Enzyme assisted extraction of polysaccharides from the fruit of Cornus officinalis

Process of enzyme assisted extraction (EAE) of polysaccharides from Cornus officinalis was optimized by response surface methodology (RSM). The influence of four different factors on the yield of C. officinalis polysaccharides (COP) was studied. Results showed that the optimal conditions were compound enzyme amount of 2.15%, extraction pH of 4.2, extraction temperature of 55 °C and extraction time of 97 min. Under these conditions, the COP yield was 9.29 ± 0.31%, which was well in agreement with the value predicted by the model. The three methods, EAE, hot water extraction (HWE), ultrasound-assisted extraction (UAE) for extracting COP by RSM were further compared. Results showed that EAE had the largest yield of polysaccharides with lower equipment cost.     

Response surface methodology for the extraction of wedelolactone from Eclipta alba using aqueous two-phase extraction

Abstract

Aqueous two-phase extraction of wedelolactone from Eclipta alba was studied using the polymer-salt system. The system consisted of polyethylene glycol (PEG) as a top phase (polymer) and sodium citrate as a bottom phase (salt). Process parameters such as PEG concentration, PEG molecular weight, salt concentration, and pH have been optimized using response surface methodology (RSM) with the help of central composite design (CCD). The optimized conditions for aqueous two-phase system (ATPS), in the case of one factor at a time approach, were found as PEG 6000, PEG concentration 18% (w/v), salt concentration 16% (w/v), and pH 7; with maximum extraction yield of 6.52?mg/g. While, RSM studies showed maximum extraction yield of 6.73?mg/g with the optimized parameters as PEG 6000, PEG concentration 18% (w/v), salt concentration 17.96% (w/v), and pH 7. ATPS was found to give a 1.3 fold increase in the extraction yield of wedelolactone as compared to other conventional extraction methods.     

Response surface method was used to optimize the ultrasonic assisted extraction of flavonoids from Crinum asiaticum

In this study, ultrasound-assisted extraction of flavonoid from Crinum asiaticum was studied through response surface methodology (RSM) to gain the best extraction process of flavonoid and enhance the extraction rate of flavonoid. In the following RSM experiment, we selected the corresponding data of every factor as the center point through the single-factor experiments, then the experimental data was subjected to multiple regression analysis. According to the statistical analysis results, the results were consistent with the polynomial regression model, the determination coefficient (R2) was 0.9769. The best conditions for maximum flavonoid yield were 60% ethanol concentration, 64 °C for extraction temperature, 1:28 (v/w) solid-to-liquid ratio with extraction time for 47 min. The best response of flavonoid yield was 1.63972%. The predicted results for best reaction conditions were in good agreement with experiment values. Ultrasound-assisted extraction method can enhance the extraction rate of flavonoid significantly. It is a powerful tool to extract of important phytochemicals from nature plant.     

Optimization of Process Conditions Using Response Surface Methodology (RSM) for Ethanol Production from Pretreated Sugarcane Bagasse: Kinetics and Modeling

Based on a five level central composite design (CCD) involving the variables substrate concentration (C), pH (P), incubation temperature (T) and fermentation time (H), a response surface methodology (RSM) for the production of ethanol from pretreated sugarcane bagasse by cellulase and yeast Kluyveromyces fragilis was standardized. The design contains a total of 31 experimental trials in which the first 24 organized in a factorial design and from 25 to 31 involving the replications of the central points. Data obtained from RSM on ethanol production were subjected to the analysis of variance (ANOVA) and analyzed using a second order polynomial equation. Maximum ethanol concentration (32.6 g/l) was obtained from 180 g/l pretreated sugarcane bagasse at the optimized process conditions (temperature 35°C, pH 5.5) in 72 h aerobic batch fermentation. Various kinetic models such as logistic model, logistic incorporated leudeking piret model and logistic incorporated modified leudeking piret model have been evaluated and the constants were predicted.     

Enzymatic synthesis of n-butyl palmitate in a solvent-free system: RSM optimization and kinetic studies

The present paper demonstrates application of biocatalysis to the synthesis of n-butyl palmitate, a cosmetic emollient ester in a solvent-free system (SFS). Fermase CALB?10000, a commercial Candida antarctica lipase B was used to accomplish the synthesis. In order to evaluate the effect of various process parameters on the synthesis, one factor at a time methodology (OFAT) and response surface methodology (RSM) complimented with central composite design (CCD) were employed. On the basis of the results obtained in one factor at one time studies, temperature, enzyme dose, and molar ratio were chosen as significant parameters and their range was selected for RSM study. The optimized factors suggested by RSM model were, temperature –60.12?°C, enzyme dose –5% w/w, and alcohol: acid ratio –2.25:1. Under these optimized factors, the experimental conversion observed was 91.25% which was in close agreement to the model predicted conversion of 92% and the enzymes were reusable up to four cycles. A separate study was carried out in order to study the effect of palmitic acid on n-butyl palmitate synthesis and to understand the kinetic profile of n-butyl palmitate synthesis reaction. Ordered bi-bi model showed a good experimental fit to the kinetic data.     

A statistical approach for optimization of R-phycoerythrin extraction from the red algae Gracilaria verrucosa by enzymatic hydrolysis using central composite design and desirability function

The aim of this research is to statistically optimize enzymatic hydrolysis parameters for the production of R-phycoerythrin (RPE) from red algae Gracilaria verrucosa. Six independent variables, incubation temperature, incubation time, ratio of buffer to raw material, cellulase loading, xylanase loading, and pH, were selected for response surface methodology studies. A central composite design was employed to maximize RPE production. A mathematical model with high determination coefficient (R ²?=?0.86) was developed and could be employed to optimize RPE extraction. The optimal extraction conditions of RPE were determined as follows: incubation temperature (48°C), incubation time (6?h), ratio of buffer to raw material (20 w/v), cellulase loading (15%), xylanase loading (5%), and pH (6.5). Under this optimal condition, the experimental yield of RPE was 6.25?mg?g^?1. Based on the result of response surface methodology and desirability function approach study, total sugar, the main by-product in RPE extraction was considered as another response. A new optimal condition was predicted as follows: incubation temperature (30°C), incubation time (12?h), ratio of buffer to raw material (20, w/v), cellulase loading (15%), xylanase loading (5%), and pH (6). Under this condition, similar RPE levels were obtained while the concentration of total sugar decreased by 40%.     

响应面优化纤维素酶辅助提取木豆叶总黄酮工艺

为探讨纤维素酶辅助提取木豆叶总黄酮最佳工艺条件,在单因素试验基础上,以酶用量、酶解温度、酶解时间和酶解pH为影响因子,总黄酮得率为响应值,采用Box-behnken中心组合设计建立4个影响因子与总黄酮得率关系的数学模型,进行响应面法分析。结果表明,纤维素酶辅助提取木豆叶总黄酮的最佳工艺条件为：酶用量为8.65 mg,酶解温度为33.88℃,酶解时间为2.02 h,酶解pH为5.02。在最优条件下总黄酮理论得率为4.86%,实测值为4.88%,拟合得到的模型与实际吻合良好。本研究建立的提取工艺条件稳定可靠,为以后木豆叶总黄酮的应用开发提供实验依据。     

Optimization of Protease Extraction from Horse Mango (Mangifera foetida Lour) Kernels by a Response Surface Methodology

Protease is one of the most important industrial enzymes with a multitude of applications in both food and non-food sectors. Although most commercial proteases are microbial proteases, the potential of non-conventional protease sources, especially plants, should not be overlooked. In this study, horse mango (Mangifera foetida Lour) fruit, known to produce latex with a blistering effect upon contact with human skin, was chosen as a source of protease, and the effect of the extraction process on its protease activity evaluated. The crude enzyme was extracted from the kernels and extraction was optimized by a response surface methodology (RSM) using a central composite rotatable design (CCRD). The variables studied were pH (x(1)), CaCl(2) (x(2)), Triton X-100 (x(3)), and 1,4-dithryeitol (x(4)). The results obtained indicate that the quadratic model is significant for all the variables tested. Based on the RSM model generated, optimal extraction conditions were obtained at pH 6.0, 8.16 mM CaCl(2), 5.0% Triton X-100, and 10.0 mM DTT, and the estimated response was 95.5% (w/w). Verification test results showed that the difference between the calculated and the experimental protease activity value was only 2%. Based on the t-value, the effects of the variables arranged in ascending order of strength were CaCl(2)< pH < DTT < Triton X-100.     

Application of Factorial Design and Box–Behnken Matrix in the Optimization of a Magnetic Nanoparticles Procedure for Copper Determination in Water and Biological Samples

This study describes the development by response surface methodology (RSM) of a procedure for copper determination by inductively coupled plasma optical emission spectrometry (ICP-OES) in water and biological samples after extraction by magnetic nanoparticles. Four variables such as, pH of solution, amount of extractant, amount of nanoparticles, and time were regarded as factors in the optimization study. Results of the two-level full factorial design (2⁴) based on an analysis of variance demonstrated that only the pH, amount of extractant (E), and amount of nanoparticles (N) were statistically significant. Optimal conditions for the extraction of copper samples were obtained by using Box–Behnken design. Optimum conditions were 5.1, 7.2 mg, and 9.6 mg, for pH of solution, amount of nanoparticles, and amount of extractant, respectively. Under the optimized experimental conditions, the detection limit of the proposed method followed by ICP-OES was found to be 0.9?µg L^?1. The method was applied to the determination of copper in water and biological samples.     

Optimization of fermentation process for the production of intracellular polysaccharide from Paecilomyces cicadae and the immuno-stimulating activity of intracellular polysaccharide

Optimization of fermentation process for the production of intracellular polysaccharide (IPS) from the fungus Paecilomyces cicadae and the immuno-stimulating activity of IPS were carried out. The quantitative effects of initial pH, fermentation temperature and time on the yield of IPS content produced by P. cicadae in submerged fermentation were investigated separately using response surface methodology (RSM). The three factors chosen for the present investigation were based on the results of a previous Plackett?CBurman (PB) design. The experimental data obtained were fitted to a second-order polynomial equation using multiple regression analysis and also analyzed by appropriate statistical methods. RSM analysis showed good correspondence between experimental and predicted values. It was found that three parameters represented significant effect. Probability value (p?<?0.0001) demonstrated a very high significance for the regression model. By solving the regression equation and analyzing the response surface contour plots, the optimal process parameters were determined, i.e. fermentation temperature 24.53?°C, initial pH 7.46 and fermentation time 73.9?h. The maximum predicted yield of IPS was 356.02???g/ml under the optimal conditions. Meanwhile, IPS from P. cicadae was found to have direct immuno-stimulating activity in vitro on murine macrophage RAW264.7 proliferative response and to stimulate nitric oxide generation in a dose-dependent manner.     

Extraction,development and validation of HPLC-UV method for rapid and sensitive determination of colchicine from Colchicum autumnale L. Bulbs

Colchicum autumnale L. also known as the autumn crocus, contains colchicine alkaloid having antifungal properties. The tuber of this plant is rich in terms of colchicine. In this research an ultrasound-assisted extraction (UAE) method was optimized for the extraction of colchicine from Colchicum autumnale L. bulbs before high-performance liquid chromatography with UV detection (HPLC-UV). Optimization of various extraction parameters was performed using response surface methodology (RSM) to evaluate the maximum colchicine yield from Colchicum autumnale L. bulbs. The Box-Behnken design (BBD) and RSM were used to investigate the effect of three key parameters (extraction time (20–60?min), extraction temperature (40–80?°C) and ultrasound power (500–700?W) on extraction efficiency. The variance analysis suggested that the dependent response variable of yield of colchicine may be expressed by a quadratic polynomial model. The optimal theoretical extraction conditions were found to be an ultrasonication power of 602.4?W, an extraction time of 42?min and a temperature of 64?°C. Under these conditions, the optimum foreseen yield was 0.237%. The experimental colchicine yield obtained by following the optimized conditions was found to be 0.238%. These values are very well compatible with each other.     

Statistical optimization of l-leucine amino peptidase production from Streptomyces gedanensis IFO 13427 under submerged fermentation using response surface methodology

Response surface methodology (RSM) employing the central composite design (CCD) was used to optimize the fermentation medium for the production of l-leucine amino peptidase (LAP) from Streptomyces gedanensis IFO13427 under submerged fermentation. The design was employed by selecting substrate concentration, NaCl concentration and initial pH as model factors by ‘one variable at a time’ experiment. A second-order quadratic model and response surface method showed that the optimum conditions (soy bean 0.3%, NaCl, 0.03 M, and initial pH 7) resulted in the improvement of LAP production (25.69 IU/ml) as compared to the initial level (12.17 ± 0.23 IU/ml) after 72 h of fermentation, whereas its value predicted by the quadratic model was 24.56 IU/ml. Analysis of variance (ANOVA) showed a high coefficient of determination (R²) value of 0.9799, ensuring a satisfactory adjustment of the quadratic model with the experimental data. This is first report on LAP production by S. gedanensis using statistical experimental design and response surface methodology in submerged fermentation.     

Optimization and economic evaluation of ultrasound extraction of lutein from Chlorella vulgaris

The main carotenoid in Chlorella vulgaris is lutein. The ultrasound alone or together with enzymatic pretreatment for the extraction of lutein from C. vulgaris was optimized using response surface methodology (RSM) to improve the extraction process. The optimal ultrasound extraction condition was: ultrasound frequency, 35 kHz; ultrasound intensity, 56.58 W/cm²; extraction temperature, 37.7°C; extraction time, 5 h; and ratio of solvent to solid, 31 mL/g, where the lutein recovery was 3.16 ± 0.03 mg/g wet C. vulgaris. The optimal enzymatic pretreatment was: reaction time, 2 h; enzyme concentration, 1.23% (v/w); pH, 4.5, and temperature 50°C. The optimal ultrasound extraction with enzymatic pretreatment was: ultrasound frequency, 35 kHz; ultrasound intensity, 56.58 W/cm²; extraction temperature, 37.7°C; extraction time, 162 min; and ratio of solvent to solid, 35.6 mL/g wet C. vulgaris, where the extraction yield of lutein was 3.36 ± 0.10 mg/g wet C. vulgaris. This was much higher than for ultrasound treatment alone. The surface areas of microalga cells treated by ultrasound with/without enzymatic pretreatment increased significantly, which might contribute to the increase in lutein yield. There were no significant differences in structure, color, and antioxidant activity of lutein between the ultrasound and conventional methods. The highest cost of the crude and lutein was obtained by the ultrasound with enzymatic pretreatment due to the complex process and liquid waste in the enzymatic pretreatment process, but the ultrasound treatment alone was the lowest. Therefore, ultrasound extraction is the most economical method for the extraction of microalgal lutein.     

响应面法优化深层发酵樟芝总三萜的提取工艺(英文)

采用响应面法对樟芝深层培养中总三萜的提取工艺进行优化。根据Box-Benhnken的中心组合实验设计原理,在单因素实验的基础上,选取溶剂浓度、提取温度和液固比为变量,应用响应面法进行三因素三水平的实验设计。以总三萜得率作为响应值,建立了樟芝总三萜提取的回归模型,对其提取条件进行进一步优化。结果表明,优化的总三萜提取条件为乙醇浓度86%,提取温度75℃,液固比37。进一步的实验也验证了该模型的有效性。     

High-efficient extraction of principal medicinal components from fresh Phellodendron bark (cortex phellodendri)

There are three key medicinal components (phellodendrine, berberine and palmatine) in the extracts of Phellodendron bark, as one of the fundamental herbs of traditional Chinese medicine. Different extraction methods and solvent combinations were investigated to obtain the optimal technologies for high-efficient extraction of these medicinal components. Results: The results showed that combined solvents have higher extracting effect of phellodendrine, berberine and palmatine than single solvent, and the effect of ultrasonic extraction is distinctly better than those of distillation and soxhlet extraction. Conclusion: The hydrochloric acid/methanol-ultrasonic extraction has the best effect for three medicinal components of fresh Phellodendron bark, providing an extraction yield of 103.12 mg/g berberine, 24.41 mg/g phellodendrine, 1.25 mg/g palmatine.     

Physicochemical Properties Analysis and Secretome of Aspergillus niger in Fermented Rapeseed Meal

The nutrient digestibility and feeding value of rapeseed meal (RSM) for non-ruminant animals is poor due to the presence of anti-nutritional substances such as glucosinolate, phytic acid, crude fiber etc. In the present study, a solid state fermentation (SSF) using Aspergillus niger was carried out with the purpose of improving the nutritional quality of RSM. The chemical composition and physicochemical properties of RSM before and after fermentation were compared. To further understand possible mechanism of solid state fermentation, the composition of extracellular enzymes secreted by Aspergillus niger during fermentation was analysed using two-dimentional difference gel electrophoresis (2D-DIGE) combined with matrix assisted laser desorption ionization—time of flight—mass spectrometer (MALDI-TOF-MS). Results of the present study indicated that SSF had significant effects on chemical composition of RSM. The fermented rapeseed meal (FRSM) contained more crude protein (CP) and amino acid (AA) (except His) than unfermented RSM. Notably, the small peptide in FRSM was 2.26 time larger than that in unfermented RSM. Concentrations of anti-nutritional substrates in FRSM including neutral detergent fiber (NDF), glucosinolates, isothiocyanate, oxazolidithione, and phytic acid declined (P < 0.05) by 13.47, 43.07, 55.64, 44.68 and 86.09%, respectively, compared with unfermented RSM. A. niger fermentation disrupted the surface structure, changed macromolecular organic compounds, and reduced the protein molecular weights of RSM substrate. Total proteins of raw RSM and FRSM were separated and 51 protein spots were selected for mass spectrometry according to 2D-DIGE map. In identified proteins, there were 15 extracellular hydrolases secreted by A. niger including glucoamylase, acid protease, beta-glucanase, arabinofuranosidase, xylanase, and phytase. Some antioxidant related enzymes also were identified. These findings suggested that A. niger is able to secrete many extracellular degradation enzymes (especially lignocellulosic hydrolyzing enzymes, acid proteases and phytase) during fermentation of RSM, thus altering chemical composition and physicochemical properties of RSM.     

Statistical optimization of medium components for the production of Antrodia cinnamomea AC0623 in submerged cultures

The nutritional medium requirement for biomass and triterpenoid production by Antrodia cinnamomea AC0623 strain was optimized. Box–Behnken was applied to optimize biomass and triterpenoid production. According to response surface methodology (RSM), the optimum concentrations of N-source were determined. The results indicate that when a submerged culture in shake flasks was operated at 28°C, initial pH 5.5, and rotation speed 105 rpm, the biomass and triterpenoid content in dry basis could be increased to 3.20% (w/w) and 31.8 mg/g, respectively. The experiments were further scaled up to 100- and 700-l fermentors. Higher content of triterpenoids (63.0 mg/g) was obtained in 700-l fermentations by means of the control of cultural conditions and the modification of medium composition based on the RSM.     

Optimization protocol for the extraction of antioxidant components from Origanum vulgare leaves using response surface methodology

In the present work, the response surface methodology (RSM) based on a central composite rotatable design (CCRD), was used to determine optimum conditions for the extraction of antioxidant compounds from Origanum vulgare leaves. Four process variables were evaluated at three levels (31 experimental designs): methanol (70%, 80%, and 90%), the solute:solvent ratio (1:5, 1:12.5, 1:20), the extraction time (4, 10, 16 h), and the solute particle size (20, 65, 110 micron). Using RSM, a quadratic polynomial equation was obtained by multiple regression analysis for predicting optimization of the extraction protocol. Analysis of variance (ANOVA) was applied and the significant effect of the factors and their interactions were tested at 95% confidence interval. The antioxidant extract (AE) yield was significantly influenced by solvent composition, solute to solvent ratio, and time. The maximum AE was obtained at methanol (70%), liquid solid ratio (20), time (16 h), and particle size (20 micron). Predicted values thus obtained were closer to the experimental value indicating suitability of the model. Run 25 (methanol:water 70:30; solute:solvent 1:20; extraction time 16 h and solute particle size 20) showed highest TP contents (18.75 mg/g of dry material, measured as gallic acid equivalents) and DPPH radical scavenging activity (IC₅₀ 5.04 μg/mL). Results of the present study indicated good correlation between TP contents and DPPH radical scavenging activity. Results of the study indicated that phenolic compounds are powerful scavengers of free radical as demonstrated by a good correlation between TP contents and DPPH radical scavenging activity.     

Optimization of ultrasound-assisted extraction conditions for total phenols with anti-hyperglycemic activity from Psidium guajava leaves

The high concentration of total phenolic compounds (TPC) in Psidium guajava leaf extracts (GvEx) is correlated to its anti-hyperglycemic activity. In this study, we established the optimum ultrasound extraction conditions for maximizing TPC yield. The response surface methodology (RSM) was employed for empirical model building. The maximum value of TPC (26.12%) was obtained at solvent to solid ratio (v/w) of 12.1, extraction temperature of 59.8 °C, and extraction time of 5.1 min. The anti-hyperglycemic activity of GvEx was compared to the commonly used diabetic drug acarbose. The IC₅₀ of GvEx for α-amylase and α-glucosidase inhibition was 50.5 μg/mL and 34.6 μg/mL, respectively. However, the IC₅₀ of acarbose for α-amylase and α-glucosidase inhibition was 95.3 μg/mL and 1075.2 μg/mL, respectively. In conclusion, GvEx obtained under optimum extraction conditions had higher anti-hyperglycemic activity than acarbose. In addition, the recommended extraction procedures for GvEx save time and are environmentally friendly.     

Statistical optimization of physical parameters for enhanced bacteriocin production by L. casei

Antimicrobial proteinaceous compounds such as bacteriocins produced from Lactobacillus sp. are widely known. They have potential antimicrobial activities towards closely related bacteria and several pathogens associated with food spoilage and hence can be a potential food bio-preservative agent. Bacteriocin production requires optimized process, complex media and well-controlled physical conditions including pH and temperature. A probiotic strain of L. casei LA-1 isolated from mango pickle was used in the present study. The influence of physical parameters viz. temperature (15 ?? 45°C), pH (4.0 ?? 7.0), incubation time (up to 48 h) and inoculum size (0.7 ?? 2.0 O.D) on bacteriocin production was analyzed. The effect of all the parameters was first investigated using the one-factor-at-a-time method (OFAT) to see the significance of these parameters on bacteriocin production and then further optimized by response surface methodology (RSM). Following OFAT analysis, all factors were found to have a significant effect on bacteriocin production. Bacteriocin production of 2,844 AU/mL was obtained at temperature 37°C, pH 6.7 and inoculum size 1.8 O.D at an incubation time of 20 h and it was produced during the stationary phase of growth. Statistical analysis showed that three variables-pH, temperature and incubation time have significant effects on bacteriocin production. RSM proved to be a powerful tool in the optimization of bacteriocin production by L. casei LA-1 with a two-fold increase, giving a production of 4652.15 AU/mL at pH 7.19, temperature 33.3°C and incubation time of 22.2 h.