combination of the relative abundances of carbon and nitrogen isotopes (¹³C- and ¹⁵N-values) in fruit juice pulps led to characteristic clusters, on the basis of which regional origin assignments of citrus juices were possible, which would not have been attained on the basis of only one of these parameters. Thus, while the ¹³C-values of juice pulps from Italy partially overlapped with those from Brazil, the ¹⁵N-values permitted an origin assignment. First attempts to extend this multi-isotope analysis to a multi-compound¹⁵N analysis, by correlating the ¹⁵N-values of fruit juice pulps to those of free amino acids, were promising. The results obtained were in line with general observation on¹⁵N abundances and biochemical correlations of secondary plant products.     

二十二碳六烯酸和花生四烯酸在婴儿配方奶粉中的应用

介绍了乳母中天然存在的两种不饱和脂肪酸二十二碳六烯酸（DHA）和花生四烯酸（AA）在婴儿生长发育中的作用及其在配方奶粉中强化对非母乳喂养婴儿营养的重要性。并介绍了各专家组织对DHA和AA在婴儿配方奶粉中补充的推荐及目前的应用情况。探讨了HDA和AA的两种来源，即天然来源和利用生物技术发酵生产。DHA和AA的天然来源主要是鱼油和蛋黄，而目前商业上大都利用生物技术以薇藻和真菌发酵生产DHA和AA。介绍了生物技术发酵生产DHA和AA的工艺，并讨论了发酵产品在婴儿配方奶粉中应用的安全性。     

The influence of different nitrogen and carbon sources on mycotoxin production in Alternaria alternata

The aim of this study was to determine the influence of different carbon and nitrogen sources on the production of the mycotoxins alternariol (AOH), alternariol monomethyl ether (AME) and tenuazonic acid (TA) by Alternaria alternata at 28 °C using a semi-synthetic medium (modified Czapek-Dox broth) supplemented with nitrogen and carbon sources. Additionally the effect of shaken and static cultivation on mycotoxin production was tested. Initial experiments showed a clear dependency between nitrogen depletion and mycotoxin production. To assess whether nitrogen limitation in general or the type of nitrogen source triggers the production, various nitrogen sources including several ammonium/nitrate salts and amino acids were tested. In static culture the production of AOH/AME can be enhanced greatly with phenylalanine whereas some nitrogen sources seem to inhibit the AOH/AME production completely. TA was not significantly affected by the choice of nitrogen source. In shaken culture the overall production of all mycotoxins was lower compared to static cultivation. Furthermore tests with a wide variety of carbon sources including monosaccharides, disaccharides, complex saccharides such as starch as well as glycerol and acetate were performed. In shaken culture AOH was produced when glucose, fructose, sucrose, acetate or mixtures of glucose/sucrose and glucose/acetate were used as carbon sources. AME production was not detected. The use of sodium acetate resulted in the highest AOH production. In static culture AOH production was also stimulated by acetate and the amount is comparable to shaken conditions. Under static conditions production of AOH was lower except when cultivated with acetate. In static cultivation 9 of 14 tested carbon sources induced mycotoxin production compared to 4 in shaken culture. This is the first study which analyses the influence of carbon and nitrogen sources in a semi-synthetic medium and assesses the effects of culture conditions on mycotoxin production by A. alternata.