Optimisation of asarone removal from Piper sarmentosum Roxburgh leaves using supercritical carbon dioxide extraction: the Box-Behnken design

Abstract Piper sarmentosum is a herbaceous shrub with numerous pharmacological benefits. However, the presence of two toxic phenylpropanoids (α- and β-asarone) limits the medicinal usage of the plant. In this study, the extraction of three asarone isomers, namely α-, β-, and -asarone was optimised using supercritical carbon dioxide extraction (SC-CO2) combined with Box-Behnken experimental design. Comparison of asarone contents in different conventional solvent extracts of P. sarmentosum leaves prior to and after SC-CO2 extraction was performed. The SC-CO2 method successfully maximised the extraction of α-, β-, and ɣ-asarone at P = 81.16 bar, T = 50.11°C, and t = 80.90 min, yielding 13.91% α-asarone, 3.43% β-asarone, and 14.95% ɣ-asarone. The SC-CO2 residue of the leaves re-extracted with conventional solvents showed a significant decrease of asarone ranging from 45% to 100% (p<0.001) compared to their counterparts without SC-CO2 treatment. α-, β-, and ɣ-asarone were completely removed in the ethanol extract of the residue. These findings suggested that the optimised SC-CO2 extraction parameters may serve as a quick treatment step for the selective removal of asarone from P. sarmentosum to develop safer extracts for the food and nutraceutical industries applications.

Optimisation of topical antibacterial preparation from Malaysian kelulut honey by using xanthan gum as polymeric agent

@#The study aims to formulate and optimise topical antibacterial preparation by using Malaysian kelulut honey as the active ingredient and xanthan gum as the polymeric agent. Response surface methodology was used to optimise the preparation. The acidity, honey concentration and xanthan gum concentration were the independent variables. The zone of inhibitions on S. aureus ATCC6538 and E. coli ATCC8739 were the response variables. The optimal preparation was evaluated on its physicochemical properties, viscosity, antibacterial efficacy and stability. The antibacterial efficacy of the optimal preparation was compared to the commercially antibacterial gel (MediHoney™, Comvita). The optimal preparation was formulated at pH of 3.5, honey concentration of 90% (w/v) and xanthan gum concentration of 1.5% (w/v) with the inhibition zones measured on S. aureus ATCC6538 was 16.2 mm and E. coli ATCC8739 was 15.8 mm respectively. The factors of acidity and honey concentration have significantly influenced the inhibition zone on S. aureus ATCC6538 and E. coli ATCC8739. The utilisation of xanthan gum as the polymeric agent was fit for the preparation which showed by adequate physicochemical properties and retained of the antibacterial effects. This was supported by constant viscosity and efficacy of the preparation within the six months of stability study indicating stable and reliable preparation. Xanthan gum is a potential polymeric agent due to its effective use in preparing stable preparation with effective antibacterial properties.

Immobilised Phytase Production from Aspergillus foetidus MTCC 11682 Using an Optimized Media

Aim: Immobilised fungal phytase production from the novel strain Aspergillus foetidus MTTC 11682 and optimisation of cultural conditions for a better and continuous economic yield. Study Design: The study was designed based on the classical method of changing one independent variable while fixing all other at a certain level- one factor at a time, a close ended system for the optimisation of fermentation process. Methodology: Physical and nutritional parameters were optimised for phytase production and subjected to statistical analysis. Adsorption and Entrapment techniques were employed to immobilise the production strain. Results: The optimum physical conditions for augmenting the yield up to 6 days incubation period were as follows: pH of 3.5, 30ºC temperatures and 5% inoculum size. Amongst the nutritional parameters, lactose and sodium nitrate were found to be the best carbon and nitrogen sources. K++, Mg++, Mn++ and Fe++ ions supported the phytase production. TritonX 100 and tween 80 showed an inducing effect on the secretion of phytase enzyme. Immobilised fungal phytase production resulted in an increased yield of 32.5% with poly urethane foam (PUF) as the matrix. A scale up fermentation resulted in an activity of 52.7 FTU/mL for immobilised cells as compared to 25.5 FTU/mL by its free counterpart. Conclusion: Phytase produced in an optimised media employing immobilised Aspergillus foetidus 11682 on poly urethane foam cubes exhibited better phytase activity, improved stability and long shelf life.

Optimisation of polymerase chain reaction conditions for detection of mineralization markers in isolated odontoblasts from human teeth

The present study aimed to determine the best polymerase chain reaction (PCR) conditions for amplification of odontoblast markers; alkaline phosphatase (ALP), dentin matrix protein 1 (DMP1), dentin sialophosphoprotein (DSPP) and osteopontin (OPN). Informed consent was obtained from the individuals prior to tooth extraction. RNA was extracted from odontoblasts obtained from extracted teeth using innuPREP RNA Mini kit (Analytik Jena, Germany). Five selected target factors in enhancing PCR: primer concentration, extension time, number of cycles, annealing time, and annealing temperature were manipulated to yield the correct size of amplicons. One step reverse transcriptase PCR reactions were performed using MyTaq One-Step RT-PCR kit (Bioline, USA) with a C1000 Thermal Cycler (Bio-Rad, USA) in a 25 µL reaction, keeping the amount of 2 ng/µL RNA, 0.25 µL reverse transcriptase, 0.5 µL RiboSafe Rnase inhibitor and 1X MyTaq One-Step Mix, constant. The optimal conditions were determined to be 400nM of primers for DMP1 and DSPP, 200 nM for ALP and OPN; 30 seconds of extension time and 35 PCR cycles for all genes; 10 seconds of annealing time for ALP, DMP1 and DSPP, 7 seconds for OPN. The annealing temperature were 56.4°C for ALP, 58.6°C for DMP1, 52.7°C for DSPP, and 56.3°C for OPN, respectively. The optimized PCR protocols produced the correct size of odontoblast markers.

A public health risk assessment for yellow fever vaccination: a model exemplified by an outbreak in the state of São Paulo, Brazil

We propose a method to analyse the 2009 outbreak in the region of Botucatu in the state of São Paulo (SP), Brazil, when 28 yellow fever (YF) cases were confirmed, including 11 deaths. At the time of the outbreak, the Secretary of Health of the State of São Paulo vaccinated one million people, causing the death of five individuals, an unprecedented number of YF vaccine-induced fatalities. We apply a mathematical model described previously to optimise the proportion of people who should be vaccinated to minimise the total number of deaths. The model was used to calculate the optimum proportion that should be vaccinated in the remaining, vaccine-free regions of SP, considering the risk of vaccine-induced fatalities and the risk of YF outbreaks in these regions.

Effects of carbon and nitrogen sources on bacteriocin-inhibitory activity of postbiotic metabolites produced by Lactobacillus plantarum I-UL4

Aims: Postbiotic metabolites are metabolic compounds produced by probiotic lactic acid bacteria. These compounds produced by Lactobacillus sp. have been shown to be effective substitutes for in-feed antibiotic in livestock due to their broad inhibitory activity. Therefore, the aim of this study was to determine the effects of various carbon and nitrogen sources on the bacteriocin-inhibitory activity of postbiotic metabolites produced by Lactobacillus plantarum I-UL4. Methodology and results: The effects of various combinations of carbon and nitrogen sources on the bacteriocininhibitory activity (expressed as modified bacteriocin activity, MAU/mL) of postbiotic metabolites produced by L. plantarum I-UL4 were determined in basal media without micronutrients. The combination of glucose (20 g/L) and yeast extract (22 g/L) gave the best bacteriocin-inhibitory activity as compared to other combinations. Maximum bacteriocininhibitory activity of 1440 MAU/mL was achieved when 36.20 g/L of yeast extract was added as the sole nitrogen source in modified de Man, Rogosa and Sharpe (MRS) medium. The glucose concentration was further optimised to enhance the bacteriocin-inhibitory activity of the postbiotic metabolites. Lower bacteriocin-inhibitory activity was observed at 5, 10, 15 and 40 g/L in comparison to 20 g/L of glucose. Conclusion, significance and impact of study: Maximum bacteriocin-inhibitory activity of postbiotic metabolites was achieved at 1440 MAU/mL when 20 g/L of glucose and 36.20 g/L of yeast extract were added as the sole carbon and nitrogen sources respectively in the modified MRS medium. Optimisation of other micronutrients present in MRS media is necessary to further enhance the bacteriocin-inhibitory activity of postbiotic metabolites produced by L. plantarum IUL4.

Application of statistical experimental design for optimisation of bioinsecticides production by sporeless Bacillus thuringiensis strain on cheap medium

In order to overproduce bioinsecticides production by a sporeless Bacillus thuringiensis strain, an optimal composition of a cheap medium was defined using a response surface methodology. In a first step, a Plackett-Burman design used to evaluate the effects of eight medium components on delta-endotoxin production showed that starch, soya bean and sodium chloride exhibited significant effects on bioinsecticides production. In a second step, these parameters were selected for further optimisation by central composite design. The obtained results revealed that the optimum culture medium for delta-endotoxin production consists of 30 g L-1 starch, 30 g L-1 soya bean and 9g L-1 sodium chloride. When compared to the basal production medium, an improvement in delta-endotoxin production up to 50% was noted. Moreover, relative toxin yield of sporeless Bacillus thuringiensis S22 was improved markedly by using optimised cheap medium (148.5 mg delta-endotoxins per g starch) when compared to the yield obtained in the basal medium (94.46 mg delta-endotoxins per g starch). Therefore, the use of optimised culture cheap medium appeared to be a good alternative for a low cost production of sporeless Bacillus thuringiensis bioinsecticides at industrial scale which is of great importance in practical point of view.

β 1,4-Xylosidase Production Potential of a Fusarium sp. on Wood Shavings.

Aims: This study was aimed at screening fungal isolates from degrading wood samples for β 1,4-xylosidase production, selecting the best isolate based on the screening test to produce the enzyme through solid state fermentation of some wood shavings and determining optimum production conditions for the fungus on best substrate. Place and Duration of Study: Microbial physiology laboratory, Department of Microbiology, University of Ibadan, Ibadan and Multi Discplinary Central laboratory University of Ibadan, Oyo State Nigeria. Between September 2010 and October 2011. Methodology: Isolates obtained from degrading wood samples were identified and screened on agar plates of para–nitrophenyl β-xyloside as carbon source. Selected isolates were used to produce β 1,4-xylosidase using shavings of Anogeissus leiocarpus, Gmelina arborea and Terminalia superba moistened with a chemically defined medium as substrates. Assay was done every 3 days for 15 days. Production dependent parameters such as pH, temperature were varied on the best wood substrate for β 1,4-xylosidase production by selected fungus. Results: Selected isolate was used for enzyme production on shavings of Anogeissus leiocarpus, Gmelina arborea and Terminalia superba as substrates, each was moistened with chemically defined medium. Assay was done every 3 days for 15 days. Production dependent parameters such as pH, temperature were varied on the best wood substrate for β 1,4-xylosidase production by fungus. Maximum β 1,4-xylosidase activity of fungus on each substrate was; Anogeissus leiocarpus, 31.620Ug-1 on 12th day, G. arborea, 8.935U g-1 on day 9 and T. superba, 6.053Ug-1 on 6th day. Optimum β 1,4-xylosidase production was obtained on A. leiocarpus at 35ºC and pH 6 with 40% and 60% aeration. 50% moisture content of substrate supplemented with soy meal as nitrogen source supported the best β 1,4-xylosidase production by fungus. Conclusion: Enzyme production was highly enhanced at optimum conditions.

Application of Box-Behnken Design for the Optimization of Citric Acid Production from Corn Starch Using Aspergillus niger.

Citric acid production from hydrolysed corn starch was optimized in this study. Response surface methodology (RSM) was employed for the analysis of the simultaneous effect of substrate concentration, broth pH and fermentation temperature on the concentration of citric acid produced during fermentation of hydrolysed corn starch. A three-variable, threelevel Box-Behnken design (BBD) comprising 15 experimental runs was used to develop a second degree statistical model for the optimisation of the fermentation conditions. The optimal fermentation conditions that resulted in the maximum citric acid concentration were substrate concentration; 50 g/L, broth pH; 2.00 and fermentation temperature; 25ºC. Under these conditions, the concentration of citric acid was obtained to be 31.96 g/L. Validation of the model indicated no difference between predicted and observed values.

Optimización de un medio de cultivo para la producción de la levadura Pichia onychis (Lv027) / Optimising culture medium for producing the yeast Pichia onychis (Lv027)

La optimización de la producción masiva de la levadura Pichia onychis fue investigada usando varios sustratos y evaluando diferentes condiciones físico-químicas de fermentación líquida. Inicialmente se realizó un screening aplicando el diseño estadístico Plackett-Burman para evaluar tres fuentes de carbono y ocho fuentes de nitrógeno (tanto orgánicas como inorgánicas) con el fin de seleccionar los factores nutricionales más influyentes sobre el crecimiento de la levadura. Posteriormente, se evaluaron cuatro fuentes nutricionales y dos variables físico-químicas utilizando un diseño factorial fraccionado como punto de partida, para llevar a cabo después un proceso de optimización aplicando un diseño estadístico Central Compuesto Rotacional. Un modelo de regresión polinomial se desarrolló usando los datos experimentales; los resultados muestran que la producción de biomasa fue afectada significativamente por condiciones tanto nutricionales como físico-químicas del medio de cultivo; el máximo rendimiento obtenido fue de 8,95 XlO9 células/mL equivalentes a una biomasa seca de 6,30 g/L, el cual se logró con las siguientes condiciones: 43,42 g/L de fuente de carbono, 0,261 g/L de fuente de nitrógeno orgánica, agitación de 110 rpm, pH = 6,0 con un tiempo de fermentación de 48 horas.

Optimising Pichia onychis yeast biomass production was evaluated using different substrates and different physicochemical conditions for liquid fermentation. The Plackett-Burman statistical design was initially applied for screening the most important nutritional variables (three carbon sources and eight nitrogen sources) affecting yeast biomass production. Four nutritional sources and two physicochemical variables were subsequently evaluated using a factorial fractionated design as the starting point for optimising the process by applying a central composite rotational design. The results obtained from employing a polynomial regression model using the experimental data showed that biomass production was strongly affected bynutritional and physicochemical conditions. The highest yield was obtained in the following conditions: 43,42 g/L carbon source, 0,261 g/L nitrogen organic source, shaking at 110 rpm, 6,0 pH, 48 h total fermentation time during which 8,95 XlO9 cells/mL were obtained, equivalent to 6,30 g/L dry biomass.