L－亮氨酸发酵过程氮源反馈与非反馈控制的初步研究

氮源可直接影响氨基酸发酵过程中菌体生长与氨基酸生成．但是关于氨基酸发酵过程中氮源控制的研究报道并不多。本文作者在L－亮氨酸发酵过程碳源流加研究及动力学特征分析基础上^〔1〕,比较了几种非反馈型、反馈型补加硫酸铵的发酵结果．提出了较佳的控制方法。     

豆粕水解液为氮源发酵产脂肪酶的研究

研究了以豆粕水解液作为氮源,假丝酵母Candida sp．99—125发酵生产脂肪酶的过程。分析水解时间对于产酶的影响,对比豆粕水解前后作为氮源发酵时的产酶规律。在30L发酵罐中批次发酵酶活最高可达6000IU／mL,采用豆油反馈流加之后,发酵脂肪酶活力可达8500IU／mL。     

山梨糖发酵产生2—酮基—L—古龙酸氮源代谢规律

对山梨糖发酵产生2-酮基-L-古龙酸氮源代谢规律进行了初步研究。通过对这一混合发酵体系蛋白和尿素代谢的研究表明,氮源代谢与单一菌体发酵相比有其特殊性,主要表现在尿素的加入有两个作用,即作为生理碱性物质调节体系pH和为菌体代谢提供部分氮源,而体系的蛋白含量随发酵时间的延续不断增加,其增加的原因是巨大芽孢杆菌由营养体转变成芽孢所致,这是该发酵体系的特点。本文还对该发酵体系各种氨基酸变化规律进行了讨论,将一共17种氨基酸按其变化规律分成了三类,较好地解释了各种氨基酸的变化情况,为进一步深入研究该体系的动力学特性提供了数据基础。     

山梨糖发酵产生2-酮基-L-古龙酸氮源代谢规律

对山梨糖发酵产生2-酮基-L-古龙酸氮源代谢规律进行了初步研究。通过对这一混合发酵体系蛋白和尿素代谢的研究表明,氮源代谢与单一菌体发酵相比有其特殊性,主要表现在尿素的加入有两个作用,即作为生理碱性物质调节体系pH和为菌体代谢提供部分氮源,而体系的蛋白含量随发酵时间的延续不断增加,其增加的原因是巨大芽孢杆菌由营养体转变成芽孢所致,这是该发酵体系的特点。本文还对该发酵体系各种氨基酸变化规律进行了讨论,将一共17种氨基酸按其变化规律分成了三类,较好地解释了各种氨基酸的变化情况,为进一步深入研究该体系的动力学特性提供了数据基础。     

鲁氏接合酵母对酱油中氨基甲酸乙酯前体物的代谢

【目的】研究鲁氏接合酵母氮源代谢特性,确定鲁氏接合酵母氮源代谢与酱油中氨基甲酸乙酯前体物瓜氨酸和尿素积累的关系。【方法】通过单一氮源培养、偏好型氮源培养和盐胁迫培养,检测不同条件下鲁氏接合酵母对精氨酸、瓜氨酸和尿素的代谢能力。【结果】通过对鲁氏接合酵母氨基酸利用能力的分析,确定了甘氨酸、丙氨酸和天冬酰胺3种氨基酸为鲁氏接合酵母的偏好型氮源。在偏好型氮源存在时,鲁氏接合酵母对尿素和瓜氨酸的利用并不受到抑制,丙氨酸和甘氨酸还能够促进对二者的利用。鲁氏接合酵母在单一氮源培养条件下不会降解精氨酸而积累尿素和瓜氨酸,反而可以大量利用氨基甲酸乙酯的前体物尿素和瓜氨酸。但在盐胁迫下,鲁氏接合酵母利用尿素和瓜氨酸受到阻遏,从而造成酱油中氨基甲酸乙酯前体物不能被充分利用而积累。【结论】盐胁迫阻遏了鲁氏接合酵母对瓜氨酸和尿素的利用,从而造成酱油发酵过程中耐盐细菌所产生的氨基甲酸乙酯前体物的积累。     

解淀粉芽孢杆菌3-2发酵羽毛产氨基酸

【目的】建立废弃羽毛液体发酵工艺,优化发酵条件,提高羽毛降解率及氨基酸产量,研发新型、高效复合氨基酸肥料。【方法】利用解淀粉芽孢杆菌3-2发酵羽毛,探究温度、发酵时间、羽毛含量、单一碳源、复合碳氮源、金属离子等对废弃羽毛降解效果以及发酵液中氨基酸种类和含量的影响。【结果】废弃羽毛降解率与氨基酸总产量呈负相关。随着发酵时间延长,羽毛降解率增加,当发酵温度为37°C、羽毛添加量为1%、以乳糖为外加碳源、添加Mg2+时,羽毛降解率最高,达到81.92%。随着羽毛添加量增加,氨基酸总含量也增加(在20%范围以内),当发酵温度为37°C、降解时间为108 h、羽毛添加量为10%、乳糖添加量为0.5%、不添加复合碳氮源、不添加金属离子时,氨基酸的种类最全(富含17种氨基酸),总含量最高,达到20.861 g/kg。【结论】利用解淀粉芽孢杆菌3-2发酵废弃羽毛生产氨基酸复合肥料是一种可靠、环保、经济的方法,获得的氨基酸肥料营养齐全。研究结果为新型复合氨基酸肥料的研发提供技术支撑。     

氮源种类及溶氧水平对锁掷酵母产类胡萝卜素组分的影响

在锁掷酵母（Sporidioboluspararoseus）发酵产类胡萝卜紊的过程中,发酵产物中类胡萝卜紊种类繁多,而且性质相似,加大了不同色素分离纯化的难度。为定向积累不同种类的类胡萝卜素,以本实验室保藏锁挪酵母JD-2为出发菌,研究了氮源种类和浓度及溶氧对锁掷酵母产类胡萝卜素的影响,并在7L发酵罐中进行了补料分批发酵试验。发现培养基中同时添加有机氮源和无机氮源且溶氧控制较低（5％）时有利于β-胡萝卜素的大量积累,最佳有机氮源和无机氮源分别为玉米浆（20g／L）、硫酸铵（5g／L）。补料分批发酵时β-胡萝卜素产量达到31．28mg／L,红酵母烯12．38mg／L。培养基中只添加有机氮源且相对溶氧控制相对较高（30％）时有利于红酵母烯的大量积累,最佳有机氮源为酵母膏（20g／L）。补料分批发酵时红酵母烯产量达到38．96mg／L,8．胡萝卜素12．36mg／L。     

食用真菌发酵豆浆过程中对豆浆中几种生物活性物质的影响

本实验以灵芝、金耳以及冬虫夏草作为发酵菌种,选用豆浆为基本原料配制培养基,在发酵过程中定时取样并测定其中几种生物活性物质的含量,分析各生物活性物质在发酵过程中的变化。研究结果表明,三种食用真菌发酵豆浆的发酵液中总游离型大豆异黄酮含量在发酵过程中均缓慢上升并分别在发酵72 h、24 h和36 h达到最高,与发酵初期相比分别增加了31.81μg/m L、19.05μg/m L和15.71μg/m L;三种食用真菌发酵液中蛋白质含量随着发酵时间的推移而降低的同时氨基酸含量均有增加,且发酵液中的肽主要是分子量范围在300 Da~1 500 Da之间的具有较高生物活性的小分子肽,发酵液中的必需氨基酸含量以及占总氨基酸含量的比值均有增加。本实验探究了食用真菌发酵豆浆过程中生物活性物质的变化,为日后的富集游离型大豆异黄酮、多肽以及氨基酸等生物活性物质实验提供依据,并为工业化生产提供实践基础。     

酿酒酵母转运蛋白Agp1p泛素化对氮源利用的影响

摘要:【目的】研究酿酒酵母细胞膜关键氮源转运蛋白Agp1p的泛素化对其氮源利用的影响。【方法】构建一个基于双分子荧光互补技术的泛素化检测载体,检测Agp1p是否受到泛素化。采取定点突变的方法对Agp1p中可能的泛素化位点进行突变,研究其泛素化的关键作用位点及其对酿酒酵母氮源利用的影响。【结果】在谷氨酰胺、精氨酸、脯氨酸及铵盐为唯一氮源的培养基中,转运蛋白Agp1p均受到泛素化调控。定点突变实验结果显示,与出发菌株相比,潜在泛素化位点突变后,菌体荧光强度均有一定程度减弱。其中四重突变体Agp1pK11,14,98,112R荧光强度最弱,表明泛素化过程被显著抑制。系列突变菌株在9种氨基酸为复合氮源以及尿素为单一氮源的发酵实验表明,四重突变体菌体氮源利用率提高效果显著。【结论】转运蛋白Agp1p的泛素化受到不同氮源的影响,泛素化位点突变可以显著影响其泛素化过程,从而改变细胞对氮源的利用过程。     

影响克拉维酸生物合成的氨基酸

发酵液的氨基酸分析显示,谷氨酸,精氨酸,天门冬氨酸,丙氨酸易被棒状链霉菌利用,发酵培养基中添加上述氨基酸后,谷氨酸,精氨酸有利于克拉维酸的生物合成,适时添加谷氨酸,精氨酸可分别提高克拉维酸的产量约25%和12%;而蛋氨酸,半胱氨酸含S氨基酸对克拉维酸生物合成不利,不同来源的黄豆粉作发酵培养基氮源,因其组成中某些氨基酸含量的差异。可使克拉维酸的产量相差百分之十几。     

Continuous citric acid fermentation by <Emphasis Type="Italic">Candida oleophila</Emphasis> under nitrogen limitation at constant C/N ratio

Summary The central aspect of this work was to investigate the influence of nitrogen feed rate at constant C/N ratio on continuous citric acid fermentation by Candida oleophila ATCC 20177. Medium ammonia nitrogen and glucose concentrations influenced growth and production. Space-time yield (STY) meaning volumetric productivity, biomass specific productivity (BSP), product concentration, product selectivity and citrate/isocitrate ratio increased with increasing residence time (RT). BSP increased in an exponential mode lowering nitrogen feed rates. Highest BSP for citric acid of 0.13 g/(g h) was achieved at lowest NH₄Cl concentration of 1.5 g/l and highest STY (1.2 g/l h) with 3 g NH₄Cl/l at a RT of 25 h. Citric acid 74.2 g/l were produced at 58 h RT and 6 g NH₄Cl/l. Glucose uptake rate seems to be strictly controlled by growth rate of the yeast cells. Optimum nitrogen concentration and adapted C/N ratio are essential for successful continuous citric acid fermentation. The biomass-specific nitrogen feed rate is the most important factor influencing continuous citric acid production by yeasts. Numerous chemostat experiments showed the feasibility of continuous citrate production by yeasts.     

毕赤酵母工程菌高密度发酵中氨态氮含量的测定

首次尝试将钠氏法应用于毕赤酵母高密度发酵中,并与靛酚蓝检测法进行了比较,证实钠氏法在0~10 mg/L的范围内,相关性好(r2=0.996 5),精确度(RSD=2.14%~5.32%)和精密度(Prec ision=97%~105%)高,更适合于毕赤酵母发酵中氨态氮含量的检测。并对其检测条件进行优化,确定检测波长为400 nm,最佳的显色反应为10 min。该方法用于毕赤酵母高密度发酵表达重组人血清白蛋白胸腺肽的结果表明,在发酵液氨基氮含量低于1.5 mg/kg,重组目的蛋白的表达出现了明显的降解,改善并控制发酵液中氨态氮的含量后,可以明显控制目的蛋白的降解,提高产品的产率。结果证实该方法用于发酵液中氨态氮的检测时,准确可靠,切实可行。     

Effect of various organic compounds on synthesis of glucoamylase by an isolated strain Penicillium italicum

Yeast extract (0.5%) stimulates the production of glucoamylase and cell synthesis while methylene blue (0.1 mM) activates the synthesis of glucoamylase. Studies on the metabolic changes during fermentation of glucoamylase in a selected medium by P. italicum show that the rate of production of glucoamylase and cellular growth are greatly accelerated between 48 and 168 hr. Rapid growth of cells during this period may account for enhanced utilization of maltose and NH4NO3 from the medium. The acid production remains constant from 48 to 144 hr. Different forms of nitrogen decrease steadily. Although methylene blue stimulates the production of glucoamylase in the broth it has practically no effect on the rate of utilization of amino and total nitrogen from the broth.     

Production of emulsan in a fermentation process using soybean oil (SBO) in a carbon-nitrogen coordinated feed

A fermentation process for the microbial production of an amphipatic lipopolysaccharide, emulsan, has been established using a triglyceride carbon source in a coordinated carbon-nitrogen feed strategy. The polysaccharide was produced by the Acinetobacter strain at a productivity of about 0.5 g emulsan/L h while utilizing only the fatty acids (FA) portion of the triglycerides or free fatty acids that were fed into the medium.A useful correlation between the utilization of the fatty acids and the consumption of the nitrogen was found and employed for the practical feed strategy using an appropriate C--N ratio (7.7 g C/g N) of the soybeanoil (SBO) (carbon source) to the ammonium hydroxide base (nitrogen source). Either the pH control or the supervising computer system could accomplish the adequate balanced feed in to the fermentor. Lipolysis slowdown was overcome by switching from a triglyceride carbon source to a free fatty acids one. A yield of about 0.2 g emulsan/g fatty acids was obtained and a final concentration of over 20 g/L was reached. The polymeric product was found to be partially associated with the cell-oil complexes in the fermentation broth unless the oily carbon source was efficiently exhausted. A fedbatch fermentation that employed a shift of the carbon source feed from triglycerides to free fatty acids appeared to be an appropriate and feasible way of producing the polymer.     

沙葱对牛瘤胃体外发酵参数和产气量的影响

目的 探究添加沙葱对牛瘤胃体外发酵和产气量的影响。 方法 实验分为对照组(以玉米秸秆为发酵底物)和沙葱处理组(在发酵底物中添加沙葱),每组设置3个重复,于不同时间节点记录2组的产气量,并在发酵72 h后对发酵液的pH值、氨态氮以及挥发性脂肪酸进行测定。 结果 12、24和72 h时,2组产气量具有一定差异,在12 h时沙葱处理组显著高于对照组(t=3.717,P=0.020 5),而24 h及72 h时沙葱组产气量极显著高于对照组(t=4.832,P=0.008 5;t=4.953,P=0.007 7)。发酵72 h时对发酵液pH值及氨态氮进行测量发现沙葱组pH值显著高于对照组(t=3.949,P=0.017 0),而对照组的氨态氮含量极显著高于沙葱组(t=-4.806,P=0.009 0)。2组挥发性脂肪酸的含量差异无统计学意义。 结论 在体外发酵条件下添加沙葱对瘤胃微生物发酵状态具有一定的调节作用,沙葱作为反刍动物使用的植物提取物饲料添加剂具有潜在价值。     

Recovery of potassium clavulanate from fermentation broth by ion exchange chromatography and desalting electrodialysis

Ion exchange chromatography (IEC) and desalting electrodialysis (DSED) processes were developed for the recovery and purification of potassium clavulanate (KCA) from fermentation broth. A strong anion exchanger, Amberlite IRA 400 resin, a potassium acetate solution as equilibrium buffer, and a potassium chloride (KCl) solution as elution buffer were used for the recovery of KCA in IEC. In order to determine optimal operating conditions, the effects of various operating parameters such as equilibrium buffer pH and concentration, elution buffer concentration, gradient length, and volumetric flow rate on KCA recovery and by-product removal were investigated using a simulated fermentation broth. In the subsequent step of DSED, employing cation (Neocepta CMS, Tokuyama, Japan) and anion (Neocepta ACS, Tokuyama, Japan) exchange membranes were carried out to remove KCl that existed in a large amount in the ion exchanged solution. The effects of operation voltage and feed composition on the performance of DSED were investigated. Based on the operating conditions determined above, IEC and DSED were applied in sequence to an ultrafiltered fermentation broth. Almost complete removal of KCl was possible with no significant loss of KCA, although the KCA recovery was slightly lower than that with the simulated fermentation broth. Based on this observation, it was concluded that IEC and DESD could be an effective process combination for the recovery of KCA from fermentation broth.     

Comparative Study of Sugar Fermentation and Protein Expression Patterns of Two Lactobacillus plantarum Strains Grown in Three Different Media

A comparative study of two strains of Lactobacillus plantarum (REB1 and MLBPL1) grown in commercial medium (MRS broth), cucumber juice, and liquid pig feed was performed to explore changes to the metabolic pathways of these bacteria, using a proteomics approach (two-dimensional electrophoresis and liquid chromatography-tandem mass spectrometry) combined with analyses of fermentable sugars and fermentation end products. The protein expression showed that even with an excess of glucose in all media, both strains could metabolize different carbohydrates simultaneously and that hexoses could also be used via a phosphoketolase pathway with preferential expression in liquid feed. Sugar analyses showed that the fermentation of sugars was homolactic for all media, with some heterolactic activity in liquid feed, as shown by the production of acetate. Cucumber juice (the medium with the highest glucose content) showed the lowest hexose consumption (10%), followed by liquid feed (33%) and MRS broth (50%). However, bacterial growth was significantly higher in cucumber juice and liquid feed than in MRS broth. This discrepancy was due to the growth benefit obtained from the utilization of the malate present in cucumber juice and liquid feed. Despite different growth conditions, the synthesis of essential cellular components and the stress response of the bacteria were unaffected. This study has improved our understanding of the mechanisms involved in the growth performance of an appropriate lactic acid bacterium strain to be used for food and feed fermentation, information that is of crucial importance to obtain a high-quality fermented product.     

A media design program for lactic acid production coupled with extraction by electrodialysis

The aim of this study was to investigate industrial media for lactic acid fermentation to reduce the cost of nitrogen sources. Corn steep liquor (CSL) was successfully used at 5% (v/v) in batch fermentations. Use of soluble CSL improved the productivity approximately 20% with an advantage of clearer fermentation broth. Yeast extract (YE)-complemented CSL media further increased the productivity. It was found that 3.1 g L(-1) yeast extract and 5% CSL could be an effective substitute for 15 g L(-1) yeast extract in 10% glucose medium. Spent brewery yeast was also used as a sole nitrogen source equivalent to 5% CSL. Lactic acid was recovered by electrodialysis from the cell free broth. Depleted cell free broth supplemented with 5 g L(-1) of yeast extract performed reasonably in batch cultures. Reuse of the fermentation broth may reduce the cost of raw materials as well as minimize the fermentation wastes.     

Critical evaluation of sampling techniques for residual glucose determination in carbon-limited chemostat culture of Saccharomyces cerevisiae

In this paper, three sampling techniques for rapid quenching of cellular metabolism and subsequent separation of cells from fermentation broth are compared: (i) quick freezing of fermentation broth directly in liquid nitrogen; (ii) quenching metabolism by exposing the fermentation broth to stainless steel beads (4-mm diameter) in a filter syringe precooled to -18 degrees C; and (iii) withdrawal of the filtrate through a 0.45 microm filter attached to a syringe and a needle inserted directly into the fermentor. It was concluded that use of liquid nitrogen as a quenching method to rapidly arrest cellular metabolism, for quantitative analysis of extracellular glucose, is not a very reliable method and that the filter syringe steel beads work very well.     

Measurement and regulation of the culture reduction state in Clostridium acetobutylicum

With a constant glucose feed concentration, the change in the continuous culture dillution rate resulted in an altered fermentation profile and the cellular NADH content. The cultures growing at high dillution rates demonstrated an oxidative metabolism low NADH and butanol concentrations. The low specific NADH flourescence (F/X) at high butanol production rates suggested that a rapid regeneration of NADH to NAD is essential for a high solventogenic culture activity. The culture florescence and butanol concentration remained constant in the solventogenic dilution rate range of D = 0.05-0.2 h(-1) with an inverse relationship between the specific flourescence (F/X) and the specific butanol production rate, q(B). Flourometric NADH observations were confirmed by enzymatic NADH determination. The stiochiometric "Fermentation Equation" was used to check the experimental data consistency and to investigate the role of the available biosynthetic and reduction energy on the culture metabolic activities under different growth conditions. The butanol concentration in the broth was stabilized in a fed-batch process when the culture NADH fluorescence was being controlled through the addition of fresh medium.