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HIGHLY EFFICIENT MALOLACTIC FERMENTATION OF RED WINE USING ENCAPSULATED BACTERIA INTO A ROBUST BIOCOMPOSITE OF SILICA-ALGINATE Guillermo Simo, Josefina Vila, Encarnación Fernández, VIOLETA RUIPEREZ, and J.M. Rodriguez-Nogales J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 05 Jun 2017 Downloaded from http://pubs.acs.org on June 8, 2017
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Journal of Agricultural and Food Chemistry
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HIGHLY EFFICIENT MALOLACTIC FERMENTATION OF RED WINE
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USING ENCAPSULATED BACTERIA INTO A ROBUST BIOCOMPOSITE OF
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SILICA-ALGINATE
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Guillermo Simó†, Josefina Vila‐‐Crespo‡, Encarnación Fernández‐‐Fernández†,
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Violeta Ruipérez‡, José Manuel Rodríguez‐‐Nogales†,*
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a
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Agronomic Engineering. Av. Madrid 44, 34071 Palencia, Spain
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b
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Food Technology Area. University of Valladolid. Technical High School of
Microbiology Area. University of Valladolid. Technical High School of Agronomic
Engineering. Av. Madrid 44, 34071 Palencia, Spain
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*Corresponding author
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Telephone number: +34 979108478
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Fax number: +34 979108302
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E‐mail address:
[email protected] 16 17
Running header: Malolactic fermentation of wine using encapsulated bacteria into
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silica-alginate gel
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ABSTRACT: Bacteria encapsulation to develop malolactic fermentation emerges as a
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biotechnological strategy that provides significant advantages over the use of free cells.
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Two encapsulation methods have been proposed embedding Oenococcus oeni, i)
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interpenetrated polymer networks of silica and Ca-alginate and ii) Ca-alginate capsules
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coated with hydrolyzed 3-aminopropyltriethoxysilane (hAPTES). Based on our results,
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only the first method was suitable for bacteria encapsulation. The optimized silica-
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alginate capsules exhibited a negligible bacteria release and an increase of 328% and
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65% in L-malic acid consumption and mechanical robustness, respectively, compared to
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untreated alginate capsules. Moreover, studies of capsule stability at different pH and
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ethanol concentrations in water solutions, and in wine indicated a better behavior of
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silica-alginate capsules than untreated ones. The inclusion of silicates and colloidal
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silica in alginate capsules containing O. oeni improved markedly their capacity to
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deplete the levels of L-malic acid in red wines, and their mechanical robustness and
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stability.
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KEY WORDS: Oenococcus oeni, encapsulation, bacteria, silicates, alginate.
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Journal of Agricultural and Food Chemistry
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INTRODUCTION
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Malolactic fermentation (MLF) in wines is an essential step to improve the
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quality of red wines. Lactic acid bacteria (LAB) and, especially Oenococcus oeni, are
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involved in the transformation of L-malic acid with final production of lactic acid, and
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significantly influence the aromatic complexity of wine.1,2 LAB immobilization has
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recently received some attention in winemaking because it offers several interesting
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advantages over the use of free cells. Some of them are the following: (i)
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immobilization support can improve bacteria protection against adverse conditions of
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MLF (e.g. extreme pH, high ethanol concentration, presence of inhibitory substances,
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and low fermentation temperature); (ii) it allows continuous fermentative processes; (iii)
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it opens the way to bacteria reutilization; (iv) it enhances productivity due to inoculation
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of higher bacteria concentration; and (v) it allows the conduction of MLF with selected
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immobilized bacteria.3–5 Despite the fact that the process of bacterial encapsulation
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increases the cost of the use of bacteria, the reutilization of the immobilized bacteria in
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repeated batches and the possibility of design continuous processes have a positive
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impact on the economic feasibility of MLF of red wine.
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Bacteria immobilization involves physical confinement of cells to a region of
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space, preserving their metabolic activity. Several biotechnological techniques have
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been developed for cell immobilization, such as (i) immobilization on the surface of a
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matrix by ionic, covalent, and/or hydrogen bond interactions; (ii) encapsulation into a
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porous matrix; (iii) cell aggregation with cross-linking reactions or by flocculation; and
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(iv) cell entrapment into organic or microporous synthetic membranes.6–8 Among these
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techniques, cell encapsulation into matrix has received a special attention due to its
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simplicity, low cost, and mild working conditions.9,10 A suitable polymeric material for
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bacteria encapsulation must be able to retain cells and maintain cell viability, and be 3 ACS Paragon Plus Environment
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permeable to gas, nutrients, and metabolites.8 Natural materials (such as alginate,
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carrageenan, chitosan, agarose, pectin, gelatin, and chitin) have been widely applied for
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cell encapsulation because they exhibit these properties and they have low cost and do
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not generally present cell toxic impurities coming from chemical reactions.9 Among
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these materials, alginate is the most common polymer used for cell10–14 and enzyme15–17
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encapsulation.
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Alginate is an ionic polymer composed of 1,4-linked β-D-mannuronic acid and
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α-L-guluronic acid residues in different sequences.18 Divalent cations, mainly Ca2+ and
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Ba2+, added to alginate solutions may lead to cross-linking of alginate molecules
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forming hydrogels compatible with the survival of entrapped cells.19 However, alginate
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gels are sensitive to chelating molecules commonly existing in food, such as citrates,
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phosphates, and lactates, and other anti-gelling cations such as Na+ and Mg2+, reducing
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their chemical stability.18 Moreover, low mechanical robustness of Ca-alginate gels
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discourages their implementation for alcohol beverages production.
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Diverse strategies have been explored to resolve disadvantages of the application
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of alginate gel for cell entrapment. Either incorporation of other polymers and/or fillers
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into alginate structure gel or application of a coating layer to alginate gel have been
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performed to enhance its mechanical and chemical stability, to increase its
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biocompatibility, and to control cell retention.20 Recently, improvement of alginate gel
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stability with silica has been reported by various authors using sol-gel process.21–25 Sol-
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gel process makes possible to design silica-alginate biocomposites, that have the
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advantages of the organic polymers (biocompatibility, flexibility, and elasticity) and the
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inorganic silica component (rigidity, chemical resistance, and thermal stability).23,26 Sol-
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gel process led by alkoxide precursors (Si(OR)4) involves formation of silicic acid
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(SiOH4) upon water hydrolysis with liberation of by-product alcohol (R-OH). Then, 4 ACS Paragon Plus Environment
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formation of siloxane (Si-O-Si) takes place between silanol groups (Si-OH) of two
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molecules. Finally, covalent silica network is obtained by polycondensation of silanols
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and siloxanes.27,28 Biocompatibility of alkoxide route may be compromised by the
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presence of by-product alcohol. An alternative aqueous route with sodium silicate and
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colloidal silica has been proposed allowing the formation of silica gel at neutral pH and
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room temperature, without generation of alcohol as by-product and therefore
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eliminating the risk of cell toxicity.27
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Silica-alginate composites for cell encapsulation have been prepared through
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sol-gel process by polycondensation of silica precursors, ionic cross-linking by
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complexation reaction between carboxyl groups of alginate and divalent cations, and
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intermolecular interaction of silanol/silica with alginate based on hydrogen link.29
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Lately, 3-aminopropyltriethoxysilane (APTES) has been proposed to form silica-coated
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alginate composites via electrostatic interaction between carboxyl groups of alginate
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from the surface of capsules and amino groups of APTES
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structure.31 Recently, alginate-silica encapsulated Oenococcus oeni have been reported
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to perform MLF in wine.24,25 Alginate capsules were first coated with ethanol/water
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solution of tetraethoxysilane (TEOS) and then with methyltriethoxylsilane (MTES) in
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gas phase. MTES-TEOS-alginate capsules loaded with bacteria showed similar
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metabolic activity, and better bacteria leakage reduction and mechanical resistance than
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untreated ones.24 These capsules allowed the development of MLF in wines with
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lysozyme added to eliminate wild LAB and their potential spoilage.25
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, so reinforcing alginate
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Unlike previous works24,25, this study explores two new approaches for O. oeni
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immobilization into silica-alginate biocomposites using aqueous routes based on (i) an
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interpenetrated polymer networks of silica and Ca-alginate and (ii) a coating for Ca-
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alginate capsules with APTES. To our knowledge, there has been no work undertaken 5 ACS Paragon Plus Environment
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to apply these strategies for LAB immobilization into silica-alginate capsules and their
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implementation in MLF. The aim of this work was to optimize the conditions of
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encapsulation of O. oeni into both silica-alginate capsules to perform MLF.
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Fermentation behavior of encapsulated O. oeni, mechanical robustness and ability to
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retain bacteria of silica-alginate capsules were evaluated. Finally, stability of silica-
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alginate capsules at different pH and ethanol concentration in water solutions and in red
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wine was also analyzed.
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MATERIAL AND METHODS
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Immobilization of O. oeni using the interpenetrated polymer network
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method. To immobilize bacteria into interpenetrated polymer networks of silica and
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alginate, a process based on the mixture of derivatives of silicon with sodium alginate
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before gelation in presence of Ca2+ was used.23,32 Sodium silicate (0.00-0.08M) (Sigma-
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Aldrich, USA) and colloidal silica (0.00-1.23M) (Ludox HS40, Sigma-Aldrich)
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concentrations, and immobilization pH (4.0-6.3) were optimized. Thirteen experiments
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were carried out using a factorial design Box-Behnken for the study of linear, quadratic
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and cross-products effects of the three factors each at three levels and three center points
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(#13 was replicated three times) for the estimation of the experimental error (Table 1).
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Response surface models were fitted to three individual responses (L-malic acid
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consumption by encapsulated bacteria in red wine (g L-1), mechanical strength of
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capsules (g), and cell release from capsules in red wine). Statgraphics® centurion XVI
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(v. 16.1.15) software was employed to fit the second-order model to factors
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(independent variables) using a second order polynomial quadratic equation. Statistical
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significance of factors was identified using Pareto diagram (p
