The role of environmental factors and medium composition on bacteriocin production by an aquaculture probiotic Enterococcus faecium MC13 isolated from fish intestine

The aim of this study was to optimize medium composition for higher yield of total viable cells and bacteriocin by Enterococcus faecium MC13. The factors such as peptone, meat extract, yeast extract, lactose, glycerol, tween 80, triammonium citrate and K₂HPO₄ were selected based on the Lactobacillus MRS medium composition. Two level factorial designs (FD) and steepest ascent path were performed to identify vital factors among the variables. Through the 2^?8 FD, peptone, yeast extract and lactose were found to be significant factors involved in the enhanced production of viable cells and bacteriocin. Therefore, these three foremost factors were further optimized by central composite design to achieve efficient yield. The optimum MRS composition was found to be peptone (40.0 g/L), meat extract (30.0 g/L), yeast extract (40.0 g/L), lactose (24.0 g/L), glycerol (5.8 g/L), Tween 80 (3.0 g/L), triammonium citrate (1.0 g/L), K₂HPO₄ (2.5 g/L), MgSO₄·7H₂O (0.10 g/L), MnSO₄·7H₂O (0.05 g/L) and dipotassium PO₄ (2.0 g/L). The optimized growth medium allowed higher amount of bacteriocin activity (36,100 AUml^?1) and total viable cells (14.22 LogCFUml^?1) production which were two-times higher than the commercial MRS medium.     

Effect of Exogenous Factors on Bacteriocin Production from Lactobacillus paracasei J23 by Using a Resting Cell System

A resting cell system was developed for bacteriocin Lac-B23 production from Lactobacillus paracasei J23. The resting cell medium contained (g/L): Glucose 20, Sodium acetate 5.0, MnSO₄ 0.25 MgSO₄ 0.5, Ammoniumhydrogencitrate 1.0, KH₂PO₄ 1.0. The resting cell incubation time and temperature were 20 h and 37 °C and the effects of exogenous factors, including amino acids, glycerol, pyruvic acid, and α-ketoglutaric acid were investigated. Cys and Gly could stimulate the production of bacteriocin, while no stimulus effect was observed for Glu, Tyr and Ala. Glycerol and pyruvic acid increased bacteriocin production and the optimum concentrations were 1% and 30 g/L, respectively. Bacteriocin could act as an inducer of its own biosynthesis. These findings are of importance for the further study of bacteriocin biosynthesis regulation and for the improvement of bacteriocin production yields.     

Optimization of medium for phenylalanine ammonia lyase production in <Emphasis Type="Italic">E. coli</Emphasis> using response surface methodology

A culture medium for phenylalanine ammonia lyase (PAL) production in E. coli was developed following preliminary studies by means of response surface methodology (RSM). The medium components having significant effect on the production were first identified by using a fractional factorial design. Then, central composite design (CCD) was used to optimize the medium constituents and explain the combined effects of four medium constituents: glucose, yeast extract, (NH₄)₂HPO₄ and MgSO₄. A quadratic model was found to fit the PAL production. CCD revealed that the optimum values of the test variables for PAL production were glucose 28.2 g/L, yeast extract 5.01 g/L, (NH₄)₂HPO₄ 7.02 g/L and MgSO₄ 1.5 g/L. PAL production of 62.85 U/g, which was in agreement with the prediction, was observed in the verification experiment. In comparison to the production of basal medium, 1.8-fold increase was obtained.     

Statistical optimization of fermentation conditions for the improved production of poly-β-hydroxybutyrate from Bacillus subtilis

Poly-β-hydroxybutyrate (PHB) has been an effective biodegradable plastic obtained by microbial fermentation. Batch fermentation of Bacillus subtilis features an attractive system for the production of PHB. Identification of appropriate media components and cultivation conditions are extremely important for the optimal production of biomass and/or PHB production. Statistical media design was utilized for the optimization of different fermentation variables (glucose, peptone, sodium chloride, K₂HPO₄, KH₂PO₄, ammonium sulfate, ammonium chloride, sodium sulfate, temperature, inoculum size, and pH). The optimized media predicted the optimal dry cell weight of 7.54?g?L^?1 and PHB production of 77.2?mg?L^?1 at 1?g?L^?1 of peptone, 1.46?g?L^?1 sodium sulfate, and pH 6.8 in 24?h. Glucose utilization, batch growth, and PHB production kinetics of B. subtilis were determined experimentally. The effect of substrate inhibition on specific growth rate was also determined experimentally for B. subtilis. The values of kinetic and substrate inhibition parameters obtained from this study shall be utilized to develop a mathematical model for PHB production for further improving the production of PHB.     

Effect of salt concentration on production of polyunsaturated fatty acids in <Emphasis Type="Italic">Thraustochytrium aureum</Emphasis> ATCC 34304

This study attempted to characterize the effects of NaCl and MgSO₄ as the primary components of sea salt affecting the yields of production of polyunsaturated fatty acids (PUFAs) by Thraustochytrium aureum ATCC 34304. Reductions in the NaCl concentration of the culture medium suppressed the formation of palmitic acid (PA, C16: 0) but enhanced the production of PUFAs, which induced an increase of the concentration of docosahexaenoic acid (DHA, C22: 6) up to 46.65% from 44.26%. MgSO₄ revealed a similar, yet more highly significant, effect on the fatty acids profile than NaCl. The yields of PUFAs and DHA showed maximum values such as 67.10% and 49.47%, respectively, at the concentration of 10 g L⁻¹ for NaCl and 0 g L⁻¹ for MgSO₄. However, the quantities of mono-and diunsaturated fatty acids, oleic acid (OA, C18: 1) and linoleic acid (LA, C18: 2) at the same concentrations, were compared with those of normal culture medium. Additionally, it was investigated that the increase of culture temperature reduced PUFAs contents but the reductions were recovered by the removal of MgSO₄ form the culture medium, which showed that concentration of salts and culture temperature affected independently the production of PUFAs in T. aureum.     

Production and characterization of laccase and manganese peroxidase from the ligninolytic fungus Fomes sclerodermeus

Fomes sclerodermeus was grown on semi‐defined media based on yeast extract, peptone and glucose (YPG). The fungus produced a minimum basal level of laccase activity irrespective of culture medium. The highest laccase production (20 U cm^?3) was obtained in cultures supplemented with CuSO₄. Manganese peroxidase (MnP) could only be detected when MnSO₄ was added to the medium. None of the aromatic compounds tested stimulated further laccase or MnP production. Laccase and MnP stimulated by Cu²⁺ or Mn²⁺ respectively were purified. Two different laccase isoenzymes with the same molecular mass (67 kDa) and N‐linked carbohydrate content (3%) and a slight difference in their pI values (3.41 and 3.48) were characterized. In addition, two different MnP isoenzymes with the same molecular mass (47 kDa) and N‐linked carbohydrate content (4%) and different pI values (3.35 and 3.45) were characterized. Both enzymes showed good stability at 25 °C and over a wide range of pH. Both laccases oxidize ABTS (2,2′‐azino‐bis(3‐ethylbenzthiazoline‐6‐sulfonic acid) more efficiently than 2,6‐dimethoxyphenol (DMP) with similar efficiency values (K_cat/K_m) while the MnP I, the major peroxidase isoenzyme in the studied conditions, oxidizes the Mn²⁺ and Mn‐mediated activity on DMP more efficiently than MnP II. Copyright © 2006 Society of Chemical Industry     

Optimization of the culture conditions for production of glutamate decarboxylase by Streptococcus salivarius ssp. thermophilus

The culture conditions for glutamate decarboxylase (GAD) production under submerged fermentation by Streptococcus salivarius ssp. thermophilus were investigated. The results indicated the optimum culture medium was composed as follows: 15.0 g L^?1 of peptone, 12.5 g L^?1 of beef extract, 12.5 g L^?1 of sucrose, 1.03 g L^?1 of dipotassium hydrogen phosphate, 5 g L^?1 of sodium acetate, 2 g L^?1 of ammonium dibasic citrate, 2.12 g L^?1 of calcium chloride, 1 g L^?1 of Tween 80, and initial pH 6.79. The optimum culture temperature and time were 37 °C and 12 h, respectively. Under these conditions, GAD production was 257.46 ± 5.12 U, which was about 1.45‐fold that of Man–Rogosa–Sharpe broth. Copyright © 2008 Society of Chemical Industry     

Optimization of salts in medium for production of carboxymethylcellulase by a psychrophilic marine bacterium, <Emphasis Type="Italic">Psychrobacter aquimaris</Emphasis> LBH-10 using two statistical methods

The concentration of four mineral salts in the medium for the production of carboxymethylcellulase (CMCase) by Psychrobacter aquimaris LBH-10 were optimized using orthogonal array method (OAM) and response surface method (RSM) and their results from two statistical methods were compared. The analysis of variance (ANOVA) of data from central composite design (CCD) based on OAM indicated that potassium phosphate gave the highest percentage participation for cell growth as well as production of CMCase. However, their relative participations of four salts for cell growth were different from those for production of CMCase. The ANOVA of results from RSM indicated that highly significant factors (“probe>F” less than 0.0001) for cell growth were K₂HPO₄ and (NH₄)₂SO₄, whereas those for production of CMCase were K₂HPO₄, NaCl, and MgSO₄·7H₂O. The optimal concentration of K₂HPO₄, NaCl, MgSO₄·7H₂O, and (NH₄)₂SO₄ for cell growth extracted by Design Expert Software based on RSM were 7.10, 0.84, 0.24, and 0.33 g/L, respectively, whereas those for production of CMCase were 3.00, 0.52, 0.34, and 0.45 g/L. The optimal concentrations of salts for cell growth and production of CMCase using RSM basically coincided with those using OAM as well as those from ‘one-factor-at-a-time’ method. The production of CMCase by P. aquimaris LBH-10 with optimized concentrations of salts was 273.0 U/mL, which was enhanced by 1.27 times higher than the previous report.     

Statistical optimization of anticholesterolemic drug lovastatin production by the red mold Monascus purpureus

Lovastatin (HMG-CoA reductase inhibitors), is an important anticholesterolemic drug which inhibits the conversion of HMG-CoA to mevalonate in the biosynthesis of cholesterol. Plackett–Burman statistical screening of 12 media components and subsequent optimization of significant parameters by response surface methodology for the biotechnological production of lovastatin by Monascus purpureus MTCC 369 was studied. In this study, the statistical analysis of Plackett–Burman experimental results showed that the medium components glucose, peptone, MnSO₄·H₂O, NaCl and NH₄Cl as the significant components influencing the lovastatin production. The most significant medium components, glucose, peptone and MnSO₄·H₂O which have confidence level of more than 95% were further optimized using a full factorial central composite design of the response surface methodology. Maximum lovastatin production of 97.5 mg l^?1 was obtained after 14 days of fermentation period in the optimized medium containing, glucose, 52.61 g l^?1 peptone, 16.65 g l^?1; NH₄Cl, 1 g l^?1; KH₂PO₄, 1 g l^?1; yeast extract, 3 g l^?1; K₂HPO₄, 1 g l^?1; KNO₃, 0.5 g l^?1; MgSO₄·7H₂O, 0.2 g l^?1; MnSO₄·H₂O, 0.418 g l^?1; NaCl, 0.5 g l^?1; CaCl₂·2H₂O, 0.1 g l^?1 and FeSO₄·7H₂O, 0.001 g l^?1 at 30 °C and 120 rpm. The production of lovastatin by M. purpureus MTCC 369 in the optimized medium was found to be four times higher than the basal medium in the submerged fermentation. The statistical experimental design serves as an efficient tool for screening large number of variables with minimum number of experiments and optimizing the significant variables for enhancing the production of lovastatin.     

Optimal culture condition for the production of phenyalanine ammonia lyase from <Emphasis Type="Italic">E. coli</Emphasis>

The effects of culturing conditions on phenylalanine ammonia lyase production by a recombinant E. coli strain were investigated by using a controlled fed-batch fermentation system. In a 5 L fermentor, the optimal composition of the batch medium was 2% glucose, 1% yeast extract, 0.7% K₂HPO₄, 0.8% KH₂PO₄, 0.5% (NH₄)₂SO₄, 0.1% MgSO₄·7H₂O. The optimal feed glucose solution was 50%. Glucose concentration and pH of the culture broth were maintained at about 2.0 g/L and 7.0 during the fed-batch phase, respectively. Following 24-h cultivation, 0.2 mmol/L isopropyl-β-D-thiogalactopyranoside (IPTG) was added and temperature was shifted from 37°C to 42°C to induce pal gene expression. Under optimal conditions, a high productivity of 300 U/g could be achieved after 48 h culture, and a cell density of OD₆₀₀ about 82 was obtained at 52 h culture at 500 r/m stirrer speed and 1 vvm, respectively.     

Optimization of Chlorella vulgaris cultivation grown in waste molasses syrup using mixture design

The aim of this study was to determine and optimize culture media for Chlorella vulgaris microalgae under mixotrophic conditions using waste molasses as a cheap carbon source containing both organic carbons and other nutrients. In the current study, at first the growth and lipid productivity of C. vulgaris were assessed in different culture media and the best media was selected for mixotrophic growth conditions. Significant medium ingredients were screened through Plackett–Burman design. Then ingredients with positive effect were considered as a mixture component and their combinations were evaluated on lipid productivity using mixture design. According to results, Zarrouk medium was considered as the base medium with the highest biomass and lipid productivity of 72 and 7.1 mg L⁻¹ d⁻¹, respectively. Based on the Plackett–Burman design, out of 11 factors, molasses, NaNO₃ and K₂HPO₄ demonstrated key roles in biomass and lipid productivity in mixotrophic conditions. Consequently, the selected three factors were investigated by mixture design. The results showed that high concentration of molasses causes decrease in biomass and lipid productivity due to high turbidity and a blend consisting of approximately 9.5 g L⁻¹ molasses, 5 g L⁻¹ NaNO₃ and 0.15 g L⁻¹ K₂HPO₄ was found as the optimum mixture with obtained lipid productivity of 115 mg L⁻¹ d⁻¹. In conclusion, waste molasses can be used as a promising feedstock for cost effective cultivation of C. vulgaris.     

Optimization of medium components for D-ribose production by transketolase-deficient <Emphasis Type="Italic">Bacillus subtilis</Emphasis> NJT-1507

Statistical experimental designs were used to optimize the composition of culture media for the production of D-ribose by Bacillus subtilis. A fractional factorial design 2^(5-2) was used to determine medium components that significantly affected D-ribose production. The concentrations of glucose and (NH₄)₂SO₄ were the significant factors. Central composite design and response surface methodology were then used to estimate the quadratic response surface and determine the factor levels for maximum production of D-ribose. Finally, the optimal medium composition was obtained (g/L): glucose, 172.75; (NH₄)₂SO₄, 13.2; yeast powder, 4; corn steep liquor, 8 and MnSO₄, 0.5. This optimization strategy increased D-ribose production from 73.21 g/L to 88.57 g/L, an increase of 22% compared with the original conditions. The D-ribose production yield to glucose concentration was also enhanced from 0.37 g/g to 0.52 g/g. Confirmatory experiments were also performed to demonstrate the accuracy of the model. Under the optimal medium using ammonia to control pH in a 5 L fermenter, the D-ribose yield was increased to 95.28 g/L after 3 days of cultivation at 37 °C.     

Effect of feed composition on PHB production from methanol by HCDC of Methylobacterium extorquens (DSMZ 1340)

BACKGROUND: Poly‐β‐hydroxybutyrate (PHB), produced by several species of bacteria, has attracted great attention as a biodegradable and biocompatible compound with similar properties to polypropylene. Unfortunately, its use is currently limited due to high production costs. One of the most common methods for overcoming this constraint is the use of inexpensive substrates, like methanol, in high cell density cultivations (HCDC). RESULTS: Fermentation was carried out with optimized feed composition (639 g l^?1 methanol, 4 g l^?1 MgSO₄.7H₂O, 41 mL L^?1 trace elements, 5.6 g L^?1 NaH₂PO₄.H₂O and 24.3 g L^?1 K₂HPO₄) and a feeding strategy based on the detection of substrate limitation by dissolved oxygen (DO). After 35 h, at which dry cell weight (DCW) reached a value of 70 g L^?1, PHB production was stimulated, and biomass and PHB productivities of 2.8 and 0.98 g L^?1 h^?1 were obtained, respectively. These results surpassed those reported in the literature for PHB production from methanol by Methylobacterium species. CONCLUSION: The proposed feed composition and feeding strategy for PHB production from methanol by Methylobacterium extorquens 1340 in fed‐batch cultivation resulted in high biomass and PHB productivity. They can be implemented for recombinant bioproducts (proteins) produced by M. extorquens due to the lack of PHB accumulation in the growth phase. Copyright © 2009 Society of Chemical Industry     

Characterization of a novel glutamate decarboxylase from Streptococcus salivarius ssp. thermophilus Y2

BACKGROUND: γ‐Aminobutyric acid with several well‐known physiological functions is biosynthesized via the irreversible α‐decarboxylation of L ‐glutamate catalysed by glutamate decarboxylase (GAD). Although Streptococcus salivarius ssp. thermophilus has been widely applied to the dairy, the characterization of its GAD has not been reported. In this paper, the purification and the characterization of S. salivarius ssp. thermophilus GAD were investigated. RESULTS: GAD was purified 22‐fold from crude protein extracts with a yield of 7.8% in five steps. The final preparation gave a single band on SDS‐PAGE. The molecular weight of GAD determined by SDS‐PAGE and gel filtration was 46.9 kDa and 103.6 kDa, respectively, indicating that the enzyme exists as a dimmer of homological subunits. The optimum temperature and pH of GAD was 55 °C and pH 4.0, respectively. The enzyme reacted only with L ‐glutamate among 19 α‐amino acids with apparent K_m at 2.3 mmol L^?1 and did not react with D ‐glutamic acid. Activity of the enzyme could significantly be activated by 5 mmol L^?1 of BaCl₂ and inhibited by FeSO₄, ZnSO₄, CuSO₄, MnSO₄, Na₂SO₄, AgNO₃, CoCl₂, LiCl and KCl, respectively. The N‐terminal amino acid sequence of GAD was NH₂‐MNEKLFREI. CONCLUSION: Both the characterization and the deduced amino sequence (ABI31651) showed the purified enzyme was a novel GAD. Copyright © 2008 Society of Chemical Industry     

Application of statistical experimental design for optimization of physiological factors and their influences on production of pullulan by <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis> HP-2001 using an orthogonal array method

Physiological factors for the production of pullulan by A. pullulans HP-2001 were optimized using orthogonal array method and their influences were compared using Qualitek-4 software. The analysis of variance (ANOVA) indicated that the most important factor for cell growth was yeast extract, whereas that for production of pullulan was glucose. The optimal conditions for cell growth were found to be 100.0 g/L glucose, 10.0 g/L yeast extract, and initial pH of 6.0, whereas those for the production of pullulan were 100.0 g/L glucose, 2.5 g/L yeast extract, and initial pH of 5.5. Among four mineral salts in the medium, potassium phosphate (K₂HPO₄) was found to be the most important factor for cell growth as well as production of pullulan. Next important salt for cell growth was (NH₄)₂SO₄, whereas that for production of pullulan was NaCl. The optimal concentrations of K₂HPO₄, NaCl, MgSO₄·7H₂O, and (NH₄)₂SO₄ for cell growth were 7.5, 1.00, 0.1, and 1.20 g/L, respectively, whereas those for production of pullulan were 2.5, 0.25, 0.8, and 0.30 g/L. The expected cell growth and the production of pullulan by A. pullulans HP-2001 under these optimized conditions were 12.61 and 11.49 g/L, respectively.     

Production of L‐lactic acid with very high gravity distillery wastewater from ethanol fermentation by a newly isolated Enterococcus hawaiiensis

BACKGROUND: A great amount of wastewater with high contents of chemical oxygen demand (COD) are produced by ethanol production. It would be useful to utilize distillery wastewater to produce L‐lactic acid, which could be a high additional value byproduct of ethanol production. The fermentation process of L‐lactic acid production by a newly isolated Enterococcus hawaiiensis CICIM‐CU B0114 is reported for the first time. RESULTS: The strain produced 56 g L^?1 of L‐lactic acid after cultivation for 48 h in optimized medium consisting of (g L^?1) 80 glucose, 10 peptone, 10 yeast extract, 1.5 Na₂HPO₄ and 0.2 MgSO₄. E. hawaiiensis CICIM‐CU B0114 was isolated and purified by subculture for growing and producing L‐lactic acid in distillery wastewater of very high gravity (VHG) from ethanol fermentation. L‐lactic acid fermentation was further studied with distillery wastewater substrate in 7 L and 15 L fermentors. The results showed that L‐lactic acid concentrations of 52 g L^?1 and 68 g L^?1 was achieved in 7 L and 15 L fermentors with the initial sugar concentrations of 67 g L^?1 and 87 g L^?1, respectively. CONCLUSION: The production of L‐lactic acid by the newly isolated E. hawaiiensis CICIM‐CU B0114 was carried out and the fermentation medium was optimized by orthogonal experimental design. This new strain holds the promise of L‐lactic acid production utilizing distillery wastewater from VHG ethanol fermentation. Copyright © 2010 Society of Chemical Industry     

Studies on cultivation and biological activities ofPleurotus nebrodensis inzenga

Environmental factors affecting mycelial growth and exo-polysaccharide production fromPleurotus nebrodensis Inzenga (PN) and biological activities of PN extractsin vitro were studied. The culture conditions for effective mycelial growth and exo-polysaccharide production were found to be 25 ‡C, 5% of inoculum size, and an initial pH from 6.5 to 7.0. When 5% of glucose was used, the maximum mycelial growth and exo-polysaccharide concentrations were 8.3 and 3.07 g/L, respectively. Among the various nitrogen sources, the mycelial growth and exo-polysaccharide production were very strong when polypeptone was used. In the case of the minerals sources, K₂HPO₄ and MgSO₄·7H₂O were found to best support for mycelial growth and exo-polysaccharide production. Under optimal conditions and methods, the maximum mycelial growth and exo-polysaccharide production were obtained after 10 days of culture, 12.84 and 4.85 g/L, respectively, in a jar fermentor. The effects of the PN extracts on the viability of three human cancer cell lines and antioxidant activity were investigatedin vitro. When cancer cells of the lung (A549), cervical region (HeLa) and colon (KM12C) were incubated at 6 mg/mL of the PN ethanol extracts, the viabilities of the HeLa and KM12C cells were slightly decreased. However, the growth of the A549 cells was remarkably inhibited when the PN ethanol extract was over 4 mg/mL. The antioxidant activity showed 46.2% at 40 μL, which was about 3.2 fold higher than that of the PN methanol extract.     

Evaluation and optimization of food-grade tannin acyl hydrolase production by a probiotic Lactobacillus plantarum strain in submerged and solid state fermentation

Tannin acyl hydrolase (tannase) production by Lactobacillus plantarum MTCC1407 was studied in submerged and solid-state fermentation process. Sequential optimization strategy using Plackett–Burman screening and response surface methodology was adopted to optimize the submerged fermentation process. Eight medium components were evaluated initially by Plackett–Burman two level factorial designs to identify the most significant parameters that affect the tannase production. The significant variables affecting tannase production were found to be tannic acid, glucose and MnSO₄·7H₂O. These factors were further optimized by response surface methodology. Maximum tannase activity of 9.13 U ml^?1 was observed at 30 h using the following medium composition (g l^?1): tannic acid, 13.16; glucose, 1.5; NH₄Cl, 1.0; CaCl₂·2H₂O, 1.0; K₂HPO₄, 0.5; KH₂PO₄, 0.5; MgSO4·7H₂O, 0.5 and MnSO₄·7H₂O, 0.03. Among the various carbon sources examined for tannase production by L. plantarum, glucose and tannic acid combination was found to be decisive for enhancing tannase yield. Solid state fermentation was conducted using various solid substrates and agricultural residues. Maximum tannase activity of 5.319 U gds^?1 was obtained using coffee husk as substrate.     

Production of polysaccharides by submerged mycelial culture of entomopathogenic fungus Cordyceps takaomontana and their apoptotic effects on human neuroblastoma cells

The effect of medium components and environmental factors on the production of mycelial biomass for the preparation of intracellular polysaccharides (IPS) and exopolysaccharides (EPS) by Cordyceps takaomontan was investigated in submerged cultures. The optimal culture condition was as follows (g/l): glucose 30, yeast extract 4, KH₂PO₄ 0.46, K₂HPO₄ 1, and MgSO₄ 0.5, and 28 °C and pH 8. When the fungus was cultivated under various agitation and aeration conditions in a 5-l stirred-tank fermenter, the maximum mycelial biomass (10.7 g/l) and EPS production (1.9 g/l) was obtained at 300 rpm and 2 vvm, respectively. The inhibitory effect of both IPS and EPS on the growth of SK-N-SH cells was studied by treating the cells with crude IPS and EPS at concentrations of 0.5, 1, and 2 mg/ml for 24, 48, 72 h, respectively. The maximum inhibitory effect on apoptosis of SK-N-SH cells (64.6%) was observed when IPS was treated at a concentration of 2 mg/ml, for 72 h. The apoptosis of SK-N-SH cells induced by IPS was evidenced by comet assay, where the number of the comet cells increased by the IPS treated. These findings suggest that IPS may therefore have therapeutic potential against neuroblastoma cells.     

Optimization of chitosanase production from Bacillus sp. RKY3 using statistical experimental designs

BACKGROUND: The culture medium and fermentation conditions for the production of constitutive chitosanase from a newly isolated Bacillus sp. RKY3 were optimized statistically. RESULTS: The variables significantly influencing both chitosanase production and cell growth were screened through the Plackett–Burman design, by which maltose, beef extract, MgSO₄, and incubation time were identified as the most significant variables. The optimum values of the selected variables and their mutual interactions were determined through the steepest ascent method and Box–Behnken experimental design. The results demonstrated that 62.30 U mL^?1 chitosanase activity was predicted with optimum conditions of maltose (30.18 g L^?1), beef extract (15.25 g L^?1), MgSO₄ (0.26 g L^?1), and incubation time (50.02 h). The predicted response was verified by the validation experiments, and the optimum conditions resulted in a maximum chitosanase activity of 63.53 ± 1.22 U mL^?1. CONCLUSION: The optimization of fermentation variables resulted in an approximately 11.3‐fold increase in chitosanase activity compared with that observed under unoptimized conditions (from 5.63 U mL^?1 to 63.53 U mL^?1). Copyright © 2009 Society of Chemical Industry