Pectinase production by Aspergillus giganteus in solid-state fermentation: optimization, scale-up, biochemical characterization and its application in olive-oil extraction

  • Gastón E. OrtizEmail author
  • María C. Ponce-Mora
  • Diego G. Noseda
  • Gabriela Cazabat
  • Celina Saravalli
  • María C. López
  • Guillermo P. Gil
  • Martín Blasco
  • Edgardo O. Albertó
Fermentation, Cell Culture and Bioengineering - Original Paper


The application of pectinases in industrial olive-oil processes is restricted by its production cost. Consequently, new fungal strains able to produce higher pectinase titers are required. The aim of this work was to study the capability of Aspergillus giganteus NRRL10 to produce pectinolytic enzymes by SSF and evaluate the application of these in olive-oil extraction. A. giganteus was selected among 12 strains on the basis of high pectinolytic activity and stability. A mixture composed by wheat bran, orange, and lemon peels was selected as the best substrate for enzyme production. Statistical analyses of the experimental design indicated that pH, temperature, and CaCl2 are the main factors that affect the production. Subsequently, different aeration flows were tested in a tray reactor; the highest activity was achieved at 20 L min−1 per kilogram of dry substrate (kgds). Finally, the pectinolytic enzymes from A. giganteus improved the oil yield and rheological characteristics without affecting oil chemical properties.


Solid-state fermentation Pectinase production Aspergillus giganteus Agricultural waste Design of experiment Olive oil Pectinolytic enzyme 



This work was supported by the PICT Start Up 2010-1312 Grant from ANPCyT and MINCyT Argentina (issued to Dr. E. Albertó). We would like to acknowledge Dr. Fernandez-Lahore (Jacobs University Bremen, Germany) for providing A. sojae ATCC 20235 strain. The NRRL strains employed in this work were kindly provided by ARS Culture Collection.

Supplementary material

10295_2016_1873_MOESM1_ESM.pdf (144 kb)
Supplementary material 1 (PDF 143 kb)
10295_2016_1873_MOESM2_ESM.pdf (36 kb)
Supplementary material 2 (PDF 35 kb)
10295_2016_1873_MOESM3_ESM.pdf (550 kb)
Supplementary material 3 (PDF 549 kb)
10295_2016_1873_MOESM4_ESM.pdf (28 kb)
Supplementary material 4 (PDF 28 kb)
10295_2016_1873_MOESM5_ESM.pdf (36 kb)
Supplementary material 5 (PDF 36 kb)
10295_2016_1873_MOESM6_ESM.pdf (481 kb)
Supplementary material 6 (PDF 481 kb)
10295_2016_1873_MOESM7_ESM.pdf (455 kb)
Supplementary material 7 (PDF 455 kb)
10295_2016_1873_MOESM8_ESM.pdf (131 kb)
Supplementary material 8 (PDF 130 kb)
10295_2016_1873_MOESM9_ESM.pdf (29 kb)
Supplementary material 9 (PDF 29 kb)
10295_2016_1873_MOESM10_ESM.pdf (11 kb)
Supplementary material 10 (PDF 10 kb)
10295_2016_1873_MOESM11_ESM.pdf (25 kb)
Supplementary material 11 (PDF 24 kb)
10295_2016_1873_MOESM12_ESM.pdf (10 kb)
Supplementary material 12 (PDF 9 kb)
10295_2016_1873_MOESM13_ESM.pdf (428 kb)
Supplementary material 13 (PDF 427 kb)
10295_2016_1873_MOESM14_ESM.pdf (14 kb)
Supplementary material 14 (PDF 13 kb)
10295_2016_1873_MOESM15_ESM.pdf (862 kb)
Supplementary material 15 (PDF 861 kb)
10295_2016_1873_MOESM16_ESM.pdf (246 kb)
Supplementary material 16 (PDF 245 kb)


  1. 1.
    Kashyap DR, Vohra PK, Chopra S, Tewari R (2001) Applications of pectinases in the commercial sector: a review. Bioresour Technol 77:215–227CrossRefPubMedGoogle Scholar
  2. 2.
    Thakur BR, Singh RK, Handa AK (1997) Chemistry and uses of pectin—a review. Crit Rev Food Sci Nutr 37:47–73CrossRefPubMedGoogle Scholar
  3. 3.
    Mercimek Takcı HA, Ucan Turkmen F (2016) Extracellular pectinase production and purification from a newly isolated Bacillus subtilis strain. Int J Food Prop 2912(10942912):1123270. doi: 10.1080/10942912.2015.1123270 Google Scholar
  4. 4.
    Hadj-Taieb N, Grati N, Ayadi M et al (2012) Optimisation of olive oil extraction and minor compounds content of Tunisian olive oil using enzymatic formulations during malaxation. Biochem Eng J 62:79–85. doi: 10.1016/j.bej.2011.04.003 CrossRefGoogle Scholar
  5. 5.
    Peres F, Martins LL, Branco C et al (2014) Laboratory-scale optimization of olive oil extraction using enzymes and microtalc. Eur J Lipid Sci Technol. doi: 10.1002/ejlt.201400060 Google Scholar
  6. 6.
    Arunachalam C, Saritha K (2009) Protease enzyme: an eco-friendly alternative for leather industry. Indian J Sci Technol 2:29–32Google Scholar
  7. 7.
    Jegannathan KR, Nielsen PH (2013) Environmental assessment of enzyme use in industrial production—a literature review. J Clean Prod 42:228–240. doi: 10.1016/j.jclepro.2012.11.005 CrossRefGoogle Scholar
  8. 8.
    Liguori R, Amore A, Faraco V (2013) Waste valorization by biotechnological conversion into added value products. Appl Microbiol Biotechnol 97:6129–6147. doi: 10.1007/s00253-013-5014-7 CrossRefPubMedGoogle Scholar
  9. 9.
    Patil SR, Dayanand A (2006) Optimization of process for the production of fungal pectinases from deseeded sunflower head in submerged and solid-state conditions. Bioresour Technol 97:2340–2344. doi: 10.1016/j.biortech.2005.10.025 CrossRefPubMedGoogle Scholar
  10. 10.
    Rojas NL, Ortiz GE, Baruque DJ et al (2010) Production of heterologous polygalacturonase I from Aspergillus kawachii in Saccharomyces cerevisiae in batch and fed-batch cultures. J Ind Microbiol Biotechnol. doi: 10.1007/s10295-010-0929-9 PubMedGoogle Scholar
  11. 11.
    Demir H, Göğüş N, Tari C et al (2012) Optimization of the process parameters for the utilization of orange peel to produce polygalacturonase by solid-state fermentation from an Aspergillus sojae mutant strain. Turk J Biol 36:394–404. doi: 10.3906/biy-1104-23 Google Scholar
  12. 12.
    Ortiz GE, Noseda DG, Ponce Mora MC et al (2016) A comparative study of new Aspergillus strains for proteolytic enzymes production by solid state fermentation. Enzyme Res 2016:3016149. doi: 10.1155/2016/3016149 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Heerd D, Yegin S, Tari C, Fernandez-Lahore M (2012) Pectinase enzyme-complex production by Aspergillus spp. in solid-state fermentation: a comparative study. Food Bioprod Process 90:102–110. doi: 10.1016/j.fbp.2011.08.003 CrossRefGoogle Scholar
  14. 14.
    Ruiz HA, Rodríguez-Jasso RM, Rodríguez R et al (2012) Pectinase production from lemon peel pomace as support and carbon source in solid-state fermentation column-tray bioreactor. Biochem Eng J 65:90–95. doi: 10.1016/j.bej.2012.03.007 CrossRefGoogle Scholar
  15. 15.
    Ortiz GE, Guitart ME, Cavalitto SF et al (2015) Characterization, optimization, and scale-up of cellulases production by Trichoderma reesei cbs 836.91 in solid-state fermentation using agro-industrial products. Bioprocess Biosyst Eng 38:2117–2128. doi: 10.1007/s00449-015-1451-2 CrossRefPubMedGoogle Scholar
  16. 16.
    Hankin L, Anagnostakis SL (1975) The use of solid media for detection of enzyme production by fungi. Mycol 67:597–607. doi: 10.2307/3758395 CrossRefGoogle Scholar
  17. 17.
    Mora-Lugo R, Madrigal M, Yelemane V, Fernandez-Lahore M (2015) Improved biomass and protein production in solid-state cultures of an Aspergillus sojae strain harboring the Vitreoscilla hemoglobin. Appl Microbiol Biotechnol 99:9699–9708. doi: 10.1007/s00253-015-6851-3 CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Miller GL (1959) Use of dinitrosalicyclic reagent for determination of reducing sugar. Anal Chem 31:426–438CrossRefGoogle Scholar
  19. 19.
    Ortiz GE, Guitart ME, Albertó E et al (2014) Microplate assay for endo-polygalacturonase activity determination based on ruthenium red method. Anal Biochem 454C:33–35. doi: 10.1016/j.ab.2014.02.028 CrossRefGoogle Scholar
  20. 20.
    Lynch JM, Barbano DM (1999) Kjeldahl nitrogen analysis as a reference method for protein determination in dairy products. J AOAC Int 82:1389–1398PubMedGoogle Scholar
  21. 21.
    Aldermann GA, Morgan DE, Harvard A, Edwards RE, Todd JR (1975) Energy allowances and feeding systems for ruminants. Minist Agric Fish Food Tech Bull 33:34–36Google Scholar
  22. 22.
    Uceda M, Frías L (1975) Epocas de recolección. Evolución del contenido graso del fruto y de la composición y calidad del aceite (Seasons of harvest. Changes on fruit oil content, oil composition and oil quality). Proceeding II Semin. Oleícola IntGoogle Scholar
  23. 23.
    International Olive Council (2015) Spectrophotometric investigation in the ultraviolet. COI/T.20/D:1–10Google Scholar
  24. 24.
    Mignani AG, Ciaccheri L, Mencaglia AA, Cimato A (2012) Optical absorption spectroscopy for quality assessment of extra virgin olive oil. Olive Oil-Const Qual Heal Prop Bioconvers. doi: 10.5772/1378 Google Scholar
  25. 25.
    Nighojkar S, Phanse Y, Sinha D et al (2006) Production of polygalacturonase by immobilized cells of Aspergillus niger using orange peel as inducer. Process Biochem 41:1136–1140. doi: 10.1016/j.procbio.2005.12.009 CrossRefGoogle Scholar
  26. 26.
    Ustok FI, Tari C, Gogus N (2007) Solid-state production of polygalacturonase by Aspergillus sojae ATCC 20235. J Biotechnol 127:322–334. doi: 10.1016/j.jbiotec.2006.07.010 CrossRefPubMedGoogle Scholar
  27. 27.
    Contreras Esquivel JC, Voget CE (2004) Purification and partial characterization of an acidic polygalacturonase from Aspergillus kawachii. J Biotechnol 110:21–28. doi: 10.1016/j.jbiotec.2004.01.010 CrossRefPubMedGoogle Scholar
  28. 28.
    Pedrolli DB, Carmona EC (2009) Pectin lyase from Aspergillus giganteus: comparative study of productivity of submerged fermentation on citrus pectin and orange waste. Appl Biochem Microbiol 45:610–616. doi: 10.1134/S0003683809060064 CrossRefGoogle Scholar
  29. 29.
    Baladhandayutham S, Thangavelu V (2011) Optimization and kinetics of solid-state fermentative production of pectinase by Aspergillus awamori. Int J ChemTech Res 3:1758–1764Google Scholar
  30. 30.
    Mrudula S, Anitharaj R (2011) Pectinase production in solid state fermentation by Aspergillus niger using orange peel as substrate. Glob J Biotechnol Biochem 6:64–71Google Scholar
  31. 31.
    Alcântara SR, da Silva FL, Leite NJ (2013) Scale up of polygalacturonase production by solid state fermentation process. doi: 10.5772/53152.
  32. 32.
    Galiotou-Panayotou M, Kapantai M, Kalantzi O (1997) Growth conditions of Aspergillus sp. ATHUM-3482 for polygalacturonase production. Appl Microbiol Biotechnol 47:425–429. doi: 10.1007/s002530050951 CrossRefPubMedGoogle Scholar
  33. 33.
    Hours RA, Voget CE, Ertola RJ (1988) Some factors affecting pectinase production from apple pomace in solid-state cultures. Biol Wastes 24:147–157. doi: 10.1016/0269-7483(88)90057-2 CrossRefGoogle Scholar
  34. 34.
    Larroche C, Moksia J, Gros J et al (1998) A convenient method for initial dry weight determination in samples from solid state cultivations. Process Biochem 33:447–451CrossRefGoogle Scholar
  35. 35.
    Zheng Z, Shetty K (2000) Solid state production of polygalacturonase by Lentinus edodes using fruit processing wastes. Process Biochem 35:825–830. doi: 10.1016/S0032-9592(99)00143-0 CrossRefGoogle Scholar
  36. 36.
    Silva D, Martins ES, Leite RSR et al (2007) Purification and characterization of an exo-polygalacturonase produced by Penicillium viridicatum RFC3 in solid-state fermentation. Process Biochem 42:1237–1243. doi: 10.1016/j.procbio.2007.05.025 CrossRefGoogle Scholar
  37. 37.
    Martínez-Trujillo A, Arreguín-Rangel L, García-Rivero M, Aguilar-Osorio G (2011) Use of fruit residues for pectinase production by Aspergillus flavipes FP-500 and Aspergillus terreus FP-370. Lett Appl Microbiol 53:202–209. doi: 10.1111/j.1472-765X.2011.03096.x CrossRefPubMedGoogle Scholar
  38. 38.
    Rodríguez-Fernández DE, Rodríguez-León JA, de Carvalho JC et al (2011) The behavior of kinetic parameters in production of pectinase and xylanase by solid-state fermentation. Bioresour Technol 102:10657–10662. doi: 10.1016/j.biortech.2011.08.106 CrossRefPubMedGoogle Scholar
  39. 39.
    Cabeza MS, Baca FL, Puntes EM et al (2011) Selection of psychrotolerant microorganisms producing cold-active pectinases for biotechnological processes at low temperature. Food Technol Biotechnol 49:187–195Google Scholar
  40. 40.
    Pedrolli DB, Gomes E, Monti R, Carmona EC (2008) Studies on productivity and characterization of polygalacturonase from Aspergillus giganteus submerged culture using citrus pectin and orange waste. Appl Biochem Biotechnol 144:191–200CrossRefPubMedGoogle Scholar
  41. 41.
    Schols HA, Geraeds CCJM, Searle-van Leeuwen MF et al (1990) Rhamnogalacturonase: a novel enzyme that degrades the hairy regions of pectins. Carbohydr Res 206:105–115. doi: 10.1016/0008-6215(90)84010-R CrossRefGoogle Scholar
  42. 42.
    Aliakbarian B, De Faveri D, Converti A, Perego P (2008) Optimisation of olive oil extraction by means of enzyme processing aids using response surface methodology. Biochem Eng J 42:34–40. doi: 10.1016/j.bej.2008.05.006 CrossRefGoogle Scholar
  43. 43.
    Najafian L, Ghodsvali A, Haddad Khodaparast MH, Diosady LL (2009) Aqueous extraction of virgin olive oil using industrial enzymes. Food Res Int 42:171–175. doi: 10.1016/j.foodres.2008.10.002 CrossRefGoogle Scholar
  44. 44.
    Clodoveo ML, Dipalmo T, Schiano C et al (2014) What’s now, what’s new and what’s next in virgin olive oil elaboration systems? A perspective on current knowledge and future trends. J Agric Eng 45:49–59. doi: 10.4081/jae.2014.193 CrossRefGoogle Scholar
  45. 45.
    Mateos RG-MJA (2006) Rapid and quantitative extraction method for the determination of chlorophylls and carotenoids in olive oil by high-performance liquid chromatography. Anal Bioanal Chem 385:1247–1254CrossRefPubMedGoogle Scholar
  46. 46.
    Vierhuis E, Korver M, Schols HA, Voragen AG (2003) Structural characteristics of pectic polysaccharides from olive fruit (Olea europaea cv moraiolo) in relation to processing for oil extraction. Carbohydr Polym 51:135–148. doi: 10.1016/S0144-8617(02)00158-3 CrossRefGoogle Scholar

Copyright information

© Society for Industrial Microbiology and Biotechnology 2016

Authors and Affiliations

  • Gastón E. Ortiz
    • 1
    Email author
  • María C. Ponce-Mora
    • 1
  • Diego G. Noseda
    • 1
  • Gabriela Cazabat
    • 2
  • Celina Saravalli
    • 2
  • María C. López
    • 2
  • Guillermo P. Gil
    • 2
  • Martín Blasco
    • 3
  • Edgardo O. Albertó
    • 1
  1. 1.Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico de Chascomús (IIB-INTECH)Universidad Nacional de San Martín (UNSAM)-Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET)San MartínArgentina
  2. 2.Centro de Investigación y Desarrollo en Tecnologías de Industrialización de AlimentosInstituto Nacional de Tecnología IndustrialSan MartínArgentina
  3. 3.Centro de Investigación y Desarrollo en Biotecnología IndustrialInstituto Nacional de Tecnología IndustrialSan MartínArgentina

Personalised recommendations