2001 AMB

Appl Microbiol Biotechnol (2001) 56:35–39 DOI 10.1007/s002530100662

MINI-REVIEW

S. Maicas

The use of alternative technologies to develop malolactic fermentation in wine

Received: 8 January 2001 / Received revision: 5 March 2001 / Accepted: 5 March 2001 / Published online: 29 May 2001 © Springer-Verlag 2001

Abstract The development of the malolactic fermentation, bioconversion of L-malic acid to L-lactic acid, is a difficult and time-consuming process that does not always proceed favorably under the natural conditions of wine. Traditional fermentations are used worldwide to produce high-quality wines, although delay or failure is not an unusual outcome. During recent years several technologies have been proposed to induce biological deacidification of wines by using malolactic bacteria, principally Oenococcus oeni and Lactobacillus sp. These alternative technologies usually involve the use of high densities of cells or enzymes, free or immobilized onto different matrices. Immobilization materials, several types of bioreactors, and the properties of many specific systems are discussed in this review.

Introduction Annual global wine production is about 28,000 metric tonnes (t) according to the statistical survey conducted by the United Nations Food and Agriculture Organization (FAO). European countries (mainly France, Italy, and Spain) lead the ranking, producing together about 20,000 t. The United States (2500 t) is the other largescale wine producer, although there are a huge number of other countries reporting wine production (http://www.fao.org).

Yeasts conduct the alcoholic fermentation in wine, mainly converting sugars to ethanol and carbon dioxide.

Malolactic fermentation (MLF) is an important secondary reaction in winemaking and generally occurs just after the alcoholic fermentation has been completed. It involves the growth of particular species of lactic acid bacteria (Lactobacillus, Pediococcus, Leuconostoc and, S. Maicas (✉) Departament de Biotecnologia, Institut d’Agroquímica i Tecnologia d’Aliments, CSIC, Paterna, P.O. Box 73, Burjassot, València, Spain 46100 e-mail: sergi.maicas@iata.csic.es Tel.: +34-6-3900022, Fax: +34-6-3636301

principally, Oenococcus). The first reason for inducing MLF is deacidification of the wine. The degradation of malic acid (dicarboxylic) during MLF to lactic acid (monocarboxylic) results in a drop in titratable acidity and a small increase in pH. In addition, MLF influences the microbiological stability and organoleptic quality of the wine (Davis et al. 1988; Kunkee 1997). Oenococcus oeni (formerly Leuconostoc oenos) (Dicks et al. 1995) is the major bacterial species found in wines during MLF, and is well adapted to the low pH and high ethanol concentration of wine (Wibowo et al. 1985). Knowledge about O. oeni and MLF has been reviewed by Wibowo et al. (1985), Henick-Kling (1993), and van Vuuren and Dicks (1993).

Discoveries of MLF in wines of various regions of the world spread with the application of technology to the practice of winemaking. From a geographical point of view, traditional MLF occurs in all wine-producing countries. It is generally related to areas of cold climate, where the decrease in acidity allows the commercialization of wines, and also to warm zones, where the pH of the wine is so high that it is difficult to prevent the fermentation (Kunkee 1967a, b). Several examples of vineries promoting MLF are found in the USA (Beelman et al. 1980; Henick-Kling et al. 1989; Delaquis et al.

2000), France (Lafon-Lafourcade 1983; de Revel et al.

1999), Spain (Sieiro et al. 1990; Pardo and Zúñiga 1992), Italy (Spettoli et al. 1987; Viti et al. 1996), and Australia (Costello et al. 1985; Davis et al. 1985). Although no official data are available about the prevalence of induction of MLF in wines, roughly 75% of commercial red wines and 40% of white wines have undergone MLF (Ough 1992). Promotion of MLF in wines requires some little extra cost for the winemaker, both in starter production and, chiefly, in fermentation time. We estimate that induction of MLF adds about 1–5% to the overall production costs, although no official data about economic aspects have been reported. Several starter cultures are available, while the time required to reduce the malic acid depends on several factors such as the ambient temperature, pH, the presence of nutrients, or initial

36 Table 1 Examples of alternative methods studied to induce malolactic fermentation in wine. Figures in parentheses represent working volumes (CSTR continuous stirred tank reactor, CCR cell recycle continuous stirred reactor, FBR fluidized bed reactor, nr no reported data) Bacterium or enzyme

Immobilization supporta

Reactor (V)

Initial L-malic Bioconversion Operation acid (g l–1) rateb (%) period (h)

Reference

Lactobacillus sp.

Lactobacillus sp.

Lactobacillus sp.

Lb. brevis Lb. casei Oenococcus oeni Oenococcus oeni Oenococcus oeni Oenococcus oeni Oenococcus oeni Oenococcus oeni Oenococcus oeni Oenococcus oeni Oenococcus oeni Oenococcus oeni Oenococcus oeni Oenococcus oeni Oenococcus oeni Oenococcus oeni Oenococcus oeni Oenococcus oeni Malolactic enzyme (O. oeni)

– Calcium alginate κ-Carrageenan κ-Carrageenan Polyacrylamide – – – – – – Calcium alginate Calcium alginate Calcium alginate Calcium alginate Calcium alginate Cellulose sponge κ-Carrageenan κ-Carrageenan Oak chips Polyacrylamide –

CSTR (350 hl) FBR (1.4 l) CSTR (0.5 l) Shake flask (5 ml) Airflow (nr) 350 hl Screw tubes (5 ml) CCR (0.3 l) Screw tubes (10 ml) CSTR (0.3 l) Screw tubes (10 ml) CSTR (2.7 l) FBR (1.4 l) CSTR (30 ml) CSTR (60 ml) CSTR (nr)d Shake flask (0.1 l) CSTR (0.5 l) Shake flask (5 ml) CSTR (0.1 l) Shake flask (5 ml) Membrane reactor (5 ml)

4.0 0.9 9.0 5.00 48.0 4.0 1.0–6.0 0.8–4.6 5.0 4.2 3.5–7.0 8.0 0.9 0.3 1.5 4.5 3.5 9.0 5.0 8.0 2.3–4.5 18.6–23.9

Caillet and Vayssier (1984) Naouri et al. (1991) Crapisi et al. (1987a) McCord and Ryu (1985) Clementi (1990) Caillet and Vayssier (1984) Gao and Fleet (1994) Gao and Fleet (1995) Lafon-Lafourcade (1970) Maicas et al. (1999b) Maicas et al. (2000) Cuenat and Villetaz (1984) Naouri et al. (1991) Shieh and Tsay (1990) Spettoli et al. (1982) Spettoli et al. (1987) Maicas et al. (2001) Crapisi et al. (1987b) McCord and Ryu (1985) Janssen et al. (1993) Rossi and Clementi (1984) Formisyn et al. (1997)

a Matrix used when immobilizing cells or enzymes b Amount of L-malic acid consumed/initial amount of L-malic

62.0–75.0 45.0 64.0 71.4 80.0–100.0 75.0–100.0 94.0–99.4 25.0–100.0 77.0 92.0–95.0 41.0–98.0 97.0–100.0 82.0 100.0 60.0 98.7 50.0 36.3 100.0 20.0–58.0 71.0 62.0–75.0

24 72 200 1 360 24 6 125 5–48 500 24–500 17 72 12 864 360 96 48 1 264 1 168

acid

numbers of bacteria. These costs are affordable to winemakers given that consumers will pay for quality wines with low levels of malic acid. Advances in starter production and the use of alternative technologies for MLF reduce these extra costs, which are absorbed into the overall costs of the wine production.

However, MLF depends upon the growth of lactic acid bacteria in the wine as a batch culture and is strongly influenced by environmental conditions, so that the process is often delayed or fails. Those problems have driven the search for alternative technologies that enable more rapid and reliable MLF to take place (Gao and Fleet 1995). The induction of MLF with high levels of cells enables better control of the process as well as guaranteeing that it takes place at the required time during the wine production (Gao and Fleet 1994; Formisyn et al. 1997; Maicas et al. 1999b). Several studies have demonstrated the possibility of controlling MLF by using a reactor with immobilized cells or enzymes (Table 1). The use of a reactor presents numerous advantages to the traditional deacidification of wines: starter cultures can be reused, the production of secondary products can be controlled, and the fermentation can be induced and halted at the moment desired by the winemaker. Moreover, the sensory quality of the wine is not adversely affected by this technology.

The overall objective of this review, therefore, is to analyze and assess the data available in the literature on the impact of processing parameters on the operational stability of free and immobilized systems for developing MLF in wine, which have been but little reviewed

(Colagrande et al. 1994; Diviès et al. 1994a). By contrast, the use of immobilized lactic acid bacteria in the dairy industry has been amply reported by Champagne et al. (1994) and Diviès et al. (1994b).

Use of high biomasses of free cells The use of high cell densities has long been employed to improve the formation of wine with low levels of malic acid (Lafon-Lafourcade 1970; Wibowo et al. 1988; Maicas et al. 2000). When O. oeni is inoculated at high suspensions (about 1×107 – 1×108 cfu ml–1), the inhibition of MLF by low pH is diminished, as bacterial development is not essential to develop the deacidification. However, the growth conditions yielding optimal biomass formation often result in lower specific productivity. This problem has forced the study of new culture media and growth conditions to ensure satisfactory production of such adapted cells (Hayman and Monk 1982; Maicas et al. 1999a). Approaches to increasing productivity in high cell density fermentations can be split into two general categories: the use of cell-recycle fermentations to reach high cell densities, and strategies for controlling continuous fermentations.

When cell-recycle bioreactors are used to induce the MLF, a tangential flow or hollow-fiber filter is used to separate the cells from the wine. In this type of continuous culture, cells remain in the vessel and reach high cell densities, whereas product (wine after MLF) is constantly removed, which can delay or prevent inhibition of cell growth by lactic acid production and low pH. Limita-

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tions of recycle bioreactors include possible shear stress on cells entering the filtration unit and potential difficulties in scale-up due to the filtration system. Two factors that must be considered when operating a cell-recycle bioreactor are the dilution rate (flow rate per volume), on which growth rate is dependent, and the recycle rate.

Gao and Fleet (1995) described an efficient membrane bioreactor system used for MLF involving high cell densities. During the 1st day of continuous operation, cells were able to degrade almost 100% of malic acid in the wine. Similar findings were described on the 2nd day of operation after storage of the reactor overnight at 4 °C. Only a slight loss (10%) in ability to degrade malic acid was recorded after 48 h, while faster losses in activity (27%) were noted by 56 h.

Strategies for controlling continuous fermentations have been recently explored by the utilization of free O. oeni cells in a continuous stirred tank reactor, which was successfully operated for 2–3 weeks to induce MLF in red wine (Maicas et al. 1999b). Operation at D=0.016 h–1 enabled 92–95% consumption of the malic acid in the wine. The advantage of using this reactor compared to the previous use of large quantities of cells is the absence of depletion of NAD+ and absence of lactic acid inhibition. This fermentation system enabled continuous growing to replace outflow of cells and MLF was successfully performed under controlled conditions.

Similar descriptions have been reported for MLF in cider by using O. oeni cells (Durieux et al. 2000).

Immobilization of cells The immobilization of microorganisms, i.e., any technique that limits the free migrations of cells, has been shown to be favorable for increasing food fermentation rates (Scott 1987). Immobilization techniques applied to induce MLF in wine include two of the four main approaches to cell immobilization (entrapment and adsorption/attachment) (Table 1). To our knowledge, neither aggregation nor encapsulation techniques have been reported for induction of MLF in wine.

Entrapment of cells represents the more definitive solution for immobilizing microorganisms that do not have a natural tendency to aggregate, e.g., lactic acid bacteria.

In this case, the cells are held either within the interstices of porous materials, such as a sponge of fibrous matrix, or by the physical restraints of membranes or encapsulating gel matrices. This approach, which is by far the most popular for bacterial immobilization, was generally used by the researchers whose publications on MLF in wine are reviewed here (Table 1). Viable cell immobilization via gel entrapment is mostly done by mixing cells with soluble gel components under sterile conditions followed by gelation, resulting in beads of a predetermined diameter range, usually 1–4 mm (Groboillot et al. 1994). The bead-entrapped cells are then exposed to wine.

Immobilization by entrapment of cells in a polyacrylamide network is the most extensively used method for

other biotechnological purposes. Early assays were performed by immobilizing Lactobacillus casei cells in polyacrylamide gels (Diviès and Siess 1976; Diviès 1977; Lee and Pack 1980; Totsuka and Hara 1981; Rossi and Clementi 1984). The properties of immobilized cells resembled those of free cells, but the immobilized cells were uniquely easy to recover and re-use (Rossi and Clementi 1984). In the presence of L-malic acid concentrations between 2.2 and 4.5 g l–1, a malolactic conversion rate of 71% was observed after 10 min incubation.

Industrial exploitation of this technique has not been undertaken, and owing to safety regulations it is practically restricted to laboratory studies for wine production.

The κ-carrageenan method has also been used for immobilization of O. oeni cells to induce wine deacidification and offers better perspectives for its industrial utilization (McCord and Ryu 1985; Crapisi et al. 1987a). κ-carrageenan is a naturally occurring polysaccharide isolated from seaweed and widely used as a food additive, being readily available and nontoxic. Crapisi et al.

(1987a) reported enhanced operational stability of Lactobacillus sp. cells immobilized in a κ-carrageenan matrix.

The combined use of this polymer together with bentonite silica provided an effective bioreactor for developing MLF in wine. The immobilized cells showed good effectiveness in decreasing L-malic acid, the conversion ratio and the decrease in titratable acidity being equivalent to about 60% of the original 8.98 g l–1 of L-malic acid.

These results were extended for several lactic acid bacteria species including Lactobacillus and O. oeni (Crapisi et al. 1987b). When Lb. brevis was immobilized the concentrations of cells remained stable for 24–48 h, while O. oeni showed a sharp decrease in the numbers of viable cells in this period of time. These findings confirmed that Lactobacillus immobilized cells had longer half-lives than those obtained with O. oeni strains.

Nevertheless, the use of Lactobacillus cells for MLF is no longer encouraged because of the better adaptation of O. oeni cells to wine stress conditions together with their controlled production of aroma compounds.

Kierstan and Bucke (1977) introduced the now frequently used calcium alginate method for cell immobilization. Seaweed is the source of this polymer, which can also be used as a food additive. Spettoli et al. (1982) used calcium alginate to immobilize O. oeni ML34, which was able to induce MLF in a red wine containing 1.5 g l–1 Lmalic acid, 21 mg l–1 SO2, 10.2% (v/v) alcohol at pH 3.15. The bacteria were able to transform up to 56% of the malic acid after 16 h. Crapisi et al. (1987b) reported the use of Lb. brevis in a continuous-flow bioreactor, the efficiency of which depended on the immobilization matrix used. Thus, the conversion ratio ranged from 34% to 45% depending on the supplier of the alginate matrix.

Naouri et al. (1991) immobilized O. oeni and Lactobacillus sp. for wine deacidification and were able to induce decarboxylation of red wine at low pH (3.0) and temperature (<20 °C), showing bioconversion rates greater than 77%. However, this fluidized bed bioreactor system had to be discarded after depletion of the NAD+ or Mn2+ in

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wine, two cofactors that are necessary for malolactic enzymes. These results were slightly improved by the introduction of a regeneration process coupled to the MLF. This regeneration was performed by placing alginate beads in a regeneration solution for 24 h, which conferred on the cells the ability to induce the malolactic process in successive fermentation solutions (Naouri et al. 1991).

Some of these entrapment techniques are sensitive to the presence of medium components which may affect their mechanical stability (e.g., phosphate in the case of alginate). Chemically cross-linked gels and adsorbents are generally much more chemically and physically stable (Freeman and Lilly 1998). Moreover, entrapmentbased procedures may be extremely powerful since they can immobilize large amounts of enzyme without chemical alteration. Immobilization via adsorption begins with a sterilized support inoculated with cell suspensions.

The biofilm subsequently developed upon exposure to growth medium is comprised of multiple cell layers located either in direct contact with the bulk medium (Janssen et al. 1993) or on the surface of inner channels in a porous structure (Maicas et al. 2001). Janssen et al. (1993) assessed the feasibility of O. oeni cells immobilized by adsorption onto oak chips for the continuous MLF of wine. This material, acceptable in vineries, enabled an effective bioconversion of malic acid. The half-life of their bioreactor was estimated at approximately 11 days when operated at 21 °C, pH 3.45, and 13% (v/v) ethanol. In terms of reactor performance, the rates of malic acid conversion were 2–3 times higher than those observed in batch experiments with freely suspended cells. Maicas et al. (2001) have recently reported the immobilization of O. oeni in positively charged cellulose sponges. In terms of bioconversion of L-malic acid, this could be efficiently achieved using a semicontinuous system based on immobilization of O. oeni cells in food-grade sponges. This system enabled L-malic acid conversion rates higher than 50% in repeated periods of 24 h without loss of activity.

Prolonged use of immobilized viable cells often involves potential continuous cell growth along with parallel cell death and lysis. Cell-supporting matrices should hence be permeable to exchange whole cells and/or cell debris with the surrounding medium, losing their mechanical stability (Freeman and Lilly 1998).

Immobilization of enzymes The study of the literature has shown numerous studies for developing MLF using different types of bioreactors with free or immobilized lactic acid bacteria. However, direct enzymatic bioconversion has scarcely been studied and only one enzymatic reactor has been reported. The malolactic enzyme (EC 1.1.1.38) of the lactic acid bacteria enables direct conversion of L-malic acid into L-lactic acid and carbon dioxide (Caspritz and Radler 1983; Battermann and Radler 1991). This process requires the availability of manganese and NAD+, which act as cofactors (Davis et al. 1985; Kunkee 1997). A membrane

reactor using free O. oeni enzyme was developed by Formisyn et al. (1997). Different conditions were tested in order to determine the best operating conditions, with promising results. MLF was accomplished in several days, yielding 62–75% with a contact time ranging from 70 to 170 min at a flow rate of 5 ml h–1. However, higher flow rates reduced the transformation and the efficiency of the reactor. These experiments demonstrated the possibility and efficiency of MLF through an enzymatic cell-free reactor, although complete and rapid consumption of the L-malic acid was not efficiently achieved.

Scale-up Scale-up in pre-existing equipment has been reported for several pilot scale bioreactors (Spettoli et al. 1987; Naouri et al. 1991; Janssen et al. 1993). Successful scaleup means a shortened cycle to full-scale production, competitive advantage, and cost savings (Reisman 1993). Basically, some problems would be expected to arise at the first stage of this process, focused on the production of sufficient levels of biomass, free or immobilized with different supports, and those inherent in control of the process with large wine volumes. The study of new immobilization matrices acceptable for wine production should improve the application of these technologies to the MLF of wine, leading to enhanced productivity.

Acknowledgements The author is grateful to I. Pardo and S. Ferrer for helpful suggestions during part of the experimental work reported in this review. The author wishes to thank J. Polaina for financial support during the write-up of this article.

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