2001 EMT

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Enzyme and Microbial Technology 28 (2001) 415– 419

www.elsevier.com/locate/enzmictec

The potential of positively-charged cellulose sponge for malolactic fermentation of wine, using Oenococcus oeni Sergi Maicas*,1, Isabel Pardo, Sergi Ferrer Departament de Microbiologia i Ecologia, Facultat de Biologia, Universitat de Vale`ncia, Burjassot, Spain Received 30 May 2000; received in revised form 31 October 2000; accepted 9 November 2000

Abstract Malolactic fermentation (MLF) is a secondary bioconversion developed in some wines involving malic acid decarboxylation. The induction of MLF in wine by cultures of free and immobilized Oenococcus oeni cells was investigated. This work reports on the effect of surface charges in the immobilization material, a recently described fibrous sponge, as well as the pH and the composition of the media where cells are suspended. A chemical treatment provided positive charge to the sponges (DE or DEAE) and gave the highest cell loadings and subsequent resistance to removal. Preculture media to grow the malolactic bacteria before the immobilization procedure were also evaluated. We have established favorable conditions for growth (Medium of Preculture), suspension solution (Tartrate-Phosphate Buffer), suspension pH (3.5–5.5) and immobilization matrix (DE or DEAE cellulose sponge) to induce MLF in red wine. The use of a semi-continuous system permitted a high-efficiency malic acid conversion by 2 ⫻ 109 cfu sponge⫺1 in at least four subsequent batch fermentations. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Cellulose sponge; Immobilization; Lactic acid bacteria; Leuconostoc oenos; Malic acid; MLF; Red wine

1. Introduction Malolactic fermentation (MLF) is a secondary process that occurs in red wine after alcoholic fermentation and consists of the conversion of L-malate to L-lactate and carbon dioxide. As a consequence of this reaction, the total acidity decreases and the organoleptic properties and biologic stability of the wine are generally improved [1,2]. Oenococcus oeni is the major bacterial species found in wines during the MLF, and is well adapted to the low pH and high ethanol concentration of wine [2,3]. Under natural conditions, the MLF is developed in weeks or months and satisfactory results are not always achieved because of physicochemical or nutritional conditions in the wine [4 – 6]. The probability of obtaining rapid and complete MLF is significantly enhanced by inoculating the wine with high levels of selected strains of O. oeni [7]. Immobilization may increase productivity due to greater * Corresponding author. Tel.: ⫹34-963900022; fax: ⫹34-963636301. E-mail address: sergi.maicas@iata.csic.es (S. Maicas). 1 Present address: Departament de Biotecnologia, Institut d’Agroquı´mica i Tecnologia d’Aliments (CSIC), P.O. Box 73. Burjassot, Vale`ncia, Spain 46100-E.

packing density or provide a more protective environment, and also improve subsequent cell separation. The improvement of immobilization techniques for wine deacidification has long been studied using alginates [8,9], polyacrylamide [6], ␬-carrageenan [10] and oak chips [11] but, nobody has yet tried to immobilize malolactic cells in ion sponge material for this purpose. For beverages, cell immobilization that involves little preparation and no additional chemicals is ideal. By using external surface adsorption, such as on to DEAE cellulose beads, support volume to biomass ratio is high and risk of attrition can restrict process conditions. With encapsulation, mass transfer limitations of nutrients in and/or metabolites out, can lead to pronounced biomass gradients, often with dead or inactive cells in the center. The sponge we have used, which is cellulose based, can be conveniently cut to size and used in packed or fluidized bed configurations. It is also autoclaved without apparent loss of activity [12] and surface properties can be tailored (i.e. made acidic or basic) by addition of functional groups [13]. Scott and O’Reilly [12] used it to co-immobilize Saccharomyces cerevisiae and Lactobacillus plantarum to induce alcoholic and malolactic fermentation in cider. To succeed by inoculating O. oeni in wine to accomplish malolactic fermentation an additional requirement must be satisfied:

0141-0229/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 1 4 1 - 0 2 2 9 ( 0 0 ) 0 0 3 3 9 - 2


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