Apresentação slaca camilo

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Apresentação slaca camilo

  1. 1. 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)
  2. 2. Introduction Aims to develop clean and efficient processes for biomass conversion into low-cost and sustainable biofuels, chemicals and pharmaceuticals.
  3. 3. Biocatalysis: Lipases and its potential applications • Triglycerides hydrolysis • Biosynthesis: Esterification, Interesterification, etc. • Detergents, Biodiesel, Cosmetics, Food processment Lipase
  4. 4. Solid state fermentation (SSF) enzyme production • Agroindustrial byproducts as Fermentation Medium; • Lower energy cost than submerged fermentation; • Considered as a green technology
  5. 5. 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;
  6. 6. Materials • 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)
  7. 7. Experimental Methodology Pre-inoculum Inoculum SSF Enzyme Extraction Enzyme Activity
  8. 8. Penicillium sp.
  9. 9. Pre-inoculum Penicillium sp. 96 hours PDA plate 30ºC Foto tubos
  10. 10. Inoculum 5mL esterilized water for slant Solid State Fermentation Inoculum Solution
  11. 11. Solid State Fermentation 250mL erlenmeyer with cotton-wool plug 1 mL of inoculum solution 10g of different mixtures of WB, SM and CSM 10g water (1:1 mass:water) Climatic Chamber 96 Hours 30ºC 90% humidity
  12. 12. Enzyme Extraction 100mL Distilled water 2 hours
  13. 13. Lipase Hydrolytic Activity (Titrimetric Method) 1 mL filtrate 30 minutes 37ºC pH 7 Olive oil + Arabic Gum + Phosphate buffer pH 7 Stopped with etanol: acetone solution NaOH 0.05 M Phenolphthalein 1% Sample – Control (without enzyme)= [fatty acid] 1 U = 1 µmol oleic acid/minute
  14. 14. Experimental Design by Simplex Centroid Mixture Method • 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$) as response variables; • Evaluate the best mixture and possible interactions with a positive synergistic effect for the response variables;
  15. 15. Experimental Matrix Wheat Bran Soybean Meal Cottonseed Meal Lipase Activity (U/mL) 1 1.00 0.00 0.00 3.31 0.45 73599.24 2 0.00 1.00 0.00 4.27 0.86 49691.91 3 0.00 0.00 1.00 6.78 0.61 111216.20 4 0.50 0.50 0.00 4.86 0.655 74215.44 5 0.50 0.00 0.50 5.72 0.53 107845.51 6 0.00 0.50 0.50 6.25 0.735 85034.01 7 0.33 0.33 0.33 6.68 0.64 104333.60 8 0.67 0.17 0.17 4.65 0.545 85274.05 9 0.17 0.67 0.17 5.45 0.75 72649.57 10 0.17 0.17 0.67 6.68 0.625 106837.61 Raw Cost Productivity (R$/kg) (U/R$)
  16. 16. Nutrients Correlation 8.00 7.00 y = -0.0021x + 5.5328 R² = 0.0003 Lipase Activity (U/mL) 6.00 5.00 4.00 y = 0.0085x + 5.0474 R² = 0.0041 y = 0.4186x + 4.1767 r=0.85 3.00 Cahrbohydrate 2.00 Protein 1.00 Lipid 0.00 0.00 10.00 20.00 30.00 40.00 Concentration (%) 50.00 60.00 70.00
  17. 17. Lipase Activity (U/mL)
  18. 18. Productivity (U/R$)
  19. 19. ANOVA Lipase Activity (U/mL) Productivity (U/R$)
  20. 20. Effects – Lipase Activity
  21. 21. Productivity (U/R$)
  22. 22. Lipase Activity (U/mL)
  23. 23. Productivity (U/R$)
  24. 24. Optimizatio n by Desirability Function
  25. 25. Experimental Validation • Triplicate in the optimum conditions: • 100% cottonseed meal, 1:1 mass:water, 30ºC, 96 hours Lipase activity (U/mL) (- 95% conf) (+95% conf) Observed 9.9 8.9 10.4 Predicted 7.46 5.7 9.2 Productivity (U/R$) (- 95% conf) (+95% conf) Observed 162295.082 145901.6393 170491.8033 Predicted 110916.7 89390.5 132442.8
  26. 26. Conclusions • Lipase Activity is directly correlated with feedstock lipid concentration; • Cottonseed Meal have the highest lipid concentration, lipase activity and productivity. • The mixture of soybean meal, cottonseed meal and wheat bran did have a small synergistic positive effect • The desirability function can optimize simultaneously the lipase activity and the productivity responses.
  27. 27. Food Biochemistry Laboratory, Food Science Dept., School of Food Engineering, UNICAMP Facebook: Bioquímica-FEA/UNICAMP

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