耐乙酸乳杆菌的筛选及其产乳酸特性

【背景】耐受乙酸的乳酸菌是传统谷物醋醋酸发酵过程中产生乳酸及其风味衍生物的重要功能微生物。【目的】从镇江香醋醋醅中分离鉴定具有耐乙酸特性的乳酸菌,并评价不同条件下该菌株的产乳酸能力。【方法】利用4%(体积比)乙酸含量的MRS培养基分离耐乙酸乳酸菌;对其进行16S rRNA基因鉴定、基因组测序、形态观察以及生理生化特性研究;考察不同乙酸浓度、葡萄糖浓度、发酵温度和时间对菌株产乳酸能力的影响。【结果】分离得到一株可耐受6%乙酸的乳杆菌Lactobacillus sp. JN500903;在厌氧静置、接种量5%、乙酸浓度5%、葡萄糖浓度40 g/L、发酵温度37°C、发酵时间10 d条件下,该菌株乳酸产量为16.1 g/L。【结论】乳杆菌JN500903能够耐受6%乙酸浓度,具有在酸性环境下合成乳酸的能力,有一定的应用潜力。     

A method for the selective enumeration and isolation of ruminal Lactobacillus and Streptococcus

Ruminal lactic acid-producing bacteria were selectively isolated and enumerated using a one hour aerobic exposure prior to incubation on a semi-selective Lactobacillus medium, MRS, under anaerobic conditions. The technique allowed growth of pure cultures of ruminal Lactobacillus spp. and Streptococcus bovis without supporting the growth of pure cultures of any of the prominent ruminal bacterial species. In mixed cultures, the one hour aerobic pre-incubation inhibited the growth of the obligate anaerobic ruminal bacteria which can otherwise grow on the MRS medium, and the subsequent anaerobic incubation permitted maximal recovery of the weakly aerotolerant ruminal lactic acid-producing Lactobacillus spp. and Streptococcus spp. The efficacy of this technique in selecting exclusively for the lactic acid-producing bacteria was also demonstrated from populations of rumen bacteria from mixed culture end-point in vitro fermentation, continuous in vitro culture and isolations from fresh ruminal samples.     

Microbiological and Biochemical Study of Coffee Fermentation

The coffee fermentation microflora were rich and mainly constituted of aerobic Gram-negative bacilli, with Erwinia and Klebsiella genuses at the highest frequencies. The best population increase was observed with lactic acid bacteria and yeasts, whereas those microorganisms that counted on a pectin medium remained constant during the fermentation step. Qualitatively, lactic acid bacteria belonged mainly to Leuconostoc mesenteroides species but the others microflora were relatively heterogeneous. The microorganisms isolated on pectin medium were Enterobacteriaceae, identified as Erwinia herbicola and Klebsiella pneumoniae, not reported as strong pectolytic strains. Throughout coffee fermentation, 60% of the simple sugars were degraded by the total microflora and not specifically by pectolytic microorganisms. Received: 21 August 2000 / Accepted: 25 September 2000     

Growth ofMethylomonas methanolica: Factors Influencing growth yield

Summary The activities of ACH¹, NAD-ICDH, NADP-ICDH and CS were determined in cell extracts of high and low citric acid-producing strains of A. niger, cultivated on molasses medium by the surface or submerged method. A high differentiation in the activities of the enzymes studied was found to occur at various accumulation stages of citric acid and during its decomposition by moulds. During intensive citric acid synthesis, the activity of ACH and that of both dehydrogenases decreased significantly (though they did not disappear completely) compared to their initial activities during the growth period of the mycelium. CS activity, however, was maintained at almost the same level over the whole fermentation period or increased slightly, particularly in the case of low citric acid-producing strains. The relationships between the activities of these enzymes were reversed during citric acid decomposition by moulds. Moreover, it was shown that in the period of mycelium growth the maximal activities of the enzymes were much higher in submerged culture than in surface culture.     

Simultaneous liquefaction,saccharification, and fermentation of l-lactic acid using aging paddy rice with hull by an isolated thermotolerant Enterococcus faecalis DUT1805

Simultaneous liquefaction, saccharification, and fermentation (SLSF) has attracted much attention for the production of bio-based chemicals, including l-lactic acid, due to its high efficiency and low cost. In this study, a lactic acid-producing bacterium with high tolerance of temperature up to 55 °C was isolated and characterized as Enterococcus faecalis DUT1805. Various strategies of stepwise controlled temperature were proposed and investigated for glucose utilization. The results indicated that E. faecalis DUT 1805 exhibited an optimal temperature at 50 °C, which could achieve temperature compatibility of enzyme, saccharification, and fermentation, and decrease the possibility of contamination by the other microorganisms during the large-scale fermentation. To reduce the cost of raw material and operation for lactic acid production, aging paddy rice with hull (APRH) was used in l-lactic acid production by simultaneous liquefaction, saccharification, and fermentation (SLSF). An open SLSF operation at 50 °C and pH 6.5, and 17% (w/v) solid loading in 5 L bioreactors was demonstrated with the lactic acid titer, yield, and productivity of 73.75 g/L, 87% to initial starch, and 2.17 g/(L h), respectively.

     

Lactic acid bacterial cell factories for gamma-aminobutyric acid

Gamma-aminobutyric acid is a non-protein amino acid that is widely present in organisms. Several important physiological functions of gamma-aminobutyric acid have been characterized, such as neurotransmission, induction of hypotension, diuretic effects, and tranquilizer effects. Many microorganisms can produce gamma-aminobutyric acid including bacteria, fungi and yeasts. Among them, gamma-aminobutyric acid-producing lactic acid bacteria have been a focus of research in recent years, because lactic acid bacteria possess special physiological activities and are generally regarded as safe. They have been extensively used in food industry. The production of lactic acid bacterial gamma-aminobutyric acid is safe and eco-friendly, and this provides the possibility of production of new naturally fermented health-oriented products enriched in gamma-aminobutyric acid. The gamma-aminobutyric acid-producing species of lactic acid bacteria and their isolation sources, the methods for screening of the strains and increasing their production, the enzymatic properties of glutamate decarboxylases and the relative fundamental research are reviewed in this article. And the potential applications of gamma-aminobutyric acid-producing lactic acid bacteria were also referred to.     

Toxicity of organic extraction reagents to anaerobic bacteria

Various forms of liquid-liquid extraction systems are being developed to separate products, such as ethanol and volatile fatty acids (VFA), from fermentation liquids, since distillation is energetically expensive. Continuous extraction is advantageous, as product inhibition of the fermentation is minimized. However, some extraction solvents may be toxic or inhibitory to microorganisms.Thirty organic chemicals were examined by means of a small scale (60 mL) batch fermentation bioassay procedure for their toxicity to a commercial inoculum (Methanobac, W.B.E. Ltd.), which was a mixed culture of facultatively anaerobic, acid-producing bacteria. Gas production, pH change of medium, and the concentrations of ethanol, VFA, and lactic acid were measured after 75 h growth. The optimum experimental conditions for toxicity testing were alfalfa as substrate (2 g), a buffered nutrient medium (pH 6.8), "Methanobac" inoculum (10 mL), and test chemicals at levels between 10 and 100 muL/mL.Thirteen chemicals were nontoxic, and included the paraffins (C(6)-C(12)), phthalates, organophosphorus compounds, Freon 113 (1,1,2-trichloro-1,2,2-trifluoro ethane), Aliquat 336 (tricaprylylmethyl ammonium chloride), di-isoamyl ether, and trioctylamine. Other amine extractants were partially toxic. Alcohols (C(5)-C(12)), ketones (C(5)-C(8)), benzene derivatives, isoamyl acetate, and di-isopropyl ether were toxic. Generally, the chemicals were not toxic unless present at levels in excess of that expected to be required to saturate the aqueous phase.Total gas production was a good indicator of toxicity even within 24 h, but the presence of homofermentative (nongas producing) lactic acid bacteria complicated interpretation."Methanobac" inoculum was compared with an inoculum derived from a rumen culture for four test chemicals. The results were essentially the same. However, the toxicity of a chemical to bacteria is likely to vary considerably between bacterial species.     

Citric acid production by Aspergillus niger ATCC 9142 from a treated ethanol fermentation co-product using solid-state fermentation

Aims:  To investigate the ability of the citric acid-producing strain Aspergillus niger ATCC 9142 to utilize the ethanol fermentation co-product corn distillers dried grains with solubles for citric acid production following various treatments.
Methods and Results:  The ability of A. niger ATCC 9142 to produce citric acid and biomass on the grains was examined using an enzyme assay and a gravimetric method, respectively. Fungal citric acid production after 240 h was higher on untreated grains than on autoclaved grains or acid-hydrolysed grains. Fungal biomass production was enhanced after autoclaving and acid-hydrolysis of the grains. Phosphate supplementation to the grains slightly stimulated citric acid production while methanol addition decreased its synthesis. Using the phosphate-supplemented grains, the optimal incubation temperature, initial moisture content of the grains and the length of fermentation time for ATCC 9142 citric acid production were determined to be 25°C, 82% and 240 h, respectively.
Conclusions:  A. niger ATCC 9142 synthesized citric acid on corn distillers dried grains with solubles. The phosphate-treated grains increased citric acid production by the strain.
Significance and Impact of the Study:  The ethanol fermentation co-product corn distillers dried grains with solubles could be useful commercially as a substrate for A. niger citric acid production.     

高产琥珀酸菌株的通量筛选

以琥珀酸放线杆菌Actinobacillus succinogenes F3—21为出发菌株,分别用吖啶黄、紫外线、紫外线．硫酸二乙酯和亚硝基胍进行诱变,产生突变菌库。用“96孔板培养－HPLC浓缩检测－厌氧瓶复筛”的模式筛选高产突变株。从1056株突变株中,筛选到一株高产菌株Ⅵ-10-C。连续传代10次,产酸水平不变。在5L发酵罐中补料分批发酵72h,Ⅵ－10－C产琥珀酸87．6g／L,生产强度1．22g/（L·h）,糖酸转化率0．66g／g;琥珀酸产量比出发菌提高了30％。代谢通量与关键酶活性分析表明：相比于F3-21,Ⅵ-10-C发酵过程中从磷酸烯醇式丙酮酸节点处流向草酰乙酸的代谢流量增加了28．9％,相对应的磷酸烯醇式丙酮酸羧化激酶（PEPCK）酶活提高了23．5％。结果表明用“96孔板培养-HPLC浓缩检测-厌氧瓶复筛”的模式能快速有效筛选高产琥珀酸菌株。     

固定化细胞利用离子交换树脂萃取发酵乳酸的研究

本文提出了利用海藻酸钙凝胶包埋固定化乳酸菌生产乳酸,用离子交换树脂从发酵液中分离出乳酸的新方法。该法成功地消除了产物乳酸对乳酸菌生长和产物乳酸形成的抑制作用,使发酵时间由１２０小时缩短到９６小时,乳酸的体积生产率由０．３２８ｇ／Ｌ·ｈ提高到０．４３２ｇ／Ｌ·ｈ。     

Method of culturing microorganisms at constant concentrations of the nutrient components

A method for batch cultivation of microorganisms in a flow medium is described, characterized by slight changes in concentrations of medium components in time and by the absence of products of vital activity of microorganisms in the fermentation medium. The conditions are achieved due to application of a fermentation installation with a microfiltrative membrane that separates the cells of cultivated microorganisms from the culture fluid and due to increasing the flow rate to a value at which the inlet and outlet concentrations of the medium components are almost equal. The cells of cultivated microorganisms under such conditions remain in the fermentation medium volume. The system was called "Ekostat". If the process is performed in "Ekostat" system, a positive deviation from the logarithmic law is observed for the growth rate of the yeast Candida utilis VSB-651 on ethanol cultivation.     

一种自下而上的合成微生物组理性构建策略，用于郫县豆瓣发酵剂设计

【目的】提出一种合成微生物组的理性构建策略,用于构建郫县豆瓣蚕豆醅初始发酵的微生物组合菌剂。【方法】采取自下而上的合成微生物组理性构建策略,以相对丰度、频率和特征向量中心度作为核心微生物属的选择指标,分析确定蚕豆醅发酵核心微生物;设计模拟原位体系的全合成培养基,并利用该培养基快速、稳定地检测核心微生物包括产香性能在内的发酵特征。基于核心微生物的产香互补性能进行双菌组合发酵实验,结合核心微生物之间的生长相互作用,设计三菌组合发酵菌剂并验证其发酵性能。【结果】本研究确定并分离了郫县豆瓣蚕豆醅发酵过程中的 9种核心微生物。检测核心微生物产生的挥发性风味化合物,发现酵母菌类、乳酸菌类和其他类微生物之间存在产香互补关系。然后,结合微生物间的生长抑制关系设计了由乳酸片球菌、肉葡萄球菌及异变假丝酵母组成的三菌组合菌剂。与企业原位发酵样品相比,三菌组合菌剂产生的挥发性化合物数量达到原位样品的63.1%,化合物种类结构较为相似。与原位样品相比,组合菌剂样品氨基酸态氮浓度提升了21.8%。【结论】本研究提出了一种自下而上的合成微生物组理性构建策略,基于此策略设计了郫县豆瓣蚕豆醅发酵组合菌剂。使用该组合菌剂作为起始发酵剂发酵的郫县豆瓣蚕豆醅具有良好的风味谱和优异的氨基酸态氮水平。本研究在合成微生物组构建与发酵食品工艺改造方面具有较大的科学与应用价值。     

高山被孢霉三阶段培养法生产花生四烯酸油脂

为提高高山被孢霉（Mortierella alpina）生物合成花生四烯酸油脂的生产效率,基于花生四烯酸油脂的积累机制,建立了一种三阶段培养法：第一阶段在全培养基中培养促进菌体生物量的快速积累,确定了60 g/L糖初始质量浓度时,最有利于生物量的快速积累;第二阶段在C源丰富而其他营养缺乏的条件下培养,促进油脂快速积累,对糖最佳初始浓度、接种时间、pH和培养时间进行了优化;第三阶段培养诱导油脂中花生四烯酸的高效积累,并确定此阶段培养时间为36 h时为最佳时间。实验结果表明：三阶段培养工艺条件下,菌体生物量、油脂量、花生四烯酸量分别为41.6、26.6和11.4 g/L,本研究相比传统分批发酵工艺在产率和花生四烯酸最终产量方面都有了显著提高。     

Possible role of acetyl-CoA-carboxylase in biosynthesis of mevalonic acid and sterols in rat liver]

Effect of citrate on acetyl-CoA incorporation into mevalonic acid, sterols and fatty acids after preliminary incubation of rat liver extracts under conditions optimal for acetyl-CoA carboxylase activation, was studied. 30 min preincubation with the citrate at 37 degrees C results in a 2--3-fold stimulation of the mevalonic acid biosynthesis from acetyl-CoA in the microsomal and soluble (140 000 g) fraction, and in that of sterols precipitated by digitonin or isolated by TLC in the mitochondria--free fraction. 2-14C-malonyl-CoA incorporation into the mevalonic acid and sterols and biosynthesis of sterols from 2-14C-mevalonic acid were not stimulated under those conditions. A correlation was shown to exist between the activity of acetyl-CoA carboxylase and the rate of acetyl-CoA incorporation into mevalonate and sterols; the activity of beta-hydroxy-beta-methylglutaryl-CoA reductase, limiting the rate of the sterol biosynthesis, was not changed. The stimulating effect of citrate was found to depend on the concentration of acetyl-CoA and NADPH in the medium. The data obtained suggest that the mevalonic acid biosynthesis in rat liver may occur in the presence of acetyl-CoA carboxylase through the formation of malonyl-CoA.     

基于微孔板的高通量筛选6-羟基烟酸转化菌方法的建立

建立了一种基于96孔板-酶标仪的双波长紫外分光光度法高通量筛选6-羟基烟酸转化菌的方法.实验以251nm为测定波长、231nm为参比波长测定转化样品的6-羟基烟酸含量,6-羟基烟酸与△A251-231在0.5～11 μg/mL浓度范围内有良好的线性关系,服从朗伯-比尔定律,平均回收率为99.11%～100.81%.利用96孔板-酶标仪,每天筛选量可达到2000～5000个反应,达到高通量筛选要求.     

Shear treatment of starter culture medium improves separation behavior of Streptococcus thermophilus cells

A central step in the production of starter cultures is the separation of the cells from the fermentation medium, which is usually achieved by disk centrifuges. In case of microorganisms which produce exopolysaccharides (e.g., various strains of lactic acid bacteria), the properties of the respective exopolysaccharides may interfere with this separation step. By using six strains of Streptococcus thermophilus the hypothesis was tested that a shear treatment of the fermented culture medium improves subsequent cell separation markedly. Depending on the type of exopolysaccharides (freely present in the medium, or as capsules around the cells) an energy input of up to 2.5 kJ/mL generated with an Ultra‐Turrax affected cell chain length of the strains and viscosity of fermentation medium differently. For bacteria producing capsular exopolysaccharides, space‐ and time‐resolved centrifugation experiments revealed an increase of sedimentation velocity after shear treatment. In general, viability of the microorganisms, detected by flow cytometry measurements and fermentation experiments, was not affected by the shearing procedure. The results therefore indicate that strain‐targeted shearing is helpful to improve the separability of cells from the fermented media.     

Sterol-like compound from sensory ganglia. Effect of a nerve growth factor and insulin on its biosynthesis

1. Sensory ganglia from 8-day-old chick embryos were incubated with a specific nerve growth factor and with insulin. 2. From the total lipid extract of the ganglia a compound with steroid characteristics was isolated. 3. The synthesis of this compound, measured spectrophotometrically, diminished after addition of the nerve growth factor and insulin to the incubation medium. 4. The incorporation of sodium [2-(14)C]acetate and dl-[2-(14)C]mevalonic acid into total lipids of the sensory ganglia was stimulated by the nerve growth factor and insulin, but the radioactivity of the sterol-like compound was slightly lower. The incorporation of labelled mevalonic acid either into total lipids or into the sterol-like compound was about 25% lower. 5. About 20% of the acetate incorporated into total lipids and about 87% of the mevalonic acid were recovered in the sterol-like compound.     

Lactic Acid Bacterial Fermentation of Burong Dalag

The fermented food, burong dalag, prepared in many Filipino homes, was studied to determine the nature of the microbiological and chemical changes that occur during fermentation. This is a lactic acid bacterial fermentation in which the species Leuconostoc mesenteroides, Pediococcus cerevisiae, and Lactobacillus plantarum played the major acid-producing role. The pH was lowered to below 4.0, and about 0.9% acid as lactic acid was attained in 1 week. It was essential to keep the product covered well to exclude air and subsequent growth of yeasts and mold.     

Biosynthesis of Abscisic Acid under Osmotic Stress: Studies Based on a Dual Labelling Technique

Sterile liquid suspension cultures from grape pericarp tissue incorporate label from mevalonic acid into abscisic acid (ABA), some of which is subsequently released into the surrounding medium. The addition of mannitol to the culture medium caused plasmolysis of individual cells and increased ABA generation and release. Levels in the medium increased up to 30 fold over a period of 8 h in the presence of mannitol (720 milliosmol). A dual labelling technique using ¹⁴C and ³H mevalonic acid was developed to distinguish between labelled compounds produced by turgid tissue and those specifically associated with the substantial increase in ABA synthesis following mannitol treatment.     

Adaptation of Lactobacillus plantarum IMDO 130201, a wheat sourdough isolate, to growth in wheat sourdough simulation medium at different pH values through differential gene expression

Sourdough is a very competitive and challenging environment for microorganisms. Usually, a stable microbiota composed of lactic acid bacteria (LAB) and yeasts dominates this ecosystem. Although sourdough is rich in carbohydrates, thus providing an ideal environment for microorganisms to grow, its low pH presents a particular challenge. The nature of the adaptation to this low pH was investigated for Lactobacillus plantarum IMDO 130201, an isolate from a laboratory wheat sourdough fermentation. Batch fermentations were carried out in wheat sourdough simulation medium, and total RNA was isolated from mid-exponential-growth-phase cultures, followed by differential gene expression analysis using a LAB functional gene microarray. At low pH values, an increased expression of genes involved in peptide and amino acid metabolism was found as well as that of genes involved in plantaricin production and lipoteichoic acid biosynthesis. The results highlight cellular mechanisms that allow L. plantarum to function at a low environmental pH.