Medium optimization of carbon and nitrogen sources for the production of spores from Bacillus amyloliquefaciens B128 using response surface methodology

The production of spores from Bacillus amyloliquefaciens B128 was investigated in shaker-flask cultivation. Optimization of the culture medium was carried out by using a two-step approach. A quick identification of a suitable source of carbon and nitrogen was obtained by a simple screening experiment, which was followed by an application of response surface methodology (RSM) for further optimization design. A five-level four-factor central composite design was employed to determine the maximum spore yield at optimum levels for lactose, tapioca, ammonium sulfate and peptone. Tapioca and peptone showed a significant linear main effect, while lactose and ammonium sulfate had no significant linear effect. The spore production was also significantly affected by lactose–ammonium sulfate and lactose–peptone. Optimum cultivation parameters were (g/L): 12.7 of lactose, 16.7 of tapioca, 1.8 of ammonium sulfate and 8.0 of peptone. The prediction spore yield was 5.93 × 10⁸ (no/mL). The actual experimental results were in agreement with the prediction.     

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.     

Evaluation of Arthrospira platensis extracellular polymeric substances production in photoautotrophic, heterotrophic and mixotrophic conditions

The kinetic study of Arthrospira platensis extracellular polymeric substances (EPS) production under different trophic modes??photoautotrophy (100???mol photons m^?2?s^?1), heterotrophy (1.5?g/L glucose), and mixotrophy (100???mol photons m^?2?s^?1 and 1.5?g/L glucose)??was investigated. Under photoautotrophic and heterotrophic conditions, the maximum EPS production 219.61?±?4.73 and 30.30?±?1.97?mg/L, respectively, occurred during the stationary phase. Under a mixotrophic condition, the maximum EPS production (290.50?±?2.21?mg/L) was observed during the early stationary phase. The highest specific EPS productivity (433.62?mg/g per day) was obtained under a photoautotrophic culture. The lowest specific EPS productivity (38.33?mg/g per day) was observed for the heterotrophic culture. The effects of glucose concentration, light intensity, and their interaction in mixotrophic culture on A. platensis EPS production were evaluated by means of 32 factorial design and response surface methodology. This design was carried out with a glucose concentration of 0.5, 1.5, and 2.5?g/L and at light levels of 50, 100, and 150???mol photons m^?2?s^?1. Statistical analysis of the model demonstrated that EPS concentration and EPS yield were mainly influenced by glucose concentration and that conditions optimizing EPS concentration were dissimilar from those optimizing EPS yield. The highest maximum predicted EPS concentration (369.3?mg/L) was found at 150???mol photons m^?2?s^?1 light intensity and 2.4?g/L glucose concentration, while the highest maximum predicted EPS yield (364.3?mg/g) was recorded at 115???mol photons m^?2?s^?1 light intensity and 1.8?g/L glucose concentration.     

Fermentation characteristics and hypoglycemic activity of an exopolysaccharide produced by submerged culture of Stropharia rugosoannulata #2

We have investigated the mycelial growth and extracellular polysaccharide (EPS) production of Stropharia rugosoannulata #2 under optimal culture conditions in a 5-L stirred-tank fermenter. Maximum biomass growth (16.35 g/L) was achieved after 5 days of cultivation, whereas EPS reached its maximum level (10.83 g/L) after 8 days. The morphological parameters (i.e., mean diameter, circularity, roughness, compactness) of the fungal pellets and broth viscosity were also characterized. The compactness of the pellets was determined to be significantly and positively correlated with EPS content. The hypoglycemic effect of the polysaccharide, investigated in streptozotocin-induced diabetic rats, included decreases in the plasma concentrations of glucose (37 %), total cholesterol (26 %), and triacylglycerol (24 %) and decreased aspartate aminotransferase activity (20 %). The results indicate the potential of this polysaccharide to prevent hyperglycemia in diabetic patients.     

Effect of aeration rates on production of extracellular polysaccharide, EPS-R, by marine bacteriumHahella chejuensis

The production of an extracellular polysaccharide, EPS-R, from the marine bacteriumHahella chejuensis was investigated at various aeration rates in a batch culture. Higher aeration rate resulted in enhanced EPS production and increased the viscosity of the culture broth. At an aeration rate of 1.5 vvm, EPS-R (12.2 g/L) was obtained with a yield (Y _P/S) of 0.6 from the STN medium after 72h of cultivation. TheH. chejuensis cells changed their rod morphology to a short-rod form in the stationary growth phase.     

桑木层孔菌液体培养条件的研究

对桑木层孔菌(Phellinus mori)液体发酵条件进行了研究,以生物量和胞外多糖为指标,通过L16(45)和L9(34)正交表进行了两次正交试验,筛选出桑木层孔菌最适液体培养条件为:麦芽糖30 g/L,酵母浸粉和蛋白胨15 g/L(质量比2 1),KH2PO4和CaCl25.5 g/L(质量比1 1),初始pH6.0;通过单因素试验筛选出最适装液量为120 mL/250 mL,最适接种量为10%。在此条件下液体发酵培养7 d后,桑木层孔菌生物量达到23.375 g/L,胞外多糖产量达到3.993 g/L。     

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。     

Enhanced production of exopolysaccharides by fed-batch culture of Ganoderma resinaceum DG-6556

The objectives of this study were to optimize submerged culture conditions of a new fungal isolate, Ganorderma resinaceum, and to enhance the production of bioactive mycelial biomass and exopolysaccharides (EPS) by fed-batch culture. The maximum mycelial growth and EPS production in batch culture were achieved in a medium containing 10 g/l glucose, 8 g/l soy peptone, and 5 mM MnCl(2) at an initial pH 6.0 and temperature 31 degrees C. After optimization of culture medium and environmental conditions in batch cultures, a fed-batch culture strategy was employed to enhance production of mycelial biomass and EPS. Five different EPS with molecular weights ranging from 53,000 to 5,257,000 g/mole were obtained from either top or bottom fractions of ethanol precipitate of culture filtrate. A fed-batch culture of G. resinaceum led to enhanced production of both mycelial biomass and EPS. The maximum concentrations of mycelial biomass (42.2 g/l) and EPS (4.6 g/l) were obtained when 50 g/l of glucose was fed at day 6 into an initial 10 g/l of glucose medium. It may be worth attempting with other mushroom fermentation processes for enhanced production of mushroom polysaccharides, particularly those with industrial potential.     

Exopolysaccharide biosynthesis and related enzyme activities of the medicinal fungus, Ganoderma lucidum, grown on lactose in a bioreactor

Exopolysaccharide (EPS) production and biosynthesis were studied in Ganoderma lucidum, a fungus used in traditional Chinese medicine, grown with lactose in a bioreactor. -Galactosidase activity, which implies the existence of a lactose permease system, was induced by lactose. Lactose feeding also increased -phosphoglucomutase activity and EPS accumulation but decreased phosphoglucose isomerase activity and lactate concentration in the culture broth. A maximum cell density of 22 g l^–1 and EPS at 1.25 g l^–1 were obtained in fed-batch bioreactor culture.     

Production and purification of a novel exopolysaccharide from lactic acid bacterium Streptococcus phocae PI80 and its functional characteristics activity in vitro

Optimum culture conditions which ease the synthesis of a novel exopolysaccharide (EPS) from a potent marine strain Streptococcus phocae was proposed in this study. The strain grows well at 35 °C, pH 7.0 and NaCl (2%) with lactose and yeast extract as best carbon and nitrogen sources. The maximum yield of EPS (11.75 and 12.14 g/L) was obtained in the presence of lactose and yeast extract at a concentration of 20 g/L respectively. EPS was refined by gel filtration chromatography using phenyl Sepharose column which revealed the presence of arabinose, fructose and galactose sugar units with molecular mass about 2.8 × 10⁵ Da. Emulsifying and flocculating stability of EPS compared with three commercial hydrocolloids. EPS exhibited better activities which are similar to that of commercial hydrocolloids. Both crude and purified EPS exhibited strong antioxidant potential by quenching hydroxyl and superoxide anion radicals. Antibiofilm activity by inhibition of Gram positive and Gram negative biofilm forming bacteria was evident in our studies. Potential antioxidant activity and biofilm inhibiting property of EPS may lead to the development of novel food grade adjuncts.     

Sequential optimization approach for enhanced production of glutamic acid from Corynebacterium glutamicum 2262 using date juice

To improve glutamic acid production from Corynebacterium glutamicum 2262 using date juice, a culture medium was screened and optimized using the statistical experimental designs of Plackett-Burman and response-surface methodology. In the first step, a two-level Plackett-Burman design was adopted to select the most important nutrients influencing the glutamic acid production, which showed that the date juice sugars, urea, peptone, and glycine betaine were the most significant ingredients (P < 0.05). Finally, response surface Box-Behnken design was employed to develop a mathematical model to identify the optimum concentrations of key components for higher glutamic acid production, which revealed the following: date juice (45 g/L), urea (16.9 g/L), peptone (15 g/L), and glycine betaine (12 g/L). The high correlation between the predicted and observed values indicated the validity of the model. Glutamic acid concentration increased significantly with optimized medium (33.2 g/L) when compared with non-optimized medium (12 g/L).     

Taxonomical Properties of a Producer of Viral-neuraminidase Inhibitor,No. 289 Strain,and Culture Conditions for Inhibitor Production

Taxonomical properties of an actinomycete, strain No. 289, a potent producer of viral-neuraminidase inhibitor, and culture conditions for the production of the inhibitor have been studied. It belongs to the genus Streptomyces and is characterized mainly by spiral formation, smooth spore surface, Gray color-series and chromogenicity. A high yield of the inhibitor was achieved when it was cultured in a medium containing lactose, peptone and yeast extract at 27° under aerobic condition; the inhibitory activity of the broth reached as high as 2⁴ deHAI units/ml at the fifth day of the main culture.     

Culture Conditions Determine the Balance between Two Different Exopolysaccharides Produced by Lactobacillus pentosus LPS26

Lactobacillus pentosus LPS26, isolated from a natural fermentation of green olives, produces a capsular polymer constituted of two exopolysaccharides (EPS): EPS A, a high-molecular-weight (high-M_w) polysaccharide (1.9 × 10⁶ Da) composed of glucose and rhamnose (3:1), and EPS B, a low-M_w polysaccharide (3.3 × 10⁴ Da) composed of glucose and mannose (3:1). Fermentation experiments in a chemically semidefined medium with different carbon sources (glucose, fructose, mannitol, and lactose) showed that all of them except fructose supported EPS A production rather than EPS B production. The influence of temperature and pH was further analyzed. As the temperature dropped, increased synthesis of both EPS was detected. The control of pH especially enhanced EPS B production. With regard to this, the maximum total EPS production (514 mg liter⁻¹) was achieved at a suboptimal growth temperature (20°C) and pH 6.0. Continuous cultures showed that EPS A, synthesized mainly at low dilution rates, is clearly dependent on the growth rate, whereas EPS B synthesis was hardly affected. EPS production was also detected in supplemented skimmed milk, but no increase on the viscosity of the fermented milk was recorded. This could be linked to the high proportion of the low-M_w polysaccharide produced in these conditions in contrast to that observed in culture media. Overall, the present study shows that culture conditions have a clear impact on the type and concentration of EPS produced by strain LPS26, and consequently, these conditions should be carefully selected for optimization and application studies. Finally, it should be noted that this is, to our knowledge, the first report on EPS production by L. pentosus.     

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.     

Production and partial characterization of the exopolysaccharide from Pleurotus sajor caju

Microbial exopolysaccharides (EPSs) are very important because they are used in biotechnological applications in different industrial areas. The aim of the study was to determine the best EPS producer Pleurotus sp., to optimize EPS production and to perform partial purification and characterization of the produced EPS. After the production conditions were optimized, the EPS was isolated and partially purified. EPS was characterized by HP-TLC, 1H-NMR, FT-IR, and TGA. Hydroxyl, superoxide, and DPPH radical scavenging activities of the EPS were also investigated spectrophotometrically. The best EPS producer and its incubation period in submerged fermentation were determined as Pleurotus sajor caju and on 5 days, respectively. Culture conditions to increase EPS production were optimized as follows (in per liter): 90 g of glucose, 10 g of yeast extract, 10 g of peptone, and 100 mM of Mg2+. The optimal initial pH, temperature, and an agitation rate of culture were determined as 5.0, 25 °C, and 150 rate min−1, respectively. The highest EPS production was determined as 33.32 ± 1.6 g L−1. After isolation of EPS, one active fraction was obtained by gel filtration chromatography. EPS is composed mainly of glucose according to HP-TLC analysis. To the results, EPS had a complex structure by having carbohydrate and protein contents. The produced EPS had high degradation temperature as well as high antioxidant activity.     

Optimization of the culture condition for an antitumor bacterium Serratia proteamacula 657 and identification of the active compounds

Exploration of novel active anti-tumor compounds from marine microbes for pharmaceutical applications has been a continuously hot spot in natural product research. Bacterial growth and metabolites may greatly vary under different culture conditions. In this study, the effects of different culture conditions and medium components on the growth and bioactive metabolites of Serratia proteamacula 657, an anti-tumor bacterium found in our previous study, were investigated. The results showed that lower temperature, weak acidic condition and solid fermentation favored the bacterial growth and the production of active compounds. Four components in the culture medium, NaCl, peptone, yeast extract and MgSO₄, were found important to the bacterial growth and active compounds production in medium optimization. Under the optimized condition of solid state fermentation at pH 6.0–7.0, 23–25 °C, with the MgSO₄-free medium containing 10.0 g/L peptone, 1.0 g/L yeast extract and 19.45 g/L NaCl, the antitumor activity of S. proteamacula 657 and the yield of crude extracts increased about 15 times and 6 times than the sample obtained in the original liquid fermentation, respectively. The active components in the metabolites of S. proteamacula 657 were identified as a homolog of prodigiosin, a red bacterial pigment, based on the analysis of the NMR and GC–MS. The bacterium S. proteamacula 657, which is adapted to lower temperature, produced prodigiosin-like pigments with highly antitumor activity, suggesting the bacterium is a potential new source for prodigiosin production.     

Production of exopolysaccharide by Lactobacillus rhamnosus R and analysis of its enzymatic degradation during prolonged fermentation

The potential of Lactobacillus rhamnosus R for producing exopolysaccharide (EPS) when grown on basal minimum medium supplemented with glucose or lactose was investigated. EPS production by L. rhamnosus R is partially growth associated and about 500 mg of EPS per liter was synthesized with both sugars. The product yield coefficient (Y(EPS/S)) was 3.15 (0.0315 g of EPS [g of lactose](-1)) and 2.88 (0.0288 g of EPS [g of glucose](-1)). It was clearly shown that the amount of EPS produced declined upon prolonged fermentation. Degradation of EPS in fermentation processes was also assessed by measuring its molecular weights and viscosities. As these reductions might have a negative effect on the yield and viscosifying properties of EPS, it was essential to examine possible causes related to this breakdown. The decrease in viscosities and molecular weights of EPS withdrawn at different cultivation times permitted us to suspect the presence of a depolymerizing enzyme in the fermentation medium. Our study on enzymatic production profiles showed a large spectrum of glycohydrolases (alpha-D-glucosidase, beta-D-glucosidase, alpha-D-galactosidase, beta-D-galactosidase, beta-D-glucuronidase, and some traces of alpha-L-rhamnosidase). These enzymes were localized, two of them (alpha-D-glucosidase and beta-D-glucuronidase) were partially purified and characterized. When incubated with EPS, these enzymes were capable of lowering the viscosity of the polymer as well as liberating some reducing sugars. Upon prolonged incubation (27 h), the loss of viscosity was increased up to 33%.     

Optimization of critical medium components using response surface methodology for biomass and extracellular polysaccharide production by <Emphasis Type="Italic">Agaricus blazei</Emphasis>

Response surface methodology (RSM) was applied to optimize the critical medium ingredients of Agaricus blazei. A three-level Box–Behnken factorial design was employed to determine the maximum biomass and extracellular polysaccharide (EPS) yields at optimum levels for glucose, yeast extract (YE), and peptone. A mathematical model was then developed to show the effect of each medium composition and its interactions on the production of mycelial biomass and EPS. The model predicted the maximum biomass yield of 10.86 g/l that appeared at glucose, YE, peptone of 26.3, 6.84, and 6.62 g/l, respectively, while a maximum EPS yield of 348.4 mg/l appeared at glucose, YE, peptone of 28.4, 4.96, 5.60 g/l, respectively. These predicted values were also verified by validation experiments. The excellent correlation between predicted and measured values of each model justifies the validity of both the response models. The results of bioreactor fermentation also show that the optimized culture medium enhanced both biomass (13.91 ± 0.71 g/l) and EPS (363 ± 4.1 mg/l) production by Agaricus blazei in a large-scale fermentation process.     

Optimization of probiotic and lactic acid production by Lactobacillus plantarum in submerged bioreactor systems

Biomass and lactic acid production by a Lactobacillus plantarum strain isolated from Serrano cheese, a microorganism traditionally used in foods and recognized as a potent probiotic, was optimized. Optimization procedures were carried out in submerged batch bioreactors using cheese whey as the main carbon source. Sequential experimental Plackett–Burman designs followed by central composite design (CCD) were used to assess the influence of temperature, pH, stirring, aeration rate, and concentrations of lactose, peptone, and yeast extract on biomass and lactic acid production. Results showed that temperature, pH, aeration rate, lactose, and peptone were the most influential variables for biomass formation. Under optimized conditions, the CCD for temperature and aeration rate showed that the model predicted maximal biomass production of 14.30 g l⁻¹ (dw) of L. plantarum. At the central point of the CCD, a biomass of 10.2 g l⁻¹ (dw), with conversion rates of 0.10 g of cell g⁻¹ lactose and 1.08 g lactic acid g⁻¹ lactose (w/w), was obtained. These results provide useful information about the optimal cultivation conditions for growing L. plantarum in batch bioreactors in order to boost biomass to be used as industrial probiotic and to obtain high yields of conversion of lactose to lactic acid.     

营养条件对光滑球拟酵母发酵生产丙酮酸的影响

丙酮酸是多种氨基酸、维生素及其它有用物质的重要前体,广泛应用于化工、制药及农用化学品工业。能够直接发酵生产丙酮酸的菌种主要有Ａｃｉｎｅｔｏｂａｃｔｅｒ[1],Ｅｎｔｅｒｏｂａｃｔｅｒ[2],Ｅｎｔｅｒｏｃｏｃｃｕｓ[3],Ｅｓｃｈｅｒｉｃｈｉａ[4],Ａｇａｒｉｃｕ?..