Optimization of Solid Fermentation Medium Composition by Simplex Centroid Mixture Design Method for Lipase Production using a New Penicillium sp. and Agroindustrial Subproducts as Substrates Camilo Teixeira, Débora Moreira, Paula Speranza, Gabriela Macedo Food Science Department, School of Food Engineering (FEA), University of Campinas (UNICAMP)
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Optimization of Solid Fermentation Medium Composition by Simplex Centroid Mixture
Design Method for Lipase Production using a New Penicillium sp. and Agroindustrial
Subproducts as Substrates
Camilo Teixeira, Débora Moreira, Paula Speranza, Gabriela MacedoFood Science Department, School of Food Engineering (FEA), University
of Campinas (UNICAMP)
Introduction
Aims to develop clean and efficient processes for biomass conversion into low-cost and
sustainable biofuels, chemicals and pharmaceuticals.
Biocatalysis: Lipases and its potential applications
• Agroindustrial byproducts as Fermentation Medium;• Lower energy cost than submerged fermentation;• Considered as a green technology
Solid state fermentation (SSF) enzyme production
Objectives
• Feedstock selection for SSF lipase production: wheat bran, soybean meal and cottonseed meal;• Evaluate the best formulation by the Simplex Centroid Mixture Design
(SCMD) • Simultaneous optimization of lipase activity and productivity by the
desirability function;
• Soybean meal and cottonseed meal were supplied by Bunge Brazil (Rondonópolis, Mato Grosso, Brazil) • Wheat bran was purchased in local Market (Campinas, São
Paulo, Brazil)• The Penicillium sp. strain was isolated by Dr. Luciana Fleuri
(UNESP – Botucatu, São Paulo, Brazil)
Materials
Experimental Methodology
Pre-inoculum
Inoculum
SSF
Enzyme Extraction
Enzyme Activity
Penicillium sp.
Pre-inoculum Penicillium sp.
Foto tubos
PDA plate 30ºC
96 hours
Inoculum
5mL esterilized water for slant
Inoculum Solution
Solid State Fermentation
Solid State Fermentation 250mL erlenmeyer with cotton-wool plug1 mL of inoculum solution10g of different mixtures of WB, SM and CSM10g water (1:1 mass:water)
Climatic Chamber96 Hours 30ºC 90% humidity
Enzyme Extraction
2 hours 100mL Distilled water
Lipase Hydrolytic Activity (Titrimetric Method)1 mL filtrate30 minutes 37ºC pH 7 Olive oil + Arabic Gum + Phosphate buffer pH 7Stopped with etanol: acetone solution NaOH 0.05 M
Phenolphthalein 1%
Sample – Control (without enzyme)= [fatty acid]1 U = 1 µmol oleic acid/minute
• Determine the best formulation for a mixture of components;• Wheat Bran, Soybean Meal and Cottonseed Meal as independent
variables • Lipase Activity (U/mL) and Productivity (U/R$) as response variables;• Evaluate the best mixture and possible interactions with a positive
synergistic effect for the response variables;
Experimental Design by Simplex Centroid Mixture Method