Sequential Experimental Design Strategy Research Articles

Spirulina (Arthrospira platensis) protein-rich extract as a natural emulsifier for oil-in-water emulsions: Optimization through a sequential experimental design strategy

Spirulina (Arthrospira platensis) proteins have been proven to present emulsifying properties. In this work, a Spirulina protein-rich extract obtained by ultrasound extraction (SpE) was tested to stabilize oil-in-water (O/W) emulsions. For this purpose, a sequential experimental design strategy (Fractional Factorial Design (FFD) 24–1 followed by a Central Composite Rotatable Design (CCRD) 22)) was applied. The effect of four variables, SpE concentration, O/W weight ratio, pH and storage time, on emulsions' zeta potential and number-mean droplet diameter was considered for the FFD 24–1, indicating SpE concentration and storage time as the relevant variables for the CCRD 22. According to zeta potential and number-mean droplet diameter evaluation, for the studied SpE concentration range (2–5 wt%), quite stable emulsions were obtained along the tested 30-days period. Even so, for 5%, visual inspection revealed extract segregation after 20-days. The optimal solution comprised 4 wt% of SpE, for an O/W weight ratio of 30/70 and a pH of 7.0 (number-mean droplet diameter of 55.66 nm and zeta potential of −43.83 mV). Overall, SpE has proven to be an excellent emulsifier, offering the potential to substitute animal-based proteins and synthetic emulsifiers. In addition, no signs of contamination by microorganisms were observed, suggesting that the SpE may also act as an antimicrobial agent.

Feasibility of bioethanol production from rice bran

Rice bran is a by-product of rice production with a high carbohydrate and starch content and the potential for bioethanol production by alcoholic fermentation. This article describes bioethanol production by Saccharomyces cerevisiae from hydrolyzed defatted rice bran (DRB) a rice by-product applying ultrasonic treatment and protease addition, as well as a sequential strategy of experimental design (SEED). In the first Central Composite Rotatable Design (CCRD), the temperature (25-30 °C) and inoculum concentration (0.5-50 g L-1) had positive effects on bioethanol production, while the effect of pH (4.0-6.0) was not significant. In the second CCRD, the temperature (28-35 °C) and inoculum concentration (10-70 g L-1) had negative and positive effects on bioethanol production (p < 0.05). Protease addition (15 µL g-1) increased the conversion of substrate into bioethanol by 76%. The optimized conditions for the production of 40.7 g L-1 bioethanol were a temperature of 31.5 °C and an inoculum concentration of 70 g L-1. Validation in a benchtop bioreactor produced 40.0 g L-1 of bioethanol from hydrolyzed DRB, and the SEED was characterized as a useful tool to improve bioethanol production from DRB. Furthermore, the DRB proved to be a by-product with great potential for bioethanol production, derived from alternative sources not commonly used in human food.

Feasibility of ultrasound-assisted optimized process of high purity rice bran protein extraction

ABSTRACT: Rice bran is a by-product of the rice milling process, found worldwide in abundance and highlighted due its protein content. This study optimized the conditions for ultrasonic-assisted extraction of protein from defatted rice bran (DRB) and characterized the rice bran protein concentrate (RBPC). A sequential strategy of experimental design was employed; the effect of pH, temperature, ultrasound exposure time, and amplitude were evaluated regarding the percentage of protein extraction by a Full factorial design (FFD) with a fixed frequencies (FFD-A: 37 kHz; FFD-B: 80 kHz). Subsequently, the percentage of protein extracted was optimized employing a Central composite rotatable design (variables: pH and ultrasound exposure time) and RBPC obtained was characterized regarding chemical and functional properties. The pH and ultrasound exposure time had positive effect (P ≤ 0.05) on percentage of protein extraction; moreover, 37 kHz frequency was more effective in protein extraction. The optimized condition (frequency: 37 kHz; temperature: 30 °C; pH: 10; ultrasound exposure time: 30 min; and amplitude: 100%) allowed 15.07% of protein recovery and the RBPC presented 84.76 g 100 g-1 of protein. Magnesium and copper were the main mineral in RBPC (34.4 and 25.5 µg g-1, respectively), while leucine was the limiting amino acid (0.42) and threonine presented the highest chemical score (1.0). The RBPC solubility was minimal at pH 4 and higher at pHs 6-10; the water and oil absorption capacity were higher than bovine serum albumin (BSA) and the emulsifying capacity was comparable to BSA, with a suitable stability. It was possible to obtain a higher purity RBPC than described in the literature, due to the optimization in the extraction process steps, with functional properties suitable for application in food products, especially emulsified ones.

Optimization of lipid profile and hardness of low-fat mortadella following a sequential strategy of experimental design.

This study aims to optimize simultaneously the lipid profile and instrumental hardness of low-fat mortadella. For lipid mixture optimization, the overlapping of surface boundaries was used to select the quantities of canola, olive, and fish oils, in order to maximize PUFAs, specifically the long-chain n-3 fatty acids (eicosapentaenoic-EPA, docosahexaenoic acids-DHA) using the minimum content of fish oil. Increased quantities of canola oil were associated with higher PUFA/SFA ratios. The presence of fish oil, even in small amounts, was effective in improving the nutritional quality of the mixture, showing lower n-6/n-3 ratios and significant levels of EPA and DHA. Thus, the optimal lipid mixture comprised of 20, 30 and 50% fish, olive and canola oils, respectively, which present PUFA/SFA (2.28) and n-6/n-3 (2.30) ratios within the recommendations of a healthy diet. Once the lipid mixture was optimized, components of the pre-emulsion used as fat replacer in the mortadella, such as lipid mixture (LM), sodium alginate (SA), and milk protein concentrate (PC), were studied to optimize hardness and springiness to target ranges of 13-16N and 0.86-0.87, respectively. Results showed that springiness was not significantly affected by these variables. However, as the concentration of the three components increased, hardness decreased. Through the desirability function, the optimal proportions were 30% LM, 0.5% SA, and 0.5% PC. This study showed that the pre-emulsion decreases hardness of mortadella. In addition, response surface methodology was efficient to model lipid mixture and hardness, resulting in a product with improved texture and lipid quality.

Optimization of the Extraction and Nutritional Value of Tiger Nut Milk by Sequential Design Strategy

Sequential experimental design strategy was applied in order to investigate the best conditions for tiger nut milk extraction process taking into account performance and physicochemical features, simultaneously. Protein, lipid, starch, calcium and total sugar composition were considered for this optimization. The tested factors were: sodium metabisulphite concentration (X1), soaking time (X2), soaking temperature (X3) and water proportion (X4). From the 24-1 fractional factorial design all independent variables were found to be statistically significant (p <0.1) and X1, X2 and X3 were chosen for the central composite design as water proportion was already in the minimum value. The best operational conditions, obtained by desirability function, were: 8 h of soaking time, 22° C for soaking temperature, 0.20% (w/v) of sodium metabisulphite and 1:1 tuber (g): water (ml) proportion. In those conditions a 78% yield was obtained for a tiger nut milk with 2.60% protein, 3.7% total sugar and 8.72% starch. The high yield obtained and the great nutritional value shows that this milk has a great potential for application in the food industry.

Protein and carbohydrate extraction from S. platensis biomass by ultrasound and mechanical agitation

Spirulina platensis is considered an alternative and excellent source of protein [46–63% dry basis (DB)], having protein levels comparable to meat and soybeans. Thus, it can be considered an adequate ingredient to supply the necessity of this compound in the food industry. Its carbohydrates (8–14% DB) may also be a useful food ingredient or a potential source of bioenergy. Thus, extracting these compounds from the microalgae biomass will maximize its exploitation. Sonication can completely or partially degrade the microalgal cell wall, providing a useful technique to extract the protein and carbohydrate. This study used a sequential strategy of experimental design (fractional factorial design and central composite rotatable design) to evaluate the protein and carbohydrate extraction from S. platensis defatted biomass using ultrasonic waves and mechanical agitation, under alkaline conditions. The optimal conditions for protein and carbohydrate co-extraction were established by selecting and maximizing the variables that significantly influenced the extraction. The optimized percentages recovery from the extraction process yielded 75.76% protein and 41.52% carbohydrate at 33–40min sonication and 40–55min agitation. The protein fraction may be further concentrated and purified for use in food formulations, and the carbohydrates may be a useful feedstock for bioethanol production.

Sequential strategy of experimental design I: Optimization of extractive distillation process of ethanol–water using [bmim][N(CN)2] as entrainer

Nowadays, one of the methods available to obtain the anhydrous ethanol is the extractive distillation process, which presents great potential depending on the solvent used. It is imperative that the solvent promotes dehydration, but low cost, the low energy consumption, and low waste generation and emissions must be taken into account. Within this context, there is high demand for new efficient solvents for extractive distillation of ethanol-water mixture, so, the ionic liquids (ILs) have some interesting properties for such applications. This work was performed in order to optimize the process using of the Plackett-Burman experimental design and the response surface methodology applied to the process of extractive distillation of azeotropic ethanol-water mixture, using the IL 1-butyl-3-methylimidazolium dicyanamide ([bmim] [N(CN)2]) as solvent. The choice of this IL as solvent was based on the data presented in the open literature.Plackett–Burman experimental design was used to evaluate the process variables. Then, these variables, called independent variables (Invar), were used in a central composite rotatable design for generating models to predict some parameters, such as the energy consumption, amount of IL used, purity and recovery of the ethanol obtained. This model was applied to an optimization algorithm, being possible to recover 99.39% of ethanol which enters the extractive distillation process with a purity of 99.76%mol, using a ratio of 0.074mol IL/mol ethanol at feed stream and consuming 3071.3kJ per kg anhydrous ethanol produced.

Soybean molasses-based bioindicator system for monitoring sterilization process: Designing and performance evaluation

A novel cost-effective Bacillus atrophaeus Sterilization Bioindicator System (BIS) with a high quality and performance was developed from a soybean byproduct and compared with the commercial BIS. It was composed of recovery medium and dry-fermented spores with sand as the support. The BIS was developed and optimized using a sequential experimental design strategy. The recovery medium contained soluble starch (1.0 g/L), soybean molasses (30.0 g/L), tryptone (40.0 g/L), and bromothymol blue (0.02 g/L) at pH 8.5. The solid-state fermentation conditions of the bioreactor and environmental humidity had no significant effects on the spore yield and dry-heat resistance. The only substrate mineral that showed a positive effect was Mn2+, allowing Mg2+, K+, and Ca2+ to be eliminated from the formulation. Validation of optimized medium indicated D160°C = 6.8±1.0 min (3.6 min more than the minimum) and spore yield = 2.3 ± 0.5 × 109 CFU/g dry sand (10,000 × initial values). BIS performance resulted in D160°C = 6.6 ± 0.1 min. Sporulation and germination kinetics allowed the sporulation process to be reduced to three days, and the growth of heat-damaged spores was sufficient to achieve visual identification of a non-sterile BIS within 21 h. Process economics was a minimum of 23.9%, and process cycle time was reduced from 29 to 15 days. The new BIS parameters demonstrated compliance to all regulatory requirements. No studies have yet described a BIS production from soybean molasses.

Glycerol-based sterilization bioindicator system from Bacillus atrophaeus: development, performance evaluation, and cost analysis

The development of new value-added applications for glycerol is of worldwide interest because of the environmental and economic problems that may be caused by an excess of glycerol generated from biodiesel production. A novel use of glycerol as a major substrate for production of a low-cost sterilization biological indicator system (BIS; spores on a carrier plus a recovery medium) was investigated. A sequential experimental design strategy was applied for product development and optimization. The proposed recovery medium enables germination and outgrowth of heat-damaged spores, promoting a D (160°C) value of 6.6 ± 0.1min. Bacillus atrophaeus spores production by solid-state fermentation reached a 2.3 ± 1.2 × 10(8) CFU/g dry matter. Sporulation kinetics results allowed this process to be restricted in 48h. Germination kinetics demonstrated the visual identification of nonsterile BIS within 24h. Performance evaluation of the proposed BIS against dry-heat and ethylene oxide sterilization showed compliance with the regulatory requirements. Cost breakdowns were from 41.8 (quality control) up to 72.8% (feedstock). This is the first report on sterilization BIS production that uses glycerol as a sole carbon source, with significant cost reduction and the profitable use of a biodiesel byproduct.

Optimization of medium formulation and seed conditions for expression of mature PsaA (pneumococcal surface adhesin A) in Escherichia coli using a sequential experimental design strategy and response surface methodology

PsaA, a candidate antigen for a vaccine against pneumonia, is well-conserved in all Streptococcus pneumoniae serotypes. A sequence of two-level experimental designs was used to evaluate medium composition and seed conditions to optimize the expression of soluble mature PsaA in E. coli. A face-centered central composite design was first used to evaluate the effects of yeast extract (5 and 23.6g/L), tryptone (0 and 10g/L), and glucose (1 and 10g/L), with replicate experiments at the central point (14.3g/L yeast extract, 5g/L tryptone, 5.5g/L glucose). Next, a central composite design was used to analyze the influence of NaCl concentration (0, 5, and 10g/L) compared with potassium salts (9.4g/L K(2)HPO(4)/2.2g/L KH(2)PO(4)), and seed growth (7 and 16h). Tryptone had no significant effect and was removed from the medium. Yeast extract and glucose were optimized at their intermediate concentrations, resulting in an animal-derived material-free culture medium containing 15g/L yeast extract, 8g/L glucose, 50μg/mL kanamycin, and 0.4% glycerol, yielding 1g/L rPsaA after 16h induction at 25°C in shake flasks at 200rpm. All the seed age and salt conditions produced similar yields, indicating that no variation had a statistically significant effect on expression. Instead of growing the seed culture for 16h (until saturation), the process can be conducted with 7h seed growth until the exponential phase. These results enhanced the process productivity and reduced costs, with 5g/L NaCl being used rather than potassium salts.

Assessment of fructooligosaccharides production from sucrose in aqueous and aqueous-organic systems using immobilized inulinase from Kluyveromyces marxianus NRRL Y-7571

This work investigated the fructooligosaccharides (FOS) synthesis by immobilized inulinase obtained from Kluyveromyces marxianus NRRL Y-7571 in aqueous and aqueous-organic systems using sucrose as substrate. The sequential strategy of experimental design was used to optimize the FOS conversion in both systems. For the aqueous-organic system, a 2(6-2) fractional design was carried out to evaluate the effects of temperature, sucrose concentration, pH, aqueous/organic ratio, enzyme activity, and polyethylene glycol concentration. For the aqueous system, a central composite design for the enzyme activity and the sucrose concentration was carried out. The highest fructooligosaccharides yield (Y FOS) for the aqueous-organic system was 18.2 ± S0.9 wt%, at 40 ºC, pH 5.0, sucrose concentration of 60% (w/w), enzyme activity of 4 U.mL-1, and aqueous/organic ratio of 25/75 wt%. The highest Y FOS for the aqueous system was 14.6 ± 0.9 wt% at 40 ºC, pH 5.0, sucrose concentration of 60 wt%, and enzyme activity of 4.0 U.mL-1.

Kinetics of Toluene Disproportionation: Modeling and Experiments

This work presents modeling and experimental studies on the kinetics of toluene disproportionation in operation ranges that include real industrial operation conditions. The influence of reaction temperature, reactor pressure, feed composition, and residence time on conversion of reactants and product selectivity was investigated. Experiments were performed according to a sequential experimental design strategy, in order to provide maximum accuracy for model predictions. Statistical treatment of parameter estimates and model adequacy was performed with the help of maximum likelihood principles. Excellent agreement between model predictions and available experimental data was obtained in the full ranges of investigated experimental conditions.

Enzymatic synthesis of ascorbyl palmitate in ultrasound-assisted system: Process optimization and kinetic evaluation

This work is focused on the optimization of reaction parameters for the synthesis of ascorbyl palmitate catalyzed by Candida antarctica lipase in different organic solvents under ultrasound irradiation. The sequential strategy of experimental design proved to be useful in determining the optimal conditions for reaction conversion in tert-butanol system using Novozym 435 as catalyst. The optimum production was achieved at 70 °C, ascorbic acid to palmitic acid molar ratio of 1:9, enzyme concentration of 5 wt% at 3 h of reaction, resulting in an ascorbyl palmitate conversion of about 27%. Reaction kinetics for ascorbyl palmitate production in ultrasound device showed that satisfactory reaction conversions (∼26%) could be achieved in short reaction times (2 h). The empirical kinetic model proposed is able to satisfactorily represent and predict the experimental data.

Optimization of the methodology for lead extraction from waste contaminated with heavy metals

A sequential experimental design strategy is used to optimize the extraction of lead from slag. The slag is composed mainly of iron (≈60%) and lead (≈6%), and cannot be disposed of in conventional landfill due to its high lead content. The extraction of lead is based on the complexing properties of ethylenediaminetetra‐acetic acid (EDTA), the iron being masked with fluoride ions. Atomic absorption spectroscopy (AAS) has been used for monitoring iron and lead concentrations. In a first step, Plackett–Burman PB‐12 experimental design was used for screening the variables and for selecting those to be used. Two central composite designs (CCDs) were then carried out to determine the optimal conditions for lead extraction. The optimized experimental conditions were established to be: contact time with the fluoride solution 30 min; particle size of slag between 150 and 600 µm; molecular concentration of fluoride solution 2.4 × 10−3 mol and of EDTA 5 × 10−4 mol; pH 1.5; 8 h contact time with the EDTA solution; and temperature 60°C. Under these experimental conditions the lead content was reduced by 57% compared with the raw sample.

Optimization of mono and diacylglycerols production from enzymatic glycerolysis in solvent-free systems

This work reports solvent-free enzymatic glycerolysis of olive oil with an immobilized lipase (Novozym 435) using Tween 40, Tween 65, Tween 80, Tween 85, Triton X-100, and soy lecithin as surfactants. The first step was the screening of two potential surfactants for Monoacylglycerol (MAG) and Diacylglycerol (DAG) production with a pre-established operating condition and 2 h of reaction time. Afterwards, a sequential experimental design strategy was carried out in order to optimize MAG and DAG production using Tween 65 and Triton X-100 as surfactants. The operating conditions that optimized MAG and DAG yields were 70 degrees C, stirring rate of 600 rpm, glycerol:olive oil molar ratio of 6:1, 16 wt% of surfactant Tween 65 and 9.0 wt% of Novozym 435, leading to a content of 26 and 17 wt% of MAG and DAG, respectively.

Use of a sequential strategy of experimental design to optimize the inulinase production in a batch bioreactor

A potential application of inulinase in the food industry is the production of fructoligosaccharides (FOS) by the transfructosilation of sucrose. The FOSs present many interesting functional properties besides their ability to increase the shelf-life and flavor of many products. The use of an industrial medium represents a good alternative to producing inulinase at low cost, since the activity may improve, or at least remain the same, as that obtained using a synthetic medium. This work was an optimization study of the inulinase production by Kluyveromyces marxianus NRRL Y-7571 using industrial pre-treated culture medium in a bioreactor employing a sequential strategy of experimental design. Initially, a Plackett-Burman (Screening Design) design was used, where the studied variables were molasses, corn steep liquor, yeast extract concentration, and agitation and aeration rates. After the analysis of the effects, a central composite rotational design (CCRD) was carried out. The optimized condition for the inulinase production was: 250 g/l of molasses, 80 g/l of corn steep liquor, 6 g/l of yeast extract, 300 rpm of agitation and 1.5 vvm aeration rate, which resulted in an enzymatic activity of 1,317 +/- 65 U/ml.

Optimization of Trehalose Production by Rhodotorula dairenensis Following a Sequential Strategy of Experimental Design

Initially, a technical screening was applied to select yeasts with a potential for trehalose production using thermal stress for the induction of synthesis. The effects of the concentrations of sugar cane molasses, corn steep liquor and a commercial yeast extract Prodex Lac SD®, and of pH and temperature on the biomass were studied using a fractional design followed by a central composite rotatable design (CCRD). The optimum values were 50 g/l for the molasses and corn steep liquor concentrations, initial pH of 5.5, and temperature of 30 °C, with no yeast extract. Under these conditions, the production of trehalose was studied using a CCRD to optimize the temperature (33 to 47 °C) and exposition time (60 to 120 min) of the cultures to the thermal stress. The maximum intracellular trehalose content reached was 20.5% (g trehalose/100 g dry cell) at temperatures of 35–40 °C and with 100–120 min of exposition time to the thermal stress.

Sequential experimental design strategy for rapid kinetic modeling of chemical synthesis

AbstractIn this work, an optimum experimental design strategy has been developed to increase the reliability of chemical reaction engineering models when few experiments are to be performed. The strategy has been used for heterogeneous liquid–liquid reactions carried out in a batch reactor. The specific case of alkaline hydrolysis of n‐amylacetate has been selected to illustrate the proposed methodology, allowing experiments to be carried out to identify the associated kinetic parameters. The kinetic parameters that arise from the model system of ordinary differential equations have been estimated by weighted least squares and a D‐optimum sequential design has been implemented to reduce the volume of the joint confidence region of the parameters. Estimates and their uncertainty obtained at the end of the strategy, as well as the generalization ability of the prediction model, are presented and discussed. © 2005 American Institute of Chemical Engineers AIChE J, 2005

Sequential experimental design strategy for kinetic parameters estimation

In this paper, a sequential experimental design strategy is developed for precise kinetic parameters estimation using the D-optimality criterion. The influence of batch or fed-batch mode on the accuracy of kinetic parameters of a consecutive competitive reaction scheme is studied. The kinetic parameters are estimated by the maximum likelihood estimator. The standard nonlinear programming SQP (Successive Quadratic Programming) ensures the determination of optimal experimental operating conditions.