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.

Lipase

• Triglycerides hydrolysis • Biosynthesis: Esterification, Interesterification, etc. • Detergents, Biodiesel, Cosmetics, Food processment

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

Experimental Matrix

Wheat BranSoybean

MealCottonseed

Meal

Lipase Activity (U/mL)

Raw Cost (R$/kg)

Productivity (U/R$)

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

Nutrients Correlation

0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.000.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

f(x) = 0.418649110910553 x + 4.17669982517546R² = 0.736504491832492

f(x) = − 0.00214428398546514 x + 5.53275313014926R² = 0.000264885852191643

f(x) = 0.00849898003736057 x + 5.04735868010881R² = 0.00411201856358012

CahrbohydrateLinear (Cahrbo-hydrate)ProteinLinear (Protein)LipidLinear (Lipid)

Concentration (%)

Lipa

se A

ctivi

ty (U

/mL)

Lipase Activity (U/mL)

Productivity (U/R$)

Lipase Activity (U/mL)

Productivity (U/R$)

ANOVA

Effects – Lipase Activity

Productivity (U/R$)

Lipase Activity (U/mL)

Productivity (U/R$)

Optimization by Desirability Function

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

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.

Food Biochemistry Laboratory, Food Science Dept., School of Food Engineering, UNICAMP

Facebook: Bioquímica-FEA/UNICAMP