Immobilization of Microbial Cells for Alcoholic and Malolactic Fermentation of Wine and Cider

  • Yiannis KourkoutasEmail author
  • Verica Manojlović
  • Viktor A. Nedović


Wine- or cider-making is highly associated with biotechnology owing to the traditional nature of must fermentation.. Nowadays, there have been considerable developments in wine- or cider-making techniques affecting all phases of wine or cider production, but more importantly, the fermentation process. It is well-known that the transformation of grape must by microbial activity results in the production of wine, and the fermentation of apples (or sometimes pears) in the production of cider. In this process, a variety of compounds affecting the organoleptic profile of wine or cider are synthesized. It is also common sense that in wine- or cider-making, the main objective is to achieve an adequate quality of the product. The technological progress and the improved quality of the wines or ciders have been associated with the control of technical parameters. Herein, cell immobilization offers numerous advantages, such as enhanced fermentation productivity, ability for cell recycling, application of continuous configurations, enhanced cell stability and viability, and improvement of quality (Margaritis and Merchant 1984; Stewart and Russel 1986; Kourkoutas et al. 2004a).

The objective of the present chapter is to analyze and assess data on the impact of immobilization technologies of viable microbial cells on the alcoholic and malolactic fermentation (MLF) of wine and cider. The immobilized biocatalysts are evaluated for their scale-up ability and their potential future impact in industrial application is highlighted and assessed. Handicaps associated with maintenance of cell viability and fermentation efficiency during preservation and storage, constraining the industrial use of immobilized cell systems are discussed.


Lactic Acid Bacterium Malic Acid Immobilize Cell Immobilize Biocatalyst Malolactic Fermentation 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. Agouridis N, Bekatorou A, Nigam P, Kanellaki M (2005) Malolactic fermentation in wine with Lactobacillus casei cells immobilized on delignified cellulosic material. J Agric Food Chem 53(7):2546–2551CrossRefGoogle Scholar
  2. Argiriou T, Kanellaki M, Voliotis S, Koutinas AA (1996) Kissiris-supported yeast cells: high biocatalytic stability and productivity improvement by successive preservations at 0°C. J Agric Food Chem 44:4028–4031CrossRefGoogle Scholar
  3. Bakoyianis V, Koutinas AA (1996) A catalytic multistage fixed-bed tower bioreactor in an industrial-scale pilot plant for alcohol production. Biotechnol Bioeng 49:197–203CrossRefGoogle Scholar
  4. Bakoyianis V, Kanellaki M, Kaliafas A, Koutinas AA (1992) Low temperature wine making by immobilized cells on mineral kissiris. J Agric Food Chem 40:1293–1296CrossRefGoogle Scholar
  5. Bakoyianis V, Kana K, Kalliafas A, Koutinas AA (1993) Low-temperature continuous wine making by kissiris-supported biocatalyst: Volatile byproducts. J Agric Food Chem 41:465–468CrossRefGoogle Scholar
  6. Bakoyianis V, Koutinas AA, Agelopoulos K, Kanellaki M (1997) Comparative study of kissiris, γ-alumina, and calcium alginate as supports of cells for batch and continuous wine-making at low temperatures. J Agric Food Chem 45:4884–4888CrossRefGoogle Scholar
  7. Bardi EP, Koutinas AA (1994) Immobilization of yeast on delignified cellulosic material for room temperature and low-temperature wine making. J Agric Food Chem 42:221–226CrossRefGoogle Scholar
  8. Bardi EP, Bakoyianis V, Koutinas AA, Kanellaki M (1996) Room temperature and low temperature wine making using yeast immobilized on gluten pellets. Process Biochem 31:425–430CrossRefGoogle Scholar
  9. Bardi E, Koutinas AA, Psarianos C, Kanellaki M (1997) Volatile by-products formed in low-temperature wine-making using immobilized yeast cells. Process Biochem 32:579–584CrossRefGoogle Scholar
  10. Brodelius P, Nilsson K (1983) Permealization of immobilized plant cells, resulting in release of intracellularly stored products with preserved cell viability. Eur J Appl Microbiol Biotechnol 17:275–280CrossRefGoogle Scholar
  11. Busova K, Magyar I, Janky F (1994) Effect of immobilized yeasts on the quality of bottle-fermented sparkling wine. Acta Alimentaria 23:9–23Google Scholar
  12. Buzas Z, Dallmann K, Szajani B (1989) Influence of pH on the growth and ethanol production of free and immobilized Saccharomyces cerevisiae cells. Biotechnol Bioeng 34:882–884CrossRefGoogle Scholar
  13. Cabranes C, Moreno J, Mangas JJ (1998) Cider production with immobilized Leuconostoc oenos. J Inst Brew 104:127–130Google Scholar
  14. Caro LH, Tettelin H, Vossen JH, Ram AF, Van den Ende H, Klis FM (1997) In silicio identification of glycosyl-phosphatidylinositol-anchored plasma-membrane and cell wall proteins of Saccharomyces cerevisae. Yeast 13:1477–1489CrossRefGoogle Scholar
  15. Chen C, Dale MC, Okos MR (1990) Minimal nutritional requirements for immobilized yeast. Biotechnol Bioeng 36:993–1001CrossRefGoogle Scholar
  16. Ciani M, Ferraro L (1996) Enhanced glycerol content in wines made with immobilized Candida stellata cells. Appl Environ Microbiol 62:128–132Google Scholar
  17. Colagrande O, Silva A, Fumi MD (1994) Recent applications of biotechnology in wine production. Biotechnol Prog 10:2–18CrossRefGoogle Scholar
  18. Crapisi A, Nuti MP, Zamorani A, Spettoli P (1987a) Improved stability of immobilized Lactobacillus sp. cells for the control of malolactic fermentation in wine. Am J Enol Vitic 38:310–312Google Scholar
  19. Crapisi A, Spettoli P, Nuti MP, Zamorani A (1987b) Comparative traits of Lactobacillus brevis, Lactobacillus fructivorans and Leuconostoc oenos immobilized cells for the control of malolactic fermentation in wine. J Appl Bacteriol 63:513–521Google Scholar
  20. Diviès C, Siess MH (1976) Étude du catabolisme de l’acide L-malique para Lactobacillus casei emprisonée dans un gel de polyacrylamide. Ann Microbiol (Paris) 127:525–539Google Scholar
  21. Diviès C, Cachon R, Cavin J-F, Prévost H (1994) Theme 4: Immobilized cell technology in wine production. Crit Rev Biotechnol 14:135–153CrossRefGoogle Scholar
  22. Doran PM, Bailey JE (1986) Effects of immobilization on growth, fermentation properties, and macromolecular composition of Saccharomyces cerevisae attached to gelatin. Biotechnol Bioeng 28:73–87CrossRefGoogle Scholar
  23. Ferraro L, Fatichenti F, Ciani M (2000) Pilot scale vinification process using immobilized Candida stellata cells and Saccharomyces cerevisiae. Process Biochem 35:1125–1129CrossRefGoogle Scholar
  24. Fumi MD, Trioli G, Colagrande O (1987) Immobilization of Saccharomyces cerevisiae in calcium alginate for sparkling wine processes. Biotechnol Lett 9:339–342CrossRefGoogle Scholar
  25. Fumi MD, Trioli G, Colombi MG, Colagrande O (1988) Immobilization of Saccharomyces cerevisiae in calcium alginate gel and its application to bottle-fermented sparkling wine production. Am J Enol Vitic 39:267–272Google Scholar
  26. Galazzo J, Bailey JE (1990) Growing Saccharomyces cerevisae in calcium alginate beads induces cell alteration, which accelerate glucose conversion to ethanol. Biotechnol Bioeng 36:417–426CrossRefGoogle Scholar
  27. Galazzo JL, Shanks JV, Bailey JE (1987) Comparision of suspended and immobilized yeast metabolism using 31P Nuclear Magnetic Resonance spectroscopy. Biotechnol Tech 1:1–6CrossRefGoogle Scholar
  28. Herrero M, Laca A, Garcia LA, Díaz M (2001) Controlled malolactic fermentation in cider using Oenococcus oeni immobilized in alginate beads and comparison with free cell fermentation. Enzyme Microb Technol 28:35–41CrossRefGoogle Scholar
  29. Hilge-Rotmann B, Rehm HJ (1990) Comparison of fermentation properties and specific enzyme activities of free and calcium-alginate-entrapped Saccharomyces cerevisiae. Appl Microbiol Biotechnol 33:54–58CrossRefGoogle Scholar
  30. Iconomopoulou M, Psarianos K, Kanellaki M, Koutinas AA (2002) Low temperature and ambient temperature wine making using freeze-dried immobilized cells on gluten pellets. Process Biochem 37:707–717CrossRefGoogle Scholar
  31. Iconomopoulou M, Kanellaki M, Soupioni M, Koutinas AA (2003) Effect of freeze-dried cells on delignified cellulosic material in low-temperature wine making. Appl Biochem Biotechnol 104:23–36CrossRefGoogle Scholar
  32. Iconomou L, Kanellaki M, Voliotis S, Agelopoulos K, Koutinas AA (1996) Continuous wine making by delignified cellulosic materials supported biocatalyst. An attractive process for industrial applications. Appl Biochem Biotechnol 60:303–313CrossRefGoogle Scholar
  33. Jamai L, Sendide K, Ettayebi K, Errachidi F, Hamdouni-Alami O, Tahri-Jouti MA, McDermott T, Ettayebi M (2001) Physiological difference during ethanol fermentation between calcium alginate-immobilized Candida tropicalis and Saccharomyces cerevisiae. FEMS Microbiol Lett 204:375–379CrossRefGoogle Scholar
  34. Kana K, Kanellaki M, Papadimitriou A, Psarianos C, Koutinas AA (1989) Immobilization of Saccharomyces cerevisiae on γ-alumina pellets and its ethanol production in glucose and raisin extract fermentation. J Ferment Bioeng 68:213–215CrossRefGoogle Scholar
  35. Kosseva MR, Kennedy JF (2004) Encapsulated lactic acid bacteria for control of malolactic fermentation in wine. Artif Cells Blood Substit Immobil Biotechnol 32:55–65CrossRefGoogle Scholar
  36. Kosseva M, Beschkov V, Kennedy JF, Lloyd LL (1998) Malolactic fermentation in Chardonnay wine by immobilized Lactobacillus casei cells. Process Biochem 33:793–797CrossRefGoogle Scholar
  37. Kourkoutas Y, Komaitis M, Koutinas AA, Kanellaki M (2001) Wine production using yeast immobilized on apple pieces at low and room temperatures. J Agric Food Chem 49:1417–1425CrossRefGoogle Scholar
  38. Kourkoutas Y, Koutinas AA, Kanellaki M, Banat IM, Marchant R (2002a) Continuous wine fermentation using a psychrophilic yeast immobilized on apple cuts at different temperatures. Food Microbiol 19:127–134CrossRefGoogle Scholar
  39. Kourkoutas Y, Douma M, Koutinas AA, Kanellaki M, Banat IM, Marchant R (2002b) Room and low temperature continuous wine making using yeast immobilized on quince pieces. Process Biochem 39:143–148CrossRefGoogle Scholar
  40. Kourkoutas Y, Kanellaki M, Koutinas AA, Banat IM, Marchant R (2003a) Storage of immobilized yeast cells for use in wine-making at ambient temperature. J Agric Food Chem 51:654–658CrossRefGoogle Scholar
  41. Kourkoutas Y, Komaitis M, Koutinas AA, Kaliafas A, Kanellaki M, Marchant R, Banat IM (2003b) Wine production using yeast immobilized on quince biocatalyst at temperatures between 30 and 0°C. Food Chem 82:353–360CrossRefGoogle Scholar
  42. Kourkoutas Y, Bekatorou A, Banat IM, Marchant R, Koutinas AA (2004a) Immobilization technologies and support materials suitable in alcohol beverages production: a review. Food Microbiol 21:377–397CrossRefGoogle Scholar
  43. Kourkoutas Y, McErlean C, Kanellaki M, Hack CJ, Marchant R, Banat IM, Koutinas AA (2004b) High-temperature wine making using the thermotolerant yeast strain Kluyveromyces cerevisiae IMB3. Appl Biochem Biotechnol 112:25–35CrossRefGoogle Scholar
  44. Koutinas AA, Bakoyianis V, Argiriou T, Kanellaki M, Voliotis S (1997) A qualitative outline to industrialize alcohol production by catalytic multistage fixed bed tower (MFBT) bioreactor. Appl Biochem Biotechnol 66:121–131CrossRefGoogle Scholar
  45. Kunkee RE (1997) A heady concoction of alcoholic and malolactic fermentations. Nat Biotechnol 15:224–225CrossRefGoogle Scholar
  46. Lodato P, Segovia De Huergo M, Buera MP (1999) Viability and thermal stability of a strain of Saccharomyces cerevisiae freeze-dried in different sugar and polymer matrices. Appl Microbiol Biotechnol 52:215–220CrossRefGoogle Scholar
  47. Lonvaud-Funel A (1995) Microbiology of the malolactic fermentation: molecular aspects. FEMS Microbiol Lett 126:209–214CrossRefGoogle Scholar
  48. Loukatos P, Kiaris M, Ligas I, Bourgos G, Kanellaki M, Komaitis M, Koutinas AA (2000) Continuous wine making by γ-alumina-supported biocatalyst. Quality of the wine and distillates. Appl Biochem Biotechnol 89:1–13CrossRefGoogle Scholar
  49. Loukatos P, Kanellaki M, Komaitis M, Athanasiadis I, Koutinas AA (2003) A new technological approach for distillate production using immobilized cells. J Biosci Bioeng 95:35–39Google Scholar
  50. Lovitt R, Jung I, Jones M (2006) The performance of a membrane bioreactor for the malolactic fermentation of media containing ethanol. Desalination 199:435–437CrossRefGoogle Scholar
  51. Lu ZX, Lu FX, Bie XM, Fujimura T (2002) Immobilization of yeast cells with polymeric carrier cross-linked using radiation techniques. J Agric Food Chem 50:2798–2801CrossRefGoogle Scholar
  52. Maicas S, Gil J-V, Pardo I, Ferrer S (1999) Improvement of volatile composition of wines by controlled addition of malolactic bacteria. Food Res Intern 32:491–496CrossRefGoogle Scholar
  53. Maicas S, Pardo I, Ferrer S (2001) The potential of positively charged cellulose sponge for malolactic fermentation of wine, using Oenococcus oeni. Enzyme Microb Technol 28:415–419CrossRefGoogle Scholar
  54. Mallios P, Kourkoutas Y, Iconomopoulou M, Koutinas AA, Psarianos C, Marchant R, Banat IM (2004) Low temperature wine-making using yeast immobilized on pear pieces. J Sci Food Agric 84:1615–1623CrossRefGoogle Scholar
  55. Mallouchos A, Reppa P, Aggelis G, Koutinas AA, Kanellaki M, Komaitis M (2002) Grape skins as a natural support for yeast immobilization. Biotechnol Lett 24:1331–1335CrossRefGoogle Scholar
  56. Mallouchos A, Komaitis M, Koutinas AA, Kanellaki M (2003) Wine fermentation by immobilized and free cells at different temperatures. Effect of immobilization and temperature on volatile by-products. Food Chem 80:109–113CrossRefGoogle Scholar
  57. Mallouchos A, Loukatos P, Bekatorou A, Koutinas A, Komaitis M (2007) Ambient and low temperature wine-making by immobilized cells on brewer’s spent grains: Effect on volatile composition. Food Chem 104(3):918–927CrossRefGoogle Scholar
  58. Margaritis A, Merchant FJA (1984) Advances in ethanol production using immobilized cell systems. Crit Rev Biotechnol 2:339–393Google Scholar
  59. Martynenko NN, Gracheva IM, Sarishvili NG, Zubov AL, El’Registan GI, Lozinsky VI (2004) Immobilization of champagne yeasts by inclusion into cryogels of polyvinyl alcohol: Means of preventing cell release from the carrier matrix. Appl Biochem Microbiol 40:158–164CrossRefGoogle Scholar
  60. McCord JD, Ryu DDY (1985) Development of malolactic fermentation process using immobilized whole cells and enzymes. Am J Enol Vitic 36:214–218Google Scholar
  61. Melzoch K, Rychtera M, Habova V (1994) Effect of immobilization upon the properties and behavior of Saccharomyces cerevisiae cells. J Biotechnol 32:59–65CrossRefGoogle Scholar
  62. Naouri P, Bernet N, Chagnaud P, Arnaud A, Galzy P (1991) Bioconversion of L-malic acid into L-lactic acid using a high compacting multiphase reactor (HCMR). J Chem Technol Biotechnol 51:81–95CrossRefGoogle Scholar
  63. Navarro JM, Durand G (1977) Modification of yeast metabolism by immobilization onto porous glass. Eur J Appl Microbiol 4:243–254CrossRefGoogle Scholar
  64. Nedovic VA, Durieux A, Van Nederveide L, Rosseels P, Vandegans J, Plaisant AM, Simon J-P (2000) Continuous cider fermentation with co-immobilized yeast and Leuconostoc oenos cells. Enzyme Microb Technol 26:834–839CrossRefGoogle Scholar
  65. Nolan AM, Barron N, Brady D, McAree T, McHale L, McHale AP (1994) Ethanol production at 45°C by an alginate-immobilized thermotolerant strain of Kluyveromyces marxianus following growth on glucose-containing media. Biotechnol Lett 16:849–852CrossRefGoogle Scholar
  66. Norton S, D’Amore T (1994) Physiological effects of yeast cell immobilization applications for brewing. Enzyme Microb Technol 16:365–375CrossRefGoogle Scholar
  67. Peinado RA, Moreno JJ, Maestre O, Mauricio JC (2005) Use of a novel immobilization yeast system for winemaking. Biotechnol Lett 27:1421–1424CrossRefGoogle Scholar
  68. Peinado RA, Moreno JJ, Villalba JM, González-Reyes JA, Ortega JM, Mauricio JC (2006) Yeast biocapsules: A new immobilization method and their applications. Enzyme Microb Technol 40:79–84CrossRefGoogle Scholar
  69. Rossi J, Clementi F (1984) L-malic acid catabolism by polyacrylamide gel entrapped Leuconostoc oenos. Am J Enol Vitic 36:100–102Google Scholar
  70. Sakurai A, Nishida Y, Saito H, Sakakibara M (2000) Ethanol production by repeated batch culture using yeast cells immobilized within porous cellulose carriers. J Biosci Bioeng 90:526–529Google Scholar
  71. Scott JA, O’Reilly AM (1996) Co-immobilization of selected yeast and bacteria for controlled flavour development in an alcoholic cider beverage. Process Biochem 31:111–117CrossRefGoogle Scholar
  72. Shindo S, Takata S, Taguchi H, Yoshimura N (2001) Development of a novel carrier using natural zeolite and continuous ethanol fermentation with immobilized Saccharomyces cerevisiae in a bioreactor. Biotechnol Lett 23:2001–2004CrossRefGoogle Scholar
  73. Shirai Y, Hashimoto K, Yamaji H, Kawahara H (1988) Oxygen uptake rate of immobilized growing hybridoma cells. Appl Microbiol Biotechnol 29:113–118CrossRefGoogle Scholar
  74. Shuler ML (1985) Immobilized whole cell bioreactors: potential tools for directing cellular metabolism. World Biotechnol Rep 2:231–239Google Scholar
  75. Silva S, Ramon-Portugal P, Silva P, Texeira MF, Strehaiano P (2002) Use of encapsulated yeast for the treatment of stuck and sluggish fermentations. J Intern des Sci de la vigne et du vin 36:161–168Google Scholar
  76. Simon JP, Durieux A, Pinnel V, Garré V, Vandegans J, Rosseels P, Godan N, Plaisant AM, Defroyennes J-P, Foroni G (1996) Organoleptic profiles of different ciders after continuous fermentation (encapsulated living cells) versus batch fermentation (free cells). In: Wijffels RH, Buitelaar RH, Bucke C, Tramper J (eds) Immobilized cells: Basics and applications. Elsevier BV, Amsterdam, pp 615–621Google Scholar
  77. Sipsas V, Kolokythas G, Kourkoutas Y, Plessas S, Nedovic VA, Kanellaki M (2009) Comparative study of batch and continuous multi-stage fixed-bed tower (MFBT) bioreactor during wine-making using freeze-dried immobized cells. J Food Eng 90:495–503CrossRefGoogle Scholar
  78. Spettoli P, Bottacin A, Nuti MP, Zamorani A (1982) Immobilization of Leuconostoc oenos ML 34 in calcium alginate gels and its application to wine technology. Am J Enol Vitic 33:1–5Google Scholar
  79. Sree NK, Sridhar M, Suresh K, Banat IM, Rao IV (2000) High alcohol production by repeated batch fermentation using an immobilized osmotolerant Saccharomyces cerevisiae. J Ind Microbiol Biotechnol 24:222–226CrossRefGoogle Scholar
  80. Stewart GG, Russel I (1986) One hundred years of yeast research and development in the brewing industry. J Inst Brew 92:537–558Google Scholar
  81. Suzzi G, Romano P, Vannini I, Turbanti L, Domizio P (1996) Cell-recycle batch fermentation using immobilized cells of flocculent Saccharomyces cerevisiae wine strains. World J Microbiol Biotechnol 12:25–27CrossRefGoogle Scholar
  82. Takaya M, Matsumoto N, Yanase H (2002) Characterization of membrane bioreactor for dry wine production. J Biosci Bioeng 93:240–244CrossRefGoogle Scholar
  83. Totsuka A, Hara S (1981) Decomposition of malic acid in red wine by immobilized microbial cells. Hako Kogaku Zasshi 59:231–237Google Scholar
  84. Tsakiris A, Bekatorou A, Psarianos C, Koutinas AA, Marchant R, Banat IM (2004a) Immobilization of yeast on dried raisin berries for use in dry white wine-makin. Food Chem 87:11–15CrossRefGoogle Scholar
  85. Tsakiris A, Sipsas V, Bekatorou A, Mallouchos A, Koutinas AA (2004b) Red wine making by immobilized cells and influence on volatile composition. J Agric Food Chem 53:1357–1363CrossRefGoogle Scholar
  86. Versari A, Parpinello GP, Cattaneo M (1999) Leuconostoc oenos and malolactic fermentation in wine: a review. J Ind Microbiol Biotechnol 23:447–455CrossRefGoogle Scholar
  87. Vijayalakshmi M, Marcipar A, Segard E, Broun GB (1979) Matrix bound transition metal for continuous fermentation tower packing. Ann NY Acad Sci 326:249–254CrossRefGoogle Scholar
  88. Volschenk H, Viljoen M, Grobler J, Petzold B, Bauer F, Subden RE, Young RA, Lonvaud A, Denayrolles M, Van Vuuren HJJ (1997) Engineering pathways for malate degradation in Saccharomyces cerevisiae. Nat Biotechnol 15:253–257CrossRefGoogle Scholar
  89. Walsh PK, Malone DM (1995) Cell growth in immobilization matrices. Biotechnol Adv 13:13–43CrossRefGoogle Scholar
  90. Webb C, Fukuda H, Alkinson B (1986) The production of cellulose in a spouted bed fermentor using cells immobilized in biomass support particles. Biotechnol Bioeng 28:41–50CrossRefGoogle Scholar
  91. Williams SA, Hodges RA, Strike TL, Snow R, Kunkee RE (1984) Cloning the gene for malolactic fermentation of wine from Lactobacillus delbrueckii in Escherichia coli and yeasts. Appl Environ Microbiol 47:288–293Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Yiannis Kourkoutas
    • 1
    Email author
  • Verica Manojlović
    • 2
  • Viktor A. Nedović
    • 3
  1. 1.Department of Molecular Biology and GeneticsDemocritus University of ThraceAlexandroupolisGreece
  2. 2.Department of Chemical EngineeringUniversity of BelgradeBelgradeSerbia
  3. 3.Department of Food Technology and BiochemistryUniversity of BelgradeBelgrade-ZemunSerbia

Personalised recommendations