响应面法优化褐藻胶降解菌Cobetia sp.20发酵培养基

为了提高褐藻胶降解菌株Cobetia sp.20产褐藻胶裂解酶的能力,利用响应面法优化其发酵产褐藻胶裂解酶的培养基。首先利用单因素法分别对发酵培养基中的不同碳源、碳源添加量、不同氮源、氮源添加量以及氯化钠添加量、磷酸二氢钾添加量、硫酸镁添加量和pH进行探究,研究各因素对产酶的影响。在单因素实验的基础上,通过Plackett-Burman试验确定Cobetia sp.20发酵培养基中影响产酶的主要因素。通过响应面试验建立回归方程。研究结果表明,Cobetia sp.20最优发酵培养基配方为褐藻胶15.00 g/L、硫酸铵7.50 g/L、氯化钠15.00 g/L、硫酸镁0.50 g/L、磷酸二氢钾5.30 g/L、硫酸亚铁0.01 g/L、pH值7.58。优化后酶活为142.79 U/mL,比优化前提高了26.36%。褐藻胶裂解酶活的提高,为褐藻胶裂解酶的工业化生产提供了参考。     

Extraction and optimization of exopolysaccharide from Lactobacillus sp. using response surface methodology and artificial neural networks

Abstract

The microbial polysaccharides secreted and produced from various microbes into their extracellular environment is known as exopolysaccharide. These polysaccharides can be secreted from the microbes either in a soluble or insoluble form.Lactobacillus sp. is one of the organisms that have been found to produce exopolysaccharide. Exo-polysaccharides (EPS) have various applications such as drug delivery, antimicrobial activity, surgical implants and many more in different fields. Medium composition is one of the major aspects for the production of EPS from Lactobacillus sp., optimization of medium components can help to enhance the synthesis of EPS . In the present work, the production of exopolysaccharide with different medium composition was optimized by response surface methodology (RSM) followed by tested for fitting with artificial neural networks (ANN). Three algorithms of ANN were compared to investigate the highest yeild of EPS. The highest yeild of EPS production in RSM was achieved by the medium composition that consists of (g/L) dextrose 15, sodium dihydrogen phosphate 3, potassium dihydrogen phosphate 2.5, triammonium citrate 1.5, and, magnesium sulfate 0.25. The output of 32 sets of RSM experiments were tested for fitting with ANN with three algorithms viz. Levenberg–Marquardt Algorithm (LMA), Bayesian Regularization Algorithm (BRA) and Scaled Conjugate Gradient Algorithm (SCGA) among them LMA found to have best fit with the experiments as compared to the SCGA and BRA.     

Optimization of exopolysaccharide overproduction by Lactobacillus confusus in solid state fermentation under high salinity stress

It is believed that high concentrations of sodium chloride (NaCl) suppress the biosynthesis of exopolysaccharide (EPS) in lactic acid bacteria (LAB). Nevertheless, overproduction of EPSs due to high salinity stress in solid state fermentation performed on an agar surface was demonstrated in this study using a response surface methodology via a central composite design (CCD). Under optimized conditions with NaCl 4.97% and sucrose 136.5 g/L at 40.79 h of incubation, the EPS yield was 259% (86.36 g/L of EPS), higher than the maximum yield produced with the modified MRS medium containing only 120 g/L of sucrose without NaCl (33.4 g/L of EPS). Biosynthesis of EPS by Lactobacillus confusus TISTR 1498 was independent of biomass production. Our results indicated that high salinity stress can enhance EPS production in solid state fermentation.     

Optimization of Exopolysaccharide Overproduction by Lactobacillus confusus in Solid State Fermentation under High Salinity Stress

It is believed that high concentrations of sodium chloride (NaCl) suppress the biosynthesis of exopolysaccharide (EPS) in lactic acid bacteria (LAB). Nevertheless, overproduction of EPSs due to high salinity stress in solid state fermentation performed on an agar surface was demonstrated in this study using a response surface methodology via a central composite design (CCD). Under optimized conditions with NaCl 4.97% and sucrose 136.5 g/L at 40.79 h of incubation, the EPS yield was 259% (86.36 g/L of EPS), higher than the maximum yield produced with the modified MRS medium containing only 120 g/L of sucrose without NaCl (33.4 g/L of EPS). Biosynthesis of EPS by Lactobacillus confusus TISTR 1498 was independent of biomass production. Our results indicated that high salinity stress can enhance EPS production in solid state fermentation.     

A novel Ganoderma lucidum G0119 fermentation strategy for enhanced triterpenes production by statistical process optimization and addition of oleic acid

A novel enhanced triterpenes fermentation production process by Ganoderma lucidum G0119 with the addition of oleic acid in the medium has been developed and optimized. All of the six exogenous additives tested were found to exhibit stimulatory effect on mycelial growth and triterpenes biosynthesis by G. lucidum. The results show that oleic acid addition had significant role in promoting triterpenes production. The optimal concentration and time of oleic acid addition were determined to be 30 mL/L and 0 h, respectively. Furthermore, three significant factors influencing triterpenes production were identified as glucose, magnesium sulfate and temperature using the Plackett–Burman design. The optimized conditions by central composite design were 27.83 g/L glucose, 1.32 g/L magnesium sulfate, 26.2°C temperature. The triterpenes fermentation yield with the optimized medium based on actual confirmatory experimental data in 6 L fermentor was 1.076 g/L versus the statistical model predicted value of 1.080 g/L. Our innovatively developed triterpenes fermentation production technology and process has been proven to produce high triterpenes productivity and yield conceivably useful for industrial production.     

Production and chemical characterization of an exopolysaccharide synthesized by psychrophilic yeast strain <Emphasis Type="Italic">Sporobolomyces salmonicolor</Emphasis> AL<Subscript>1</Subscript> isolated from Livingston Island,Antarctica

The exopolysaccharide (EPS) production by psychrophilic Antarctic yeast Sporobolomyces salmonicolor AL₁ reached the maximum yield in medium containing sucrose (50 g/L) and diammonium sulfate (2.5 g/L) after a 5-d fermentation (5.64 g/L) at 22 °C, the dynamic viscosity of the culture broth reaching (after 5 d) 15.4 mPa s. EPS showed a mannan-like structure and high molar mass, and did not affect cellular viability and proliferation of murine macrophages. It exhibited also a protective effect against the toxic activity of Avarol.     

植物乳杆菌ZJ316生产细菌素

[目的]研究植物乳杆菌ZJ316生长和产细菌素的最佳培养基成分和发酵条件,以提高该菌产plantaricin ZJ316的能力.[方法]改变培养基成份和发酵条件,考察不同氮源、碳源等培养基成分和不同的发酵温度等条件对ZJ316生长和产细菌素的影响.[结果]最佳培养基为MRS培养基;优化后的培养基配方为葡萄糖10 g/L,麦芽糖10 g/L,酵母提取物10 g/L,蛋白胨10 g/L,柠檬酸三铵2 g/L,吐温80为1 Ml/L,K2HPO4·3H2O 6 g/L,乙酸钠5 g/L,硫酸镁0.2 g/L,硫酸锰0.05 g/L.培养基初始Ph6.5,30℃静置培养24 h.[结论]通过培养基成分和发酵条件的优化,细菌素产量提高了2.3倍,为进一步研究和规模化生产奠定基础.     

Screening of Ganoderma strains with high polysaccharides and ganoderic acid contents and optimization of the fermentation medium by statistical methods

Polysaccharides and ganoderic acids (GAs) are the major bioactive constituents of Ganoderma species. However, the commercialization of their production was limited by low yield in the submerged culture of Ganoderma despite improvement made in recent years. In this work, twelve Ganoderma strains were screened to efficiently produce polysaccharides and GAs, and Ganoderma lucidum 5.26 (GL 5.26) that had been never reported in fermentation process was found to be most efficient among the tested stains. Then, the fermentation medium was optimized for GL 5.26 by statistical method. Firstly, glucose and yeast extract were found to be the optimum carbon source and nitrogen source according to the single-factor tests. Ferric sulfate was found to have significant effect on GL 5.26 biomass production according to the results of Plackett–Burman design. The concentrations of glucose, yeast extract and ferric sulfate were further optimized by response surface methodology. The optimum medium composition was 55 g/L of glucose, 14 g/L of yeast extract, 0.3 g/L of ferric acid, with other medium components unchanged. The optimized medium was testified in the 10-L bioreactor, and the production of biomass, IPS, total GAs and GA-T enhanced by 85, 27, 49 and 93 %, respectively, compared to the initial medium. The fermentation process was scaled up to 300-L bioreactor; it showed good IPS (3.6 g/L) and GAs (670 mg/L) production. The biomass was 23.9 g/L in 300-L bioreactor, which was the highest biomass production in pilot scale. According to this study, the strain GL 5.26 showed good fermentation property by optimizing the medium. It might be a candidate industrial strain by further process optimization and scale-up study.     

Production of extracellular polysaccharide, EPS WN9, fromPaenibacillus sp. WN9 KCTC 8951P and its usefulness as a cement mortar admixture

The production of extracellular polysaccharide, EPS WN9, fromPaenibacillus sp. and its suitability as a viscosity modifying admixture for cement mortar mixing were investigated. After 48 h culture in an optimized medium, cell growth and EPS production were 1,2 g/L and 4.0 g/L, respectively. By adding EPS WN9 to mortar, it was possible to prepare a homogeneous mortar without material segregation and excess air entrapment. The optimal amount of EPS addition to mortar was found to be 0.02 to 0.05%(w/w) of the cement used. Increasing the dosage of EPS WN9 from 0 to 0.05%(w/w) resulted in a setting retardation of 0.14 h to 0.8 h and an increase in the compressive strength of mortar of 10 to 20%.     

Study of mycelial growth and bioactive polysaccharide production in batch and fed-batch culture of Grifola frondosa

The fermentation of Grifola frondosa was investigated in the shake flasks and a 5-L jar fermenter in batch and fed-batch modes. In the shake-flask experiments, the preferable mycelial growth and exopolysaccharide (EPS) production was observed at relatively low pH; maltose and glucose were preferred carbon sources for high mycelial production. The EPS was doubled after 13 d of cultivation when glucose was increased from 2% to 4%. Yeast extract (YE) (0.4%) in combination with corn steep powder (CSP) (0.6%) and YE (0.8%) in combination with CSP (1.2%) were preferred nitrogen sources for high mycelial production and EPS production, respectively. All plant oils tested significantly stimulate cell growth of G. frondosa but they failed to enhance EPS production. The EPS products usually consisted of two fractions of different molecular sizes varied by the plant oils used. The fed-batch fermentation by glucose feeding was performed when the glucose concentration in the medium was lower than 0.5% (5g/L), which greatly enhanced the accumulation of mycelial biomass and EPS; the mycelial biomass and EPS were 3.97g/L and 1.04g/L before glucose feeding, which reached 8.23g/L and 3.88g/L at 13 d of cultivation. In contrast, the mycelial biomass and EPS in the batch fermentation were 6.7g/L and 3.3g/L at 13 d of cultivation.     

Production, characterization and antioxidant activity of exopolysaccharides from submerged culture of Morchella crassipes

The aim of this work was to investigate the fermentation optimization, molecular characterization, and antioxidant activity in vitro of exopolysaccharides (EPS) from Morchella crassipes in submerged culture. Firstly, an optimal medium for EPS production was obtained by single-factor experiment and central composite design as follows: maltose 44.79?g/L and tryptone 4.21?g/L. Then, one fraction of EPS was obtained from the culture filtrates by size exclusion chromatography and the molecular characteristics were examined by a multi-angle laser light scattering and refractive index detector system. The weight-average molar mass and the polydispersity ratio of the EPS fraction were revealed to be 1.961?×?10(4)?g/mol and 1.838, respectively. FT-IR spectroscopy was used for obtaining vibrational spectra of the purified EPS fraction. Finally, the antioxidant activity of EPS was investigated and the relationship with molecular properties was discussed as well.     

OPTIMIZATION OF FERMENTATION MEDIUM FOR ACETOIN PRODUCTION BY Bacillus subtilis SF4-3 USING STATISTICAL METHODS

To improve the acetoin-producing ability of Bacillus subtilis SF4-3, isolated from “natto,” a Japanese traditional food, the fermentation medium was optimized in shake-flask fermentation by statistically designed methods. Based on results of the single-factor experiment, orthogonal experiment, and Plackett–Burman design, yeast extract, corn steep liquor, and urea were identified as showing significant influence on the acetoin production. Subsequently, the optimum combination of the three factors was investigated by the Box–Behnken design (BBD) of response surface methodology (RSM) in order to further enhance the acetoin production. The maximum acetoin yield of 45.4 g/L was predicted when the concentrations of yeast extract, corn steep liquor, and urea were 8.5 g/L, 14.6 g/L, and 3.8 g/L, respectively. The results were further confirmed in triplicate experiments using the optimized medium (glucose 160 g/L, yeast extract 8.5 g/L, corn steep liquor 14.6 g/L, urea 3.8 g/L, manganese sulfate 0.05 g/L, ferrous sulfate 0.05 g/L), and an acetoin yield of 46.2 g/L was obtained in the validation experiment, which was in agreement with the prediction. After the optimization of medium components, an increase of 36.28% in acetoin production was achieved in comparison to that at the initial medium levels.     

Bioconversion of biodiesel refinery waste in the bioemulsifier by Trichosporon mycotoxinivorans CLA2

ABSTRACT: BACKGROUND: The microbial bioemulsifiers was surface active compounds, are more effective in stabilizing oil-in-water emulsions. The yeasts have been isolated to produce bioemulsifiers from vegetable oils and industrial wastes. RESULTS: Trichosporon mycotoxinivorans CLA2 is bioemulsifier-producing yeast strain isolated from effluents of the dairy industry, with ability to emulsify different hydrophobic substrates. Bioemulsifier production (mg/L) and the emulsifying activity (E24) of this strain were optimized by response surface methodology using mineral minimal medium containing refinery waste as the carbon source, which consisted of diatomaceous earth impregnated with esters from filters used in biodiesel purification. The highest bioemulsifier production occurred in mineral minimal medium containing 75 g/L biodiesel residue and 5 g/L ammonium sulfate. The highest emulsifying activity was obtained in medium containing 58 g/L biodiesel refinery residue and 4.6 g/L ammonium sulfate, and under these conditions, the model estimated an emulsifying activity of 85%. Gas chromatography and mass spectrometry analysis suggested a bioemulsifier molecule consisting of monosaccharides, predominantly xylose and mannose, and a long chain aliphatic groups composed of octadecanoic acid and hexadecanoic acid at concentrations of 48.01% and 43.16%, respectively. The carbohydrate composition as determined by GC-MS of their alditol acetate derivatives showed a larger ratio of xylose (49.27%), mannose (39.91%), and glucose (10.81%). 1 H NMR spectra confirmed by COSY suggested high molecular weight, polymeric pattern, presence of monosaccharide's and long chain aliphatic groups in the bioemulsifier molecule. CONCLUSIONS: The biodiesel residue is an economical substrate, therefore seems to be very promising for the low-cost production of active emulsifiers in the emulsification of aromatics, aliphatic hydrocarbons, and kerosene.     

Effects of ascorbic acid and uracil on exo-polysaccharide production with Hericium erinaceus in liquid culture

Hericium erinaceus is a well known edible and medicinal mushroom used in East-Asia. Recently, H. erinaceus has attracted a lot of attention owing to its antitumor, immuno-modulatory, and cytotoxic effect. It has been postulated that the fruiting body of H. erinaceus contains a polysaccharide that is similar to β-D-glucan, which is known to have antitumor activity against Sarcoma 180. However, optimized liquid culture conditions for enhanced polysaccharide productivity have yet to be developed, which is a necessary step for industrial applications. Therefore, the aim of this study was to determine the optimal liquid culture conditions for maximum polysaccharide production. In shake flask cultures, the optimal concentration of ascorbic acid was found to be 2.0 g/L, which prevented the broth from changing color from yellow to black. The optimal culture conditions were determined to be 23°C, 200 rpm, and a 10% inoculum size, at an uncontrolled initial pH. In addition, the modified medium contained 20 g/L glucose, 10 g/L yeast extract, and 2.0 g/L ascorbic acid. The maximum mycelial biomass and exo-polysaccharide (EPS) production in the modified medium containing uracil was 13.43 and 1.26 g/L, respectively.     

galU基因在乳酸乳球菌中的克隆及其对胞外多糖产生的影响

从EcoliBL21克隆到UDP-葡萄糖焦磷酸化酶（UGPase）基因galU,与pNZ8048载体连接构建重组表达质粒pNZ8048-galU,进而导入乳酸乳球菌L.lactisL18中,得到重组菌L.lactisL18／pNZS048-galU,研究galU插入对该菌产生胞外多糖的影响。结果显示,在含葡萄糖和乳糖（20：20g／L）的MRS培养基中,重组菌L.lactisL18／pNZ8048-galU在30℃,pH6．5的条件下培养26h,EPS产量最高,为1489．54mg／L;而相同条件下,L．lactisL18培养28h产量最高,为848．93mg／L。二者相比,EPS产量增加了1．75倍。     

Optimization of acetic acid production from synthesis gas by chemolithotrophic bacterium--Clostridium aceticum using statistical approach

Efforts in optimizing reducing agents, cysteine-HCl.H2O and sodium sulfide in order to attain satisfactory responses during acetic acid fermentation have been carried out in this study. Cysteine-HCl.H2O each with five concentrations (0.00-0.50 g/L) was optimized one at a time and followed by sodium sulfide component (0.00-0.50 g/L). Response surface methodology (RSM) was used to determine the optimum concentrations of cysteine-HCl.H2O and sodium sulfide. The statistical analysis showed that the amount of cells produced and efficiency in CO conversion were not affected by sodium sulfide concentration. However, sodium sulfide is required as it does influence the acetic acid production. The optimum reducing agents for acetic acid fermentation was at 0.30 g/L cysteine-HCl.H2O and sodium sulfide respectively and when operated for 60 h cultivation time resulted in 1.28 g/L acetic acid production and 100% CO conversion.     

Optimization of fermentation conditions and rheological properties of exopolysaccharide produced by deep-sea bacterium Zunongwangia profunda SM-A87

Zunongwangia profunda SM-A87 isolated from deep-sea sediment can secrete large quantity of exopolysaccharide (EPS). Response surface methodology was applied to optimize the culture conditions for EPS production. Single-factor experiment showed that lactose was the best carbon source. Based on the Plackett–Burman design, lactose, peptone and temperature were selected as significant variables, which were further optimized by the steepest ascent (descent) method and central composite design. The optimal culture conditions for EPS production and broth viscosity were determined as 32.21 g/L lactose, 8.87 g/L peptone and an incubation temperature of 9.8°C. Under these conditions, the maximum EPS yield and broth viscosity were 8.90 g/L and 6551 mPa•s, respectively, which is the first report of such high yield of EPS from a marine bacterium. The aqueous solution of the EPS displayed high viscosity, interesting shearing thinning property and great tolerance to high temperature, a wide range of pH, and high salinity.     

利用扁桃斑鸠菊叶发酵生产蛹虫草胞外多糖的条件及其抗氧化活性

蛹虫草胞外多糖具有增强免疫力、抗疲劳等药理活性,有极高的保健价值。为高效地获取蛹虫草胞外多糖,本研究通过向发酵培养基中添加适量的扁桃斑鸠菊叶粉末,来提高蛹虫草发酵液中胞外多糖的产量,并对优化得到的胞外多糖红外吸收光谱和化学抗氧化活性进行了研究。实验结果表明,液体发酵最优条件为:扁桃斑鸠菊叶粉末添加量8 g/L、发酵时间9 d、pH 6.5、接种量5.0 mL,在此条件下,蛹虫草胞外多糖的产量可达(5.24±0.28) mg/mL,与未添加扁桃斑鸠菊叶的空白组相比,胞外多糖产量提高了约205.20%;红外分析与抗氧化活性实验结果显示,扁桃斑鸠菊叶对蛹虫草生产的胞外多糖结构和活性影响较小。该研究结果表明扁桃斑鸠菊叶能够有效地提高蛹虫草胞外多糖的产量,为蛹虫草胞外多糖的高效生产提供了新思路。     

Variations in exopolysaccharide production by Rhizobium tropici

Rhizobium tropici, a legume-symbiont soil bacterium, is known for its copious production of exopolysaccharide (EPS). Many aspects of this organism’s growth and EPS production, however, remain uncharacterized, including the influence of environment and culturing conditions upon EPS. Here, we demonstrate that R. tropici EPS chemical composition and yield differ when grown with different substrates in a defined minimal medium in batch culture. Exopolysaccharide was quantified from R. tropici grown using arabinose, glucose, sucrose, mannitol, fructose, or glutamate as a sole carbon source. All tested substrates produced plenteous amounts of exopolysaccharide material. Variations in pH and carbon-to-nitrogen (C/N) ratio also resulted in assorted cell growth and exopolysaccharide production differences. We found that optimizing the C/N ratio has a greater impact upon R. tropici EPS production than upon R. tropici growth. A maximum EPS yield of 4.08 g/L was realized under optimized conditions, which is large even in comparison with other known rhizobia. We provide evidence that the chemical composition of R. tropici EPS can vary with changes to the growth environment. The composition of glucose-grown EPS contained rhamnose-linked residues that were not present in arabinose-grown EPS.     

Optimization of culture conditions and medium components for the production of mycelial biomass and exo-polysaccharides with Cordyceps militaris in liquid culture

Both crude exo-biopolymers and mycelial biomass, produced by liquid culture of Cordyceps species, are believed to possess several potential health benefits. As a result of its known biological activities, Cordyceps militaris has been extensively characterized in regards to potential medicinal applications. However, optimized liquid culture conditions for enhanced polysaccharide productivity have yet to be developed, which is a necessary step for industrial applications. Therefore, in this study, the liquid culture conditions were optimized for maximal production of mycelial biomass and exo-polysaccharide (EPS) by C. militaris. The effects of medium composition, environmental factors, and C/N ratio were investigated. Among these variables 80 g, glucose; 10 g, yeast extract; 0.5 g, MgSO₄·7H₂O; and 0.5 g, KH₂PO₄ in 1 L distilled water were found to be the most suitable carbon, nitrogen, and mineral sources, respectively. The optimal temperature, initial pH, agitation, and aeration were determined to be 24°C, uncontrolled pH, 200 rpm, and 1.5 vvm, respectively. Under these optimal conditions, mycelial growth in shake flask cultures and 5 L jar bioreactors was 29.43 and 40.60 g/L, respectively, and polysaccharide production in shake flask cultures and 5 L jar bioreactors was 2.53 and 6.74 g/L, respectively.