Optimization of the fermentation media for sophorolipid production from Candida bombicola ATCC 22214 using a simplex centroid design

This article describes the use of a simplex centroid mixture experimental design to optimize the fermentation medium in the production of sophorolipids (SLs) using Candida bombicola. In the first stage, 16 media ingredients were screened for the ones that have the most positive influence on the SL production. The sixteen ingredients that were chosen are five different carbohydrates (fructose, glucose, glycerol, lactose, and sucrose), five different nitrogen sources (malt extract, peptone extract, soytone, urea, and yeast extract), two lipid sources (mineral oil and oleic acid), two phosphorus sources (K₂HPO₄ and KH₂PO₄), MgSO₄, and CaCl₂. Multiple regression analysis and centroid effect analysis were carried out to find the sugar, lipid, nitrogen source, phosphorus source, and metals having the most positive influence. Sucrose, malt extract, oleic acid, K₂HPO₄, and CaCl₂ were selected for the second stage of experiments. An augmented simplex centroid design for five ingredients requiring 16 experiments was used for the optimization stage. This produced a quadratic model developed to help understand the interaction amongst the ingredients and find the optimal media concentrations. In addition, the top three results from the optimization experiments were used to obtain constraints that identify an optimal region. The model together with the optimal region constraints predicts the maximum production of SLs when the fermentation media is composed of sucrose, 125 g/L; malt extract, 25 g/L; oleic acid, 166.67 g/L; K₂HPO₄, 1.5 g/L; and CaCl₂, 2.5 g/L. The optimal media was validated experimentally and a yield of 177 g/L was obtained. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

大桑菊合剂的提取工艺研究

目的：大桑菊舍刺为医院制剂,由桑叶、菊花、薄荷、连翘、鱼腥草等中药经乙醇提取制成,为完善大桑菊合剂的制备工艺,对该制剂的制备工艺进行初步的研究。方法：①实验设计：以加水量、提取时间、提取次数和醇沉浓度为考察因素,各因素均取3水平,采用正交试验L4（3^4）优选制备工艺。②含量测定方法：以大桑菊饮合剂中总黄酮的含量为指标,以芦丁为标准品,利用紫外分光光度计于503nm下测定吸光值,计算样品中总黄酮的含量,以此作为筛选大桑菊合剂最佳制备工艺的重要指标。结果：最佳制备工艺为不浸泡,加8倍水,提取3次,每次1小时。结论：经正交设计优化的大桑菊合剂的中药制备工艺合理、可行。     

Mixture design as first step for improved glutaminase production in solid-state fermentation by isolated Bacillus

Aim: Investigation of mixture‐design impact on glutaminase production by isolated Bacillus sp. Methods and Results: An augmented simplex centroid design was used to optimize a three (wheat bran, Bengal gram husk and palm seed fibre) component mixture for glutaminase production. Selected substrate materials showed impact on glutaminase production values at individual level by Bengal gram husk [2789 U gds^?1 (gram dry substrate] and in two‐level combination with wheat bran and Bengal gram husk (maximum of 3300 U gds^?1). Conclusion: Bengal gram husk is the most suitable substrate medium for glutaminase production by Bacillus sp. Maximum glutaminase production is achieved using solid‐substrate mixture at two‐level combinations in the ratio of 66 : 34 for Bengal gram husk and wheat bran, respectively. Significance and Impact of the Study: The present study has significance in large‐scale production of glutaminase at commercial level with the use of multi‐substrate rather than single‐substrate/support material.     

Design Efficiency and Estimation for Component Amount Models

Component amount models were introduced by Piepel and Cornell (1985) to describe mixture experiments where the response depends not only on the proportions of the components but also on the total amount. The extended simplex centroid design supported by the barycenters corresponding to the regression functions in the model is D-optimal or fails to be D-optimal depending on the regression model under consideration. For two special component amount models, namely the ν-tic polynomial and the minimum polynomial of degree ν, the efficiency of the simplex centroid design was investigated. This covers the presentation of new results concerning the D-optimality as well as the assessment of the simplex centroid design in non-optimal situations by means of the G-efficiency. Formulae for the least squares estimates as well as their variances are presented.     

Mixture design of starchy substrates hydrolysis by an immobilized glucoamylase from Aspergillus brasiliensis

Starch has great importance in human diet, since it is a heteropolymer of plants, mainly found in roots, as potato, cassava and arrowroots. This carbohydrate is composed by a highly-branched chain: amylopectin; and a linear chain: amylose. The proportion between the chains varies according to the botanical source. Starch hydrolysis is catalyzed by enzymes of the amilolytic system, named amylases. Among the various enzymes of this system, the glucoamylases (EC 3.2.1.3 glucan 1,4-alpha-glucosidases) are the majority because they hydrolyze the glycosidic linkages at the end of starch chains releasing glucose monomers. In this work, a glucoamylase secreted in the culture medium, by the ascomycete Aspergillus brasiliensis, was immobilized in Dietilaminoetil Sepharose-Polyethylene Glycol (DEAE-PEG), since immobilized biocatalysts are more stable in long periods of hydrolysis, and can be recovered from the final product and reused for several cycles. Glucoamylase immobilization has shown great thermal stability improvement over the soluble enzyme, reaching 66% more activity after 6?h at 60?°C, and 68% of the activity after 10 hydrolysis cycles. A simplex centroid experimental mixture design was applied as a tool to characterize the affinity of the immobilized enzyme for different starchy substrates. In assays containing several proportions of amylose, amylopectin and starch, the glucoamylase from A. brasiliensis mainly hydrolyzed the amylopectin chains, showing to have preference by branched substrates.     

Field‐based comparison of ligand and coactivator binding sites of nuclear receptors

A structure‐based comparison of the ligand‐binding domains of 35 nuclear receptors from five different subfamilies is presented. Their ligand and coactivator binding sites are characterized using knowledge‐based contact preference fields for hydrophobic and hydrophilic interactions implemented in the MOE modeling environment. Additionally, for polar knowledge‐based field points the preference for negative or positive electrostatic interactions is estimated using the Poisson‐Boltzmann equation. These molecular‐interaction fields are used to cluster the nuclear receptor family based on similarities of their binding sites. By analyzing the similarities and differences of hydrophobic and polar fields in binding pockets of related receptors it is possible to identify conserved interactions in ligand and coactivator binding pockets, which support e.g. design of specific ligands during lead optimization or virtual screening as docking filter. Examples of remarkable similarities between ligand binding sites of members from phylogenetically different nuclear receptor families (RXR, RAR, HNF4, NR5) and differences between closely related subtypes (LXR, RAR, TR) are discussed in more detail. Significant similarities and differences of coactivator binding sites are shown for NR3Cs, LXRs and PPARs. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 884–894, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

An exploratory analysis of the suitability of diets fed to a flightless insectivore,the North Island brown kiwi (Apteryx mantelli), in New Zealand

The ingredients and the macro‐ and micro‐nutrient contents of diets that are fed to captive kiwi at seven New Zealand holding facilities were subject to analysis. The nutrient compositions were compared across facilities and with an estimate of the nutrient composition of the diets of wild kiwi based on the intake of various dietary ingredients reported in the literature. A total of 20 ingredients were used, the number and proportion of these ingredients varied greatly between facilities. Six of the diets were based primarily on meat and three comprised a mixture of meat and cat biscuits. Just one included a proprietary insectivore mix. Nutrient content varied greatly between the seven diets, organic matter ranging from 91.8 to 95.6%, crude protein from 41.9 to 62.9%, and crude fat from 9.0 to 28.7% of dry matter content. Large variations were found in the total content and profile of amino acids and of fatty acids of the diets. The nutrient content of all seven diets differed from that of the natural diet of wild kiwi. Hence, the presence of nutrient deficiencies in the current formulations cannot be excluded. Zoo Biol 29:537–550, 2010. © 2009 Wiley‐Liss, Inc.     

Nitrogen and harvest management of Conservation Reserve Program (CRP) grassland for sustainable biomass feedstock production

The Biomass Regional Feedstock Partnership has identified grasslands planted under the Conservation Reserve Program (CRP) as a potential source for herbaceous bioenergy feedstock. The goal of this project is to assess the yield potential of CRP grasslands across diverse regions. Consistent with that goal, the objective of this project was to establish yield potential and quality parameters for several different CRP grasslands, representative of different growing environments. Standard field scale agricultural practices were used as management guidelines at each location. The test locations were identified and established based on known regions containing concentrated tracts of CRP grassland and represented variable climatic parameters and production histories. Biomass production potential for CRP land dominated by either warm‐ or cool‐season grass mixtures in each location was evaluated over the course of three growing seasons (2008, 2009, and 2010). Specifically, a mixture of warm‐season perennial grasses was evaluated in North Dakota, Kansas, and Oklahoma, while a cool‐season mixture was evaluated in Montana, Georgia, and Missouri. Maximum biomass yields for the three warm‐season CRP sites ranged from 4.0 to 7.2 Mg ha^?1 and for the three cool‐season CRP sites 3.4–6.0 Mg ha^?1. Our results demonstrate that CRP grassland has potential as a bioenergy feedstock resource if the appropriate management practices are followed.     

Efficient high‐throughput biological process characterization: Definitive screening design with the Ambr250 bioreactor system

The burgeoning pipeline for new biologic drugs has increased the need for high‐throughput process characterization to efficiently use process development resources. Breakthroughs in highly automated and parallelized upstream process development have led to technologies such as the 250‐mL automated mini bioreactor (ambr250?) system. Furthermore, developments in modern design of experiments (DoE) have promoted the use of definitive screening design (DSD) as an efficient method to combine factor screening and characterization. Here we utilize the 24‐bioreactor ambr250? system with 10‐factor DSD to demonstrate a systematic experimental workflow to efficiently characterize an Escherichia coli (E. coli) fermentation process for recombinant protein production. The generated process model is further validated by laboratory‐scale experiments and shows how the strategy is useful for quality by design (QbD) approaches to control strategies for late‐stage characterization. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1388–1395, 2015     

High throughput multiplexed method for evaluation of enantioselective performance of chiral selectors by HPLC‐ESI‐MS and dynamic titration: Cinchona alkaloid carbamates discriminating N‐blocked amino acids

Because of the generally different interaction of enantiomers with biological systems, there has been an ever increasing demand for artificial highly enantioselective systems that can facilitate separation processes involved in the research and development of enantiomerically pure drugs. Such systems may be discovered by large‐scale screening of compound libraries which warrants rapid and cost‐efficient screening methods. Here, we demonstrate enantioselectivity determination for systems of cinchona alkaloid carbamates and N‐blocked amino acids using HPLC‐MS and the recently developed dynamic titration technique (Fry?ák P, Schug KA. Anal Chem 2008;80:1385‐1393). A mixture of nine N‐blocked amino acids (either D or L enantiomers) was separated on a reversed‐phase column with cinchona alkaloid carbamates added postcolumn. Dissociation constants of the observed noncovalent complexes were determined from the HPLC‐MS data. Enantioselectivity was then calculated from the dissociation constants, pointing out the best performing systems. For these systems, apparent dissociation constants were measured for the whole range of enantiomeric composition and were shown to obey a proposed theoretical model. Chirality, 2009. © 2009 Wiley‐Liss, Inc.     

Development of a minimal defined medium for recombinant human interleukin-3 production by Streptomyces lividans 66

A systematic approach was developed to identify and optimize the essential amino acids in defined minimal medium for the production of recombinant human interleukin 3 (rHuIL-3) by Streptomyces lividans. Starvation trials were carried out initially to narrow down the number of probable essential amino acids from an initial number of 20 to 8. Then a screening mixture experiment was designed and performed with the eight identified amino acids and distance-based multivariate analysis was employed to rank the probable essential amino acids regarding both growth and product formation. Following this procedure, the search was narrowed to four amino acids (Asp, Leu, Met, and Phe). Finally, a mixture design experiment known as the simplex lattice design was carried out and the composition of the optimum minimal medium was found (Asp 53%, Met 5%, and Phe 42%).     

Identification of tubulins as substrates of serine protease HtrA1 by mixture‐based oriented peptide library screening

Serine protease HtrA1 belongs to a family of chymotrypsin‐like proteases that were first identified in bacteria and later in mammalian systems. These proteases were identified as components of protein quality control in prokaryotic systems and as regulators of diverse signaling pathways in mammalian systems. In particular, HtrA1 is implicated in trophoblast cell migration and invasion, tumor progression, chemotherapy‐induced cytotoxicity, osteoarthritis, age‐related macular degeneration, and pathogenesis of Alzheimer's disease. However, systematic analysis of its potential substrates in biological system is still lacking. Therefore, we performed a mixture‐based oriented peptide library screening to identify putative substrates of HtrA1. We identified [AEGR]‐[LAGR]‐[IAMLR]‐[TVIAL] as consensus residues for P1 to P4 sites. We identified several putative substrates of HtrA1 involved in the pathogenesis of various diseases. In this study, we report on the identification of tubulins as potential substrates of HtrA1, and validated tubulins as in vitro and intracellular substrates of HtrA1. These results provide initial insights into substrate identification and functional characterization of HtrA1 in pathogenesis of various diseases. J. Cell. Biochem. 107: 253–263, 2009. © 2009 Wiley‐Liss, Inc.     

High‐throughput FTIR spectroscopy of intact HepG2 cells reveals additive and non‐additive effects of individual fatty acids when given as mixtures

In the present study we investigated the ability of high‐throughput FTIR spectroscopy in combination with multivariate data analysis to reveal if any combinatory effects of fatty acids in mixture are present in liver HepG2 cell cultures after three days of exposure. For this investigation we used an experimental mixture design containing three different octadecenoic acids (oleic acid: C18 : 1 cis‐ 9, elaidic acid: C18 : 1 trans‐ 9 and vaccenic acid: C18 : 1 trans‐ 11) of a total concentration of 100 μM. The results obtained revealed both additive and non‐additive effects of individual fatty acids when combined in mixtures. Furthermore, we demonstrate by use of scanning electron microscopy that cells are preserved as intact structures ensuring that FTIR measurements are obtained on whole cell keeping cell compounds in their natural surroundings during measurements. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Model‐free feedback design for a mixed cancer therapy

In this article, a model‐free feedback control design is proposed for the drug administration in mixed cancer therapy. This strategy is very attractive because of the important issue of parameter uncertainties unavoidable when dealing with biological models. The proposed feedback scheme use past measurements to update an on‐line simplified model. The control design is then based on model predictive control in which a suitable switching is performed between two different cost functions. The effectiveness of the proposed model‐free control strategy is validated using a recently developed model (unknown to the controller) governing the cancer growth on a cells population level under combined immune and chemotherapy and using real human data. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

大桑菊合剂的提取工艺研究

目的:大桑菊合剂为医院制剂,由桑叶、菊花、薄荷、连翘、鱼腥草等中药经乙醇提取制成,为完善大桑菊合剂的制备工艺,对该制剂的制备工艺进行初步的研究。方法:①实验设计:以加水量、提取时间、提取次数和醇沉浓度为考察因素,各因素均取3水平,采用正交试验L(934)优选制备工艺。②含量测定方法:以大桑菊饮合剂中总黄酮的含量为指标,以芦丁为标准品,利用紫外分光光度计于503nm下测定吸光值,计算样品中总黄酮的含量,以此作为筛选大桑菊合剂最佳制备工艺的重要指标。结果:最佳制备工艺为不浸泡,加8倍水,提取3次,每次1小时。结论:经正交设计优化的大桑菊合剂的中药制备工艺合理、可行。     

A centroid model of species distribution with applications to the Carolina wren Thryothorus ludovicianus and house finch Haemorhous mexicanus in the United States

Drastic shifts in species distributions are a cause of concern for ecologists. Such shifts pose great threat to biodiversity especially under unprecedented anthropogenic and natural disturbances. Many studies have documented recent shifts in species distributions. However, most of these studies are limited to regional scales, and do not consider the abundance structure within species ranges. Developing methods to detect systematic changes in species distributions over their full ranges is critical for understanding the impact of changing environments and for successful conservation planning. Here, we demonstrate a centroid model for range‐wide analysis of distribution shifts using the North American Breeding Bird Survey. The centroid model is based on a hierarchical Bayesian framework which models population change within physiographic strata while accounting for several factors affecting species detectability. Yearly abundance‐weighted range centroids are estimated. As case studies, we derive annual centroids for the Carolina wren and house finch in their ranges in the U.S. We further evaluate the first‐difference correlation between species’ centroid movement and changes in winter severity, total population abundance. We also examined associations of change in centroids from sub‐ranges. Change in full‐range centroid movements of Carolina wren significantly correlate with snow cover days (r = ?0.58). For both species, the full‐range centroid shifts also have strong correlation with total abundance (r = 0.65, and 0.51 respectively). The movements of the full‐range centroids of the two species are correlated strongly (up to r = 0.76) with that of the sub‐ranges with more drastic population changes. Our study demonstrates the usefulness of centroids for analyzing distribution changes in a two‐dimensional spatial context. Particularly it highlights applications that associate the centroid with factors such as environmental stressors, population characteristics, and progression of invasive species. Routine monitoring of changes in centroid will provide useful insights into long‐term avian responses to environmental changes.     

Screening and selection of media components for lactic acid production using Plackett–Burman design

Plackett–Burman design was used to efficiently select important media components influencing lactic acid production in a two step screening procedure. A total of 36 screening experiments were conducted for studying the effect of various media components such as carbon and nitrogen (simple and complex) sources, minerals/buffering agents and a specific inducer for the production of lactic acid by Lactobacillus plantarum NCIM 2084. The eleven ingredients chosen after the first screening experiments were further screened by a Plackett-Burman design consisting of 12 experiments. Liquefied starch, wheat bran extract, ammonium nitrate, manganese sulphate and sodium acetate were chosen as promising ingredients for further optimisation studies. The highest yield of 41.9?g/l of lactic acid was obtained at the end of 24 hours of fermentation which corresponded to 90% conversion, on the basis of sugar supplied.     

Confidence Intervals for Relative Risks in Disease Mapping

Several analysis of the geographic variation of mortality rates in space have been proposed in the literature. Poisson models allowing the incorporation of random effects to model extra‐variability are widely used. The typical modelling approach uses normal random effects to accommodate local spatial autocorrelation. When spatial autocorrelation is absent but overdispersion persists, a discrete mixture model is an alternative approach. However, a technique for identifying regions which have significant high or low risk in any given area has not been developed yet when using the discrete mixture model. Taking into account the importance that this information provides to the epidemiologists to formulate hypothesis related to the potential risk factors affecting the population, different procedures for obtaining confidence intervals for relative risks are derived in this paper. These methods are the standard information‐based method and other four, all based on bootstrap techniques, namely the asymptotic‐bootstrap, the percentile‐bootstrap, the BC‐bootstrap and the modified information‐based method. All of them are compared empirically by their application to mortality data due to cardiovascular diseases in women from Navarra, Spain, during the period 1988–1994. In the small area example considered here, we find that the information‐based method is sensible at estimating standard errors of the component means in the discrete mixture model but it is not appropriate for providing standard errors of the estimated relative risks and hence, for constructing confidence intervals for the relative risk associated to each region. Therefore, the bootstrap‐based methods are recommended for this matter. More specifically, the BC method seems to provide better coverage probabilities in the case studied, according to a small scale simulation study that has been carried out using a scenario as encountered in the analysis of the real data.     

Production of cellulase-free xylanase by a thermotolerant Streptomyces sp. grown on agricultural waste and media optimization using mixture design and Plackett–Burman experimental design methods

Cellulase-free xylanase was produced by Streptomyces sp. Ab106 on finely ground cane bagasse at 55 °C. The optimal medium composition was developed by applying the mixture design and linear mathematical program, and evaluated using the Plackett–Burman experimental design. The best composition of basal medium was found by using the mixture design method. The highest xylanase activity, 10.6 IU, was obtained after 6 days of fermentation in shaked flask at 100 rpm, 55 °C, pH 7. Both experimental designs showed that trace elements induced xylanase production. With fermentation in a 5-l fermenter, xylanase activity of 12.5 IU was achieved.     

Direct prediction of profiles of sequences compatible with a protein structure by neural networks with fragment‐based local and energy‐based nonlocal profiles

Locating sequences compatible with a protein structural fold is the well‐known inverse protein‐folding problem. While significant progress has been made, the success rate of protein design remains low. As a result, a library of designed sequences or profile of sequences is currently employed for guiding experimental screening or directed evolution. Sequence profiles can be computationally predicted by iterative mutations of a random sequence to produce energy‐optimized sequences, or by combining sequences of structurally similar fragments in a template library. The latter approach is computationally more efficient but yields less accurate profiles than the former because of lacking tertiary structural information. Here we present a method called SPIN that predicts Sequence Profiles by Integrated Neural network based on fragment‐derived sequence profiles and structure‐derived energy profiles. SPIN improves over the fragment‐derived profile by 6.7% (from 23.6 to 30.3%) in sequence identity between predicted and wild‐type sequences. The method also reduces the number of residues in low complex regions by 15.7% and has a significantly better balance of hydrophilic and hydrophobic residues at protein surface. The accuracy of sequence profiles obtained is comparable to those generated from the protein design program RosettaDesign 3.5. This highly efficient method for predicting sequence profiles from structures will be useful as a single‐body scoring term for improving scoring functions used in protein design and fold recognition. It also complements protein design programs in guiding experimental design of the sequence library for screening and directed evolution of designed sequences. The SPIN server is available at http://sparks‐lab.org . Proteins 2014; 82:2565–2573. © 2014 Wiley Periodicals, Inc.