Food and Bioprocess Technology

, Volume 12, Issue 9, pp 1559–1572 | Cite as

Multi-enzymatic Systems Immobilized on Chitosan Beads for Pomegranate Juice Treatment in Fluidized Bed Reactor: Effect on Haze-Active Molecules and Chromatic Properties

  • Ilaria Benucci
  • Caterina Mazzocchi
  • Claudio LombardelliEmail author
  • Ilaria Cacciotti
  • Marco Esti
Original Paper


In this study, two different food-grade enzymes (i.e., bromelain from a pineapple stem (protease) and Pectinex® BE XXL (pectinase)) were successfully immobilized on chitosan beads and their application in pomegranate juice clarification was evaluated. The immobilization procedure was optimized for maximizing the specific activity of biocatalysts, and the best performance was reached using an immobilization solution containing 1.0 mgBSAeq/mL (for protease) and 1.8 mgBSAeq/mL (for pectinase). The biocatalysts were combined in a multi-enzymatic system and used in a fluidized bed reactor, varying the protease-to-pectinase ratio (1:2 or 1:4) and the treatment time (4 h or 8 h). The process carried out using the protease-to-pectinase ratio 1:2, for 8 h, was the most suitable in terms of immediate (− 49%) and potential (− 70%) turbidity depletion compared with the untreated juice, after 21 days. At the end of the storage period, this biotechnological approach allowed a significant reduction of haze-active molecules. All the enzymatically treated juices better preserved the anthocyanin pattern compared with the untreated juice over time. The best supplied treatment allowed better retaining the native chromatic properties of juice, preserving it from colloidal instability as well as from the possible related color degradation tendency.


Pomegranate juice clarification Covalent immobilization Chitosan from A. niger Multi-enzymatic treatment Fluidized bed reactor 


Funding Information

This work was financially supported by the BioEnBi project “Biotecnologie enzimatiche innovative per processi di chiarifica sostenibili nel settore birrario” (Grant 85-2017-15362), funded by Lazio Innova Spa, Lazio Region (Italy), in the context of Progetti Gruppi di Ricerca, Lazio Innova 2018–2020.


  1. Albersheim, P. (1966). Pectin lyase from fungi. Methods in Enzymology, 8, 628–631.CrossRefGoogle Scholar
  2. Alper, N., & Acar, J. (2004). Removal of phenolic compounds in pomegranate juices using ultrafiltration and laccase-ultrafiltration combinations. Nahrung/Food, 48(3), 184–187.CrossRefPubMedGoogle Scholar
  3. Aviram, M., Rosenblat, M., Gaitini, D., Nitecki, S., Hoffman, A., Dornfeld, L., Volkova, N., Presser, D., Attias, J., Liker, H., & Hayek, T. (2004). Pomegranate juice consumption for 3 years by patients with carotid artery stenosis reduces common carotid intima-media thickness, blood pressure and LDL oxidation. Clinical Nutrition, 23(3), 423–433.CrossRefPubMedGoogle Scholar
  4. Baiano, A., Terracone, C., Gambacorta, G., & La Notte, E. (2009). Phenolic content and antioxidant activity of Primitivo wine: comparison among winemaking technologies. Journal of Food Science, 74, 258–267.CrossRefGoogle Scholar
  5. Bavaro, T., Cattaneo, G., Serra, I., Benucci, I., Pregnolato, M., & Terreni, M. (2016). Immobilization of neutral protease from Bacillus subtilis for regioselective hydrolysis of acetylated nucleosides: application to capecitabine synthesis. Molecules, 21, 1621.Google Scholar
  6. Bell, C., & Hawthorne, S. (2008). Ellagic acid, pomegranate and prostate cancer: a minireview. Journal of Pharmacy and Pharmacology, 60(2), 139–144.CrossRefPubMedGoogle Scholar
  7. Benucci, I., Lombardelli, C., Cacciotti, I., Liburdi, K., Nanni, F., & Esti, M. (2016). Chitosan beads from microbial and animal sources as enzyme supports for wine application. Food Hydrocolloids, 61, 191–200.CrossRefGoogle Scholar
  8. Birò, E., Nemeth, A. S., Sisak, C., Feczko, T., & Gyenis, J. (2008). Preparation of chitosan particles suitable for enzyme immobilization. Journal of Biochemical and Biophysical Methods, 70(6), 1240–1246.CrossRefPubMedGoogle Scholar
  9. Bradford, M. M. (1976). A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binging. Analytical Biochemistry, 72(1-2), 248–254.CrossRefGoogle Scholar
  10. Busto, M. D., García-Tramontín, K. E., Ortega, N., & Perez-Mateos, M. (2006). Preparation and properties of an immobilized pectinlyase for the treatment of fruit juices. Bioresource Technology, 97(13), 1477–1483.CrossRefPubMedGoogle Scholar
  11. Cacciotti, I., Lombardelli, C., Benucci, I., & Esti, M. (2019). Clay/chitosan biocomposite systems as novel green carriers for covalent immobilization of food enzymes. Journal of Materials Research and Technology, In press, 8(4), 3644–3652.CrossRefGoogle Scholar
  12. Cao, L. (2005). Covalent enzyme immobilization (2 Eds). In Carrier-bound immobilized enzymes (pp. 169–316). KGaA: WILEY-VCH Verlag GmbH & Co.CrossRefGoogle Scholar
  13. Cappannella, E., Benucci, I., Lombardelli, C., Liburdi, K., Bavaro, T., & Esti, M. (2016). Immobilized lysozyme for the continuous lysis of lactic bacteria in wine: bench-scale fluidized-bed reactor study. Food Chemistry, 210, 49–55.CrossRefPubMedGoogle Scholar
  14. Cerreti, M., Liburdi, K., Benucci, I., & Esti, M. (2016). The effect of pectinase and protease treatment on turbidity and on haze active molecules in pomegranate juice. LWT-Food Science and Technology, 73, 326–333.CrossRefGoogle Scholar
  15. Cerreti, M., Liburdi, K., Benucci, I., Emiliani Spinelli, S., Lombardelli, C., & Esti, M. (2017). Optimization of pectinase and protease clarification treatment of pomegranate juice. LWT -Food Science and Technology, 82, 58–65.CrossRefGoogle Scholar
  16. Chen, H., Liu, L., Lv, S., Liu, X., Wang, M., Song, A., & Ji, X. (2010). Immobilization of Aspergillus niger xylanase on chitosan using dialdehyde starch as a coupling agent. Applied Biochemistry and Biotechnology, 162(1), 24–32.CrossRefPubMedGoogle Scholar
  17. Chuang, W. Y., Young, T. H., Yao, C. H., & Chiu, W. Y. (1999). Properties of the poly(vinyl alcohol)/chitosan blend and its effect on the culture of fibroblast in vitro. Biomaterials, 20(16), 1479–1487.CrossRefPubMedGoogle Scholar
  18. Deng, Z., Wang, F., Zhou, B., Li, J., Li, B., & Liang, H. (2019). Immobilization of pectinases into calcium alginate microspheres for fruit juice application. Food Hydrocolloids, 89, 691–699.CrossRefGoogle Scholar
  19. Di Cosimo, R., McAuliffe, J., Poulose, A. J., & Bohlman, G. (2013). Industrial use of immobilized enzymes. Chemical Society Reviews, 42, 6437–6474.CrossRefGoogle Scholar
  20. Di Stefano, R., & Guidoni, S. (1989). Metodi per lo studio dei polifenoli dei vini. L' Enotecnico, 25, 81–89.Google Scholar
  21. Erkan, M. (2011). Pomegranate (Punica granatum L.). In E. M. Yahia (Ed.), Postharvest biology and technology of tropical and subtropical fruits (pp. 287–311). Oxford: Woodhead Publishing Limited.CrossRefGoogle Scholar
  22. Erkan-Koç, B., Türkyılmaz, M., Yemis¸, O., & Ozkan, M. (2015). Effects of various protein- and polysaccharide-based clarification agents on antioxidative compounds and colour of pomegranate juice. Food Chemistry, 184, 37–45.CrossRefPubMedGoogle Scholar
  23. Gardossi, L., Ebert, C., Ferrario, V., Braiuca, P., Basso, A., & Vaccari, L. (2008). Immobilizzazione di enzimi: ottimizzazione di biocatalizzatori industriali. La chimica e l’industria, 1, 94–100.Google Scholar
  24. Glories, Y. (1984). La couleur des vins rouges. Mesure, origine et interprétation. Partie I. Connaissance de la Vigne et du Vin, 18, 195–217.Google Scholar
  25. Grassin, C., & Fauquembergue, P. (1996). Application of pectinases in beverages. Progress in Biotechnology, 14, 453–462.Google Scholar
  26. Gulec, H. A., Bagci, P. O., & Bagci, U. (2017). Clarification of apple juice using polymeric ultrafiltration membranes: a comparative evaluation of membrane fouling and juice quality. Food and Bioprocess Technology, 10(5), 875–885.CrossRefGoogle Scholar
  27. Hale, L. P., Greer, P. K., Trinh, C. T., & James, C. L. (2005). Proteinase activity and stability of natural bromelain preparations. International Immunopharmacology, 5, 783–793.CrossRefPubMedGoogle Scholar
  28. Jana, A., Halder, S. K., Ghosh, K., Paul, T., Vágvölgyi, C., Mondal, K. C., & Mohapatra, P. K. D. (2015). Tannase immobilization by chitin-alginate based adsorption-entrapment technique and its exploitation in fruit juice clarification. Food and Bioprocess Technology, 8(11), 2319–2329.CrossRefGoogle Scholar
  29. Jiang, D. S., Long, S. Y., Huang, J., Xiao, H. Y., & Zhou, J. Y. (2005). Immobilization of Pycnoporus sanguineus laccase on magnetic chitosan microspheres. Biochemical Engineering Journal, 25(1), 15–23.CrossRefGoogle Scholar
  30. Kalaycıoğlu, Z., & Erim, F. B. (2016). Total phenolic contents, antioxidant activities, and bioactive ingredients of juices from pomegranate cultivars worldwide. Food Chemistry, 221, 496–507.CrossRefPubMedGoogle Scholar
  31. Karangwa, E., Hayat, K., Rao, L., Nshimiyimana, D. S., Foh, M. B. K., Li, L., Ntwali, J., Raymond, L. V., Xia, S., & Zhang, X. (2012). Improving blended carrot-orange juice quality by the addition of cyclodextrins during enzymatic clarification. Food and Bioprocess Technology, 5(6), 2612–2617.CrossRefGoogle Scholar
  32. Kashyap, D. R., Chandra, S., Kaul, A., & Tewari, R. (2000). Production, purification and characterization of pectinase from a Bacillus sp. DT7. World Journal of Microbiology and Biotechnology, 16(3), 277–282.CrossRefGoogle Scholar
  33. Laudani, C. G., Habulin, M., Knez, Z., Della Porta, G., & Reverchon, E. (2007). Immobilized lipase-mediated long-chain fatty acid esterification in dense carbon dioxide: bench-scale packed-bed reactor study. The Journal of Supercritical Fluids, 41(1), 74–81.CrossRefGoogle Scholar
  34. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227(5259), 680–685.Google Scholar
  35. Miao, C., Zhonghong, L., Jianlong, W., Wupeng, G., Tianli, Y., Ronghua, L., et al. (2012). Food related applications of magnetic iron oxide nanoparticles: enzyme immobilization, protein purification, and food analysis. Trends in Food Science & Technology, 27, 47–56.CrossRefGoogle Scholar
  36. Miller, G. L. (1959). Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry, 31(3), 426–428.CrossRefGoogle Scholar
  37. Mirzaaghaei, M., Goli, S. A. H., & Fathi, M. (2016). Application of sepiolite in clarification of pomegranate juice: changes on quality characteristics during process. International Journal of Food Science & Technology, 51(7), 1666–1673.CrossRefGoogle Scholar
  38. O’Halloran, J., O’Sullivan, M., & Casey, E. (2019). Production of whey-derived DPP-IV inhibitory peptides using an enzymatic membrane reactor. Food and Bioprocess Technology, 12(5), 799–808.CrossRefGoogle Scholar
  39. Onsekizoglu, P. (2013). Production of high-quality clarified pomegranate juice concentrate by membrane processes. Journal of Membrane Science, 442, 264–271.CrossRefGoogle Scholar
  40. Petenzi, M., Bavaro, T., Cornaggia, C., Ubiali, D., Pregnolato, M., & Pasini, D. (2012). Synthesis, post-modification and characterization of linear polystyrene-based supports for interaction with immobilized biocatalysts. Polymer International, 61, 1611–1618. Google Scholar
  41. Pinelo, M., Zeuner, B., & Meyer, A. S. (2010). Juice clarification by protease and pectinase treatments indicates new roles of pectins and proteins in cherry juice turbidity. Food and Bioproducts Processing, 88(2-3), 259–265.CrossRefGoogle Scholar
  42. Reddy, M. K., Gupta, S. K., Jacob, M. R., Khan, S. I., & Ferreira, D. (2007). Antioxidant, antimalarial and antimicrobial activities of tannin-rich fractions, ellagitannins and phenolic acids from Punica granatum L. Planta Medica, 53, 461–467.CrossRefGoogle Scholar
  43. Ribéreau-Gayon, P., Glories, Y., Maujean, A., & Dubourdieu, D. (2000). The chemistry of wine. Stabilization and treatments. In Handbook of enology volume 2 (2nd ed.). Chichester: John Wiley & Sons Ltd..Google Scholar
  44. Sakurai, K., Maegawa, T., & Takahashi, T. (2000). Glass transition temperature of chitosan and miscibility of chitosan/poly (N-vinyl pyrrolidone) blends. Polymer, 41(19), 7051–7056.CrossRefGoogle Scholar
  45. Saponjić, S., Knežević-Jugović, Z. D., Bezbradica, D. I., Zuza, M. G., Saied, O. A., Bosković Vragolović, N., & Mijin, D. Z. (2010). Use of Candida rugosa lipase immobilized on sepabeads for the amylcaprylate synthesis: batch and fluidized bed reactor study. Electronic Journal of Biotechnology, 13, 1–15.CrossRefGoogle Scholar
  46. Sepúlveda, E., Sáenz, C., Peña, A., Robert, P., Bartolomé, B., & Gómez-Cordovés, C. (2010). Influence of the genotype on the anthocyanin composition, antioxidant capacity and color of Chilean pomegranate (Punica granatum L.) juices. Chilean Journal of Agricultural Research, 70, 50–57.CrossRefGoogle Scholar
  47. Siebert, K. J. (2006). Haze formation in beverages. LWT - Food Science and Technology, 39(9), 987–994.CrossRefGoogle Scholar
  48. Siebert, K. J., Carrasco, A., & Lynn, P. Y. (1996). Formation of protein-polyphenol haze in beverages. Journal of Agricultural and Food Chemistry, 44(8), 1997–2005.CrossRefGoogle Scholar
  49. Sorrivas, V., Genovese, D. B., & Lonzano, J. E. (2006). Effect of pectinolytic and amylolitic enzymes on apple juice turbidity. Journal of Food Processing and Preservation, 36, 118–133.CrossRefGoogle Scholar
  50. Tischer, W., & Wedekind, F. (1999). Immobilised enzymes: methods and applications. In W. D. Fessner et al. (Eds.), Biocatalysis-from discovery to application. Topics in current chemistry (pp. 95–126). Berlin: Springer.CrossRefGoogle Scholar
  51. Turfan, O., Turkyilmaz, M., Yemis, O., & Ozkan, M. (2011). Anthocyanin and colour changes during processing of pomegranate (Punica granatum L. cv. Hicaznar) juice from sacs and whole fruit. Food Chemistry, 129(4), 1644–1651.CrossRefGoogle Scholar
  52. Turfan, O., Turkyilmaz, M., Yemis, O., & Ozkan, M. (2012). Effects of clarification and storage on anthocyanins and color of pomegranate juice concentrates. Journal of Food Quality, 35(4), 272–282.CrossRefGoogle Scholar
  53. Tzulker, R., Glazer, I., Bar-Ilan, I., Holland, D., Aviram, M., & Amin, R. (2007). Antioxidant activity, polyphenol content and related compounds in different fruit juices and homogenates prepared from 29 different pomegranate accessions. Journal of Agriculture and Food Chemestry, 55(23), 9559–9570.CrossRefGoogle Scholar
  54. Vardin, H., & Fenercioglu, H. (2003). Study on the development of pomegranate juice processing technology: clarification of pomegranate juice. Food/Nahrung, 47(5), 300–303.CrossRefPubMedGoogle Scholar
  55. Vincenzi, S., Marangon, M., Tolin, S., & Curioni, A. (2011). Protein evolution during the early stages of white winemaking and its relations with wine stability. Australian Journal of Grape and Wine Research, 17(1), 20–27.CrossRefGoogle Scholar
  56. Zeng, M., Fang, Z., & Xu, C. (2004). Effect of compatibility on the structure of the microporous membrane prepared by selective dissolution of chitosan/synthetic polymer blend membrane. Journal of Membrane Science, 230(1-2), 175–181.CrossRefGoogle Scholar
  57. Zhao, L., Wang, Y., Qiu, D., & Liao, X. (2014). Effect of ultrafiltration combined with high-pressure processing on safety and quality features of fresh apple juice. Food and Bioprocess Technology, 7(11), 3246–3258.CrossRefGoogle Scholar
  58. Zhou, G. X., Chen, G. Y., & Yan, B. B. (2014). Biodiesel production in a magnetically stabilized, fluidized bed reactor with an immobilized lipase in magnetic chitosan microspheres. Biotechnology Letters, 36(1), 63–68.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Agriculture and Forestry Science (DAFNE)Tuscia UniversityViterboItaly
  2. 2.Department of EngineeringUniversity of Rome “Niccolo Cusano”RomeItaly

Personalised recommendations