Response surface designs for experiments in bioprocessing

Many processes in the biological industries are studied using response surface methodology. The use of biological materials, however, means that run-to-run variation is typically much greater than that in many experiments in mechanical or chemical engineering and so the designs used require greater replication. The data analysis which is performed may involve some variable selection, as well as fitting polynomial response surface models. This implies that designs should allow the parameters of the model to be estimated nearly orthogonally. A class of three-level response surface designs is introduced which allows all except the quadratic parameters to be estimated orthogonally, as well as having a number of other useful properties. These subset designs are obtained by using two-level factorial designs in subsets of the factors, with the other factors being held at their middle level. This allows their properties to be easily explored. Replacing some of the two-level designs with fractional replicates broadens the class of useful designs, especially with five or more factors, and sometimes incomplete subsets can be used. It is very simple to include a few two- and four-level factors in these designs by excluding subsets with these factors at the middle level. Subset designs can be easily modified to include factors with five or more levels by allowing a different pair of levels to be used in different subsets.     

裂褶菌F17锰过氧化物酶酶活力影响因素的响应面优化(英文)

锰过氧化物酶是真菌分泌的一种糖基化的含有血红素辅基的胞外蛋白,在染料降解和脱色过程中起着重要作用。本实验利用本实验室保存的的白腐真菌裂褶菌Schizophyllum sp.F17产锰过氧化物酶(MnP),研究MnP的酶学性质,并对酶活条件进行优化。实验通过超滤浓缩、DEAE-纤维素、DE52离子交换层析和Sephadex G-75凝胶过滤等步骤,分离纯化得到电泳纯的锰过氧化物酶。该酶蛋白含量为23μg/mL,分子量大小为49.2kDa,在0.1mmol/L H2O2中半衰期为5~6min。Mn2+、H2O2以及酶的用量可以影响MnP酶促反应的效率,在单因子分析法的基础上,通过全因子中心组合设计响应面分析表明:H2O2以及H2O2与酶用量之间的交互作用对酶促反应的作用是最显著的。在优化条件下,酶对偶氮染料金橙G、刚果红显示出较强的脱色能力。     

Optimization of a medium for enhancing nicotine biodegradation by Ochrobactrum intermedium DN2

AIMS: To optimize a medium for nicotine degradation by Ochrobactrum intermedium DN2 in presence of yeast extract, glucose and Tween 80 using response surface methodology (RSM). METHODS AND RESULTS: In this study, the effects of yeast extract, glucose and Tween 80 on nicotine degradation were investigated in flasks using a novel nicotine-degrading bacterium, O. intermedium DN2. A full factorial central composite design was applied in the design of experiments and in the analysis of the experimental data. The results showed that the most significant variable influencing nicotine degradation was yeast extract, followed by glucose, and then Tween 80. Moreover these three factors interacted with each other and combined to produce positive effects on nicotine degradation. The experimental data also allowed the development of an empirical model (P < 0.0001) describing the inter-relationship between independent and dependent variables. By solving the regression equation, the optimal values of the variables were determined as: yeast extracts 0.094%, glucose 0.101% and Tween 80 0.080%. Using the medium obtained, about 1,220 mg l(-1) of nicotine was degraded (95.55%) within 10 h at the specific biodegradation of 116.59 mg l(-1) h(-1) in 30-l bioreactor containing 25-l tobacco extract. CONCLUSIONS: An optimal medium of nicotine degradation by the strain DN2 was obtained. SIGNIFICANCE AND IMPACT OF THE STUDY: RSM proved to be reliable in developing the model, optimizing factors and analysing interaction effects. The results provide better understanding on the interactions between yeast extract, glucose and Tween 80 for nicotine biodegradation.     

Optimization of a culture medium for xylanase production by Bacillus sp. using statistical experimental designs

The concentrations of oat spelt xylan, casein hydrolysate and NH4Cl in the culture medium for production of xylanase from Bacillus sp. I-1018 were optimized by means of response surface methods. The path of steepest ascent was used to approach the optimal region of the medium composition. The optimum composition of the nutrient medium was then easily determined by using a central composite design and was found to be 3.16g/l of xylan, 1.94g/l casein hydrolysate, 0.8g/l of NH4Cl. The xylanase production was increased by 135% when the strain was grown in the optimized medium compared to initial medium.     

Estimation of scopoletin from a polyherbal formulation composition using HPLC coupled with fluorescence detector through the concept of Design of Experiment

High-performance liquid chromatography (HPLC) coupled with a fluorescence detector was used to analyse bioactive phytoconstituent scopoletin from a polyherbal composition derived from the extract prepared from roots of Argyreia nervosa, roots of Withania somnifera, and fruits of Tribulus terrestris. This analytical method was developed as a quality control tool for standardization of the composition to be formulated to enhance spermatogenesis. Chromatographic separation was achieved using Luna® (250 mm × 4.6 mm, 100 Å, 5 μm) C₁₈ column as a stationary phase, and water (0.01 M glacial acetic acid):methanol: acetonitrile (60:20:20, %v/v/v) as the mobile phase; passed through the column at a set flow rate of 1.0 ml min⁻¹. The elute in the flow cell was excited at 345 nm and the chromatogram was recorded at 444 nm as the emission wavelength. As a part of the analytical Quality by Design approach, systemic studies were conducted to identify potential risks affecting the critical attributes (area, resolution, retention time) of the analytical method, and mitigating the potential risks after optimizing the chromatographic parameters with the help of the Design of Experiment approach. The developed analytical method was subjected to the validation studies, which showed a linear relationship (r² = 0.9982) between the concentration and the area corresponding to scopoletin peak in the concentration range 10–130 ng ml⁻¹. The method was found selective, sensitive, and precise. The recovery of the scopoletin was found in a range 99.53–102.13%; confirming the accuracy of the analytical method. The amount of scopoletin was estimated to be 0.146%w/w from the polyherbal composition.     

Optimization of the extraction of polyphenols and antioxidant activity from Malva parviflora L. leaves using Box–Behnken design

Abstract

Box–Behnken Design (BBD) was used to optimize the extraction conditions of polyphenols from Malva (Malva parviflora L.) leaves. The effect of ethanol concentration (20–80%), solvent/leaf powder ratio (10:1 to 30:1, v/w) and extraction time (5–45?min) on the polyphenols yield and the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of the obtained extracts were investigated. Quadratic models fit well. The optimal conditions (53.40% ethanol, solvent/leaf powder ratio 20:1 (v/w), and 15?min) resulted in an extract with a maximum yield of polyphenols (1098.4?mg GAE/100?g leaf powder) and high inhibition percentage of DPPH radical (33.31%) with desirability 0.742. High Performance Liquid Chromatography (HPLC) analysis indicated that the major identified polyphenol compounds extracted at the optimal conditions were naringenin, ρ-coumaric acid, apigenin-7-glucoside, luteolin, and cinnamic acid. These findings indicate that M. parviflora leaf extracts possess DPPH radical scavenging activity and could be used as a natural source for bioactive products.     

Cost-effective optimization of gellan gum production by Sphingomonas paucimobilis using corn steep liquor

Abstract

Gellan gum, produced by Sphingomonas paucimobilis, is increasingly used in food and pharmaceutical industries as stabilizing, emulsifying, texturing and gelling agents. However, its high production costs may limit its full commercial potential. Therefore, in this study, we investigated ways to reduce gellan gum production costs and improve yields. We first revealed corn steep liquor (CSL) as a cost-effective nutrient source that can improve gellan gum yields. We then systematically optimized culture conditions even further, and revealed that the addition of Triton X-100 surfactant and selected inorganic nitrogen sources improved gellan gum production. Under our optimized conditions (glucose 33.75?g/L, CSL 10?g/L, urea 2.5?g/L, MgSO₄ 1.08?g/L, KH₂PO₄ 3.24?g/L, K₂SO₄ 1?g/L and Triton X-100 0.75?g/L), we yielded a maximum concentration of 14.41?g/L, which was about 1.5-fold higher than non-optimized CSL-based medium. Our findings highlight the use of CSL as a cost effective and promising nutrient source for industrial production of gellan gum.     

Optimization of carotenoid production by Umbelopsis ramanniana

Umbelopsis ramanniana was investigated to increase carotenoid production. Nine different carbon sources and six different nitrogen sources were evaluated for the maximum carotenoid production. The most effective nitrogen and carbon sources were KNO₃ and lactose, respectively. Then, the optimization of medium components for enhancement of carotenoid production by Umbelopsis ramanniana was achieved using Plackett–Burman design. Box–Behnken response surface methodology was applied to further optimize carotenoid and biomass production. Carbon to nitrogen ratio, lactose concentration, and shaking speed were studied as variables in Box–Behnken design. The optimum conditions for carotenoid and biomass production were determined as 32.42 g/L of lactose concentration, 20:1 of carbon to nitrogen ratio, and shaking speed of 130 rpm. The maximum carotenoid and biomass production under optimized conditions were 1141 μg/L (β-carotene-Eq) and 13.14 g/L, respectively. When compared to the control fermentation, carotenoid, and biomass production were increased by about 2 and 1.3 folds, respectively.     

Synthesis of geranyl propionate in a solvent-free medium using Rhizomucor miehei lipase covalently immobilized on chitosan–graphene oxide beads

The chemical route of producing geranyl propionate involves the use of toxic chemicals, liberation of unwanted by-products as well as problematic separation process. In view of such problems, the use of Rhizomucor miehei lipase (RML) covalently bound onto activated chitosan–graphene oxide (RML-CS/GO) support is suggested. Following analyses using Fourier transform infrared spectroscopy, field emission scanning electron microscopy, transmission electron microscopy, and thermogravimetry, properties of the RML-CS/GO were characterized. A response surface methodological approach using a 3-level-four-factor (incubation time, temperature, substrate molar ratio, and stirring rate) Box–Behnken design was used to optimize the experimental conditions to maximize the yield of geranyl propionate. Results revealed that 76?±?0.02% of recovered protein had yielded 7.2?±?0.04?mg?g^?1 and 211?±?0.3%?U?g^?1 of the maximum protein loading and esterification activity, respectively. The actual yield of geranyl propionate (49.46%) closely agreed with the predicted value (49.97%) under optimum reaction conditions (temperature: 37.67°C, incubation time: 10.20?hr, molar ratio (propionic acid:geraniol): 1:3.28, and stirring rate: 100.70?rpm) and hence, verifying the suitability of this approach. Since the method is performed under mild conditions, the RML-CS/GO biocatalyst may prove to be an environmentally benign alternative for producing satisfactory yield of geranyl propionate.     

Statistical optimization of culture conditions for bacterial cellulose production using Box-Behnken design

Culture conditions in a jar fermentor for bacterial cellulose (BC) production from A. xylinum BPR2001 were optimized by statistical analysis using Box-Behnken design. Response surface methodology was used to predict the levels of the factors, fructose (X1), corn steep liquor (CSL) (X2), dissolved oxygen (DO) (X3), and agar concentration (X4). Total 27 experimental runs by combination of each factor were carried out in a 10-L jar fermentor, and a three-dimensional response surface was generated to determine the effect of the factors and to find out the optimum concentration of each factor for maximum BC production and BC yield. The fructose and agar concentration highly influenced the BC production and BC yield. However, the optimum conditions according to changes in CSL and DO concentrations were predicted at almost central values of tested ranges. The predicted results showed that BC production was 14.3 g/L under the condition of 4.99% fructose, 2.85% CSL, 28.33% DO, and 0.38% agar concentration. On the other hand, BC yield was predicted in 0.34 g/g under the condition of 3.63% fructose, 2.90% CSL, 31.14% DO, and 0.42% agar concentration. Under optimized culture conditions, improvement of BC production and BC yield were experimentally confirmed, which increased 76% and 57%, respectively, compared to BC production and BC yield before optimizing the culture conditions.     

INVESTIGATION OF THE GIBBERELLIC ACID OPTIMIZATION WITH A STATISTICAL TOOL FROM Penicillium variable IN BATCH REACTOR

The culture conditions for gibberellic acid (GA₃) production by the fungus Penicillium variable (P. variable) was optimized using a statistical tool, response surface methodology (RSM). Interactions of culture conditions and optimization of the system were studied using Box–Behnken design (BBD) with three levels of three variables in a batch flask reactor. Experimentation showed that the model developed based on RSM and BBD had predicted GA₃ production with R ²  = 0.987. The predicted GA₃ production was optimum (31.57 mg GA₃/kg substrate) when the culture conditions were at 7 days of incubation period, 21% v/w of inoculum size, and 2% v/w of olive oil concentration as a natural precursor. The results indicated that RSM and BBD methods were effective for optimizing the culture conditions of GA₃ production by P. variable mycelia.     

Optimization of methazolamide-loaded solid lipid nanoparticles for ophthalmic delivery using Box–Behnken design

The purpose of the present study was to optimize methazolamide (MTZ)-loaded solid lipid nanoparticles (SLNs) which were used as topical eye drops by evaluating the relationship between design factors and experimental data. A three factor, three-level Box–Behnken design (BBD) was used for the optimization procedure, choosing the amount of GMS, the amount of phospholipid, the concentration of surfactant as the independent variables. The chosen dependent variables were entrapment efficiency, dosage loading, and particle size. The generated polynomial equations and response surface plots were used to relate the dependent and independent variables. The optimal nanoparticles were formulated with 100?mg GMS, 150?mg phospholipid, and 1% Tween80 and PEG 400 (1:1, w/v). A new formulation was prepared according to these levels. The observed responses were close to the predicted values of the optimized formulation. The particle size was 197.8?±?4.9?nm. The polydispersity index of particle size was 0.239?±?0.01 and the zeta potential was 32.7?±?2.6?mV. The entrapment efficiency and dosage loading were about 68.39% and 2.49%, respectively. Fourier transform infrared spectroscopy (FT-IR) study indicated that the drug was entrapped in nanoparticles. The optimized formulation showed a sustained release followed the Peppas model. MTZ-SLNs showed significant prolonged decreasing intraocular pressure effect comparing with MTZ solution in vivo pharmacodynamics studies. The results of acute eye irritation study indicated that MTZ-SLNs and AZOPT both had no eye irritation. Furthermore, the MTZ-SLNs were suitable to be stored at low temperature (4?°C).     

An approach toward optimization of the influential growth determinants of opportunistic yeast isolate Pichia guilliermondii

The present study reports statistical optimization of growth conditions of an opportunistic fungal strain Pichia guilliermondii, isolated from the blood of patients suffering from bancroftian filariasis. Seven key determinants, namely, primary inoculums size (%), volume (mL) and pH of media, serum proportion, temperature (°C), incubation time (hr), and agitation speed (rpm) that influence in vitro growth of the pathogen were optimized statistically using response surface methodology (RSM). RSM with seven factors and two-level Box–Behnken design was employed for designing experimental run, prediction of case statistics, suitable exploration of quadratic response surfaces, and constructing a second-order polynomial equation. Analysis of variance (ANOVA) showed that primary inoculums size, volume of culture media, temperature, incubation time, and agitation speed exert most significant influence over fungal growth. The RSM study predicted that optimum fungal growth can be obtained using 10% primary inoculums size in 100 mL culture media with pH 6.0, 6.28% serum, 32.5°C temperature, and 24 hr of incubation, alongside agitation speed at 400 rpm. The desirability of the optimized growth model for P. guilliermondii is 99.123%, which indicated its accuracy and acceptability. Finally, the optimized growth module illustrated in the study could be useful in improving in vitro growth of clinically important P. guilliermondii.     

Optimization of culture media for large‐scale lutein production by heterotrophic Chlorella vulgaris

Lutein is a carotenoid with a purported role in protecting eyes from oxidative stress, particularly the high‐energy photons of blue light. Statistical optimization was performed to growth media that supports a higher production of lutein by heterotrophically cultivated Chlorella vulgaris. The effect of media composition of C. vulgaris on lutein was examined using fractional factorial design (FFD) and central composite design (CCD). The results indicated that the presence of magnesium sulfate, EDTA‐2Na, and trace metal solution significantly affected lutein production. The optimum concentrations for lutein production were found to be 0.34 g/L, 0.06 g/L, and 0.4 mL/L for MgSO₄·7H₂O, EDTA‐2Na, and trace metal solution, respectively. These values were validated using a 5‐L jar fermenter. Lutein concentration was increased by almost 80% (139.64 ± 12.88 mg/L to 252.75 ± 12.92 mg/L) after 4 days. Moreover, the lutein concentration was not reduced as the cultivation was scaled up to 25,000 L (260.55 ± 3.23 mg/L) and 240,000 L (263.13 ± 2.72 mg/L). These observations suggest C. vulgaris as a potential lutein source. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:736–743, 2014     

α-Amylase immobilization onto functionalized graphene nanosheets as scaffolds: Its characterization,kinetics and potential applications in starch based industries

α-Amylase is imperative for starch and its deriviatized industries. Functionalized graphene sheets were tailored and optimized as scaffold for α-amylase immobilization using Response Surface Methodology based on Box–Behnken design, with an overall immobilization efficiency of 85.16%. Analysis of variance provided adequacy to the mathematical model for further studies. Native and immobilized functionalized graphene were characterized using transmission and scanning electron microscopy, followed by Fourier transform infrared (FTIR) spectroscopy. Wheat α-amylase conjugated with functionalized graphene sheets were visually evident on transmission and scanning micrographs while the FTIR spectra showed interplay of various chemical interactions and bonding, during and after immobilization. Optimum pH and optimum temperature for immobilized enzyme though remained unchanged but showed broader range whereas K_m showed a slight decrease (1.32 mg/mL). It also showed enhanced thermal and storage stability and retained 73% residual activity after 10 uses. These ensemble of properties and non-toxic nature of functionalized graphene, makes it viable to be absorbed commercially in starch processing industries.     

Diacerein niosomal gel for topical delivery: development,in vitro and in vivo assessment

The purpose of this study was to load diacerein (DCR) in niosomes by applying response surface methodology and incorporate these niosomes in gel base for topical delivery. Box–Behnken design was used to investigate the effect of charge-inducing agent (X₁), surfactant HLB (X₂) and sonication time (X₃) on the vesicle size (Y₁), entrapment efficiency (Y₂) and cumulative drug released (Y₃). DCR niosomal formulations were prepared by thin film hydration method. The optimized formula was incorporated in different gel bases. DCR niosomal gels were evaluated for homogeneity, rheological behavior; in vitro release and pharmacodynamic activity by carrageenan-induced hind paw edema method in the rat compared with DCR commercial gel. The results revealed that the mean vesicle sizes of the prepared niosomes ranged from 7.33 to 23.72?µm and the entrapment efficiency ranged from 9.52% to 58.43% with controlled release pattern over 8?h. DCR niosomal gels exhibited pseudoplastic flow with thixotropic behavior. The pharmacodynamic activity of DCR niosomal gel in 3% HPMC showed significant, 37.66%, maximum inhibition of edema size in comparison with 20.83% for the commercial gel (p?相似文献   

基于全细胞催化合成NADP重组细胞复合通透剂的筛选

本文以重组大肠杆菌E. coli BL21(DE3) p ET30α(+)-Nmnat为研究对象,考察化学通透剂处理重组菌对其催化合成NADP的影响。实验显示,在单因素筛选实验中,四种通透剂曲拉通X-100、吐温80、二甲基亚砜和十六烷基三甲基溴化铵(CTAB)对细胞进行通透化处理均可提高全细胞催化产率,且提高幅度差距并不显著。进一步采用析因设计考察四种通透剂的复合效应,除了曲拉通X-100与吐温80和曲拉通X-100与CTAB交互作用的P大于0. 05为不显著,其余所有交互作用项P值均小于0. 05,呈现显著性,且复合通透剂中,仅单一增加CTAB量,复合体系的协同作用将减少,制约作用增加,不适宜用于复合配方。最后采用中心组合设计进一步对复配效果进行优化,通过拟合得到最佳通透剂配比为曲拉通X-100(1. 64%),吐温80(2. 53%)以及二甲基亚砜(1. 06%),此时NADP预测产率达到77. 46%,与实际验证产率均值77. 05%相差甚微,说明实验优化模型具有良好预测性,优化结果与未添加通透剂相比提升近47%,表明优化复合通透剂配方更有利于提高重组菌催化合成NADP。     

MODEL DEVELOPMENT AND PROCESS OPTIMIZATION FOR SOLVENT EXTRACTION OF POLYPHENOLS FROM RED GRAPES USING BOX–BEHNKEN DESIGN

The objective of the present study is to find out the optimum extraction conditions for extraction of polyphenols from red grapes using Box–Behnken design. Red grapes polyphenols were extracted using acid–ethanol solvent at various extraction temperature (40–60°C), extraction time (20–100 min) and different solid–liquid ratio (1:5–1:15 g:ml). The effect (main and interactive) of extraction conditions on total anthocyanin, phenolic and flavonoid content were studied using Box–Behnken design (three factors at three levels). The results showed that the contribution of the quadratic model was significant for all the responses. Second-order mathematical regression models were developed and were found to fit well with observed data. Derringer's desirability function methodology was performed to find out the optimal conditions based on both individual and combinations of all responses (extraction temperature: 57°C, time: 61 min, and solid–liquid ratio: 1:8.7 g:ml) were established. At this optimal condition, the anthocyanin yield, total phenolic and flavonoid content were 73.92 mg/100 g, 221.4 mg GAE/100 g, and 79.08 mg CE/100 g, respectively. A desirability value of 0.902 was achieved at this point.