Controlled malolactic fermentation in cider usingOenococcus oeniimmobilized in alginate beads and comparison with free

cell fermentation

Monica Herrero, Adriana Laca, Luis A. Garcı´a, Mario Dıaz*Department of Chemical Engineering and Environmental Technology, (I.U.B.A). University of Oviedo, Oviedo, Spain

Received 20 April 1999; received in revised form 22 May 2000; accepted 3 July 2000

Abstract

Cells ofOenococcus oeni(formerlyLeuconostoc oenos) immobilized in alginate beads were used as starter culture to conduct malolacticfermentation in cider production. Concentrations of major organic acids and volatile compounds were monitored during the process, andresults were compared to those obtained when using free cells in the same conditions. The rates of malic acid consumption were similarbut lower ethanoic acid content and higher concentration of alcohols were detected with immobilized cells. These features have beneficialeffects on the organoleptic properties of cider. A comparison between the kinetic behavior in immobilized and free cells, based on the dataobtained for the malic acid consumption, has been developed solving the homogeneous diffusion model when it is applied to the systemwith immobilized cells. © 2001 Elsevier Science Inc. All rights reserved.

Keywords:Cider fermentation; Immobilization; Alginate; Malolactic

1. Introduction

Malolactic fermentation in cider making is difficult tocontrol and a complex process, because many nutritionaland physicochemical factors affect the growth and metab-olism of lactic acid bacteria. Essential growth factors in themust promote, whereas products of yeast metabolism suchas fatty acids and ethanol inhibit the growth of lactic acidbacteria. To ensure the malolactic fermentation, the malo-lactic bacteria population should reach, at least, 106 colonyforming units (CFU)/ml.

The use of starter cultures in cider fermentation mightallow cider makers to produce a uniformly high qualityproduct to be maintained during successive processes andseasons. Although the use of starter cultures to controlindustrial fermentations is well established in brewing andwine production, it has not been widely adopted in cidermaking. Several lyophilized starter cultures for malolacticfermentation are available in the market, but previous stud-ies [1,2] reported that indigenous malolactic bacteria were

better adapted to musts and yielded better results in malicacid degradation.

The use of immobilized cells in fermentation processesoffers some very well known advantages over the use offree cells [3]. For example, the concentration of the catalyticactivity in a reduced volume allows producers to reduce thesize of reactors and recover final products more easily incontinuous or batch production systems. Gel entrapment isa popular method of cell immobilization because usuallyuses non-toxic reagents compatible with food production[4,5,6]. The gel matrix, when alginate is used as support, haspores less than 90 nm in diameter [7] allowing retention ofcells and diffusion of nutrient molecules and metabolites.Alginate beads have been used on an industrial scale inbrewing and wine making [8,9]. Previous work [10] usingfree cells led to the conclusion that sequential inoculation ofyeast at 15°C followed by inoculation of lactic acid bacteriaand fermentation at 22°C are optimum for metabolizingmalic acid.

This paper reports the use of starter cultures of a strain ofO. oeni, isolated from the cellar of a cider industry, forinducing malolactic fermentation in cider on a laboratoryscale. The progress of the fermentations induced by addingfree or immobilized cells was compared.

* Corresponding author. Tel.:134-98-5103439; fax: 134-98-5103434.

E-mail address: mdf@sauron.quimica.uniovi.es (M. Diaz).

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0141-0229/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved.PII: S0141-0229(00)00265-9

2. Materials and methods

2.1. Microorganisms

A commercial active-dried yeast strain ofSaccharo-myces bayanus (oviformis)(strain Pasteur Institute, Paris1969, “Champagne” and supplied by Novo Ferment (Swit-zerland)) was used for primary fermentations. The malolac-tic bacteria (strain Lc2) was previously isolated in the cellarof the cider industry Escanciador, S.A. (Villaviciosa, Astu-rias, Spain) and was identified as aOenococcus oenistrain(formerly Leuconostoc oenos), which was selected on thebasis of its ability to perform malic acid degradation.

2.2. Experimental conditions

Concentrated apple juice, supplied by an industrial ciderfactory, was reconstituted with distilled water (1:6), yield-ing a final density of aprox. 1060 g/liter. Then, the juice wassterilized in a tangential flow filtration device (Filtron Ome-gacell 150TM) connected to a peristaltic pump, using poly-ethersulfone membranes (0.33mm pore diameter).

Fermentations were carried out in sterilized 250 ml Er-lenmeyer flasks containing 100 ml of the culture mediumand placed in an orbital shaker (New Brunswick, G25), at100 r.p.m, at the assay temperatures.

Yeast active-dried preparation was rehydrated in sterileapple juice and grown under aerobic conditions shaken at250 r.p.m., 28°C, for 18 h. The apple must was then inoc-ulated with yeasts at a final concentration of 106 CFU/ml.Alcoholic fermentation by yeast was carried out at 15°C.

Malolactic bacteria were previously grown in applejuice, prepared as described, supplemented with yeast ex-tract 0.5% (w/v), incubated at 30°C without shaking, due tothe microaerophilic nature of this bacteria, for 6 days untilstationary phase was reached.

To start malolactic transformation, once the alcoholicfermentation was completed and must density (withoutcells) reached approximately 1005 g/liter (11 days wereneeded) as a result of yeast sugar metabolism, the immobi-lized malolactic bacteria were added to the flasks, that wereincubated at 22°C, in the same conditions. Every flask wasinoculated with beads containing the immobilizedO. oeni(aprox. 5 3 107 CFU/ml). Alternatively, the malolacticfermentation was performed using free cells (107 CFU/ml)in the same conditions.

2.3. Cell immobilization technique

Cell entrapment in calcium alginate beads [11] was car-ried out under sterile conditions. Cells grown as describedwere mixed with 2.1% (w/v) sodium alginate (JanssenChimica) in the proportion 2.9 108 CFU/ml alginate. Themixture obtained was then allowed to drop at a constant rateonto a 3% (w/v) CaCl2 solution kept in continuous agitation.To complete the gelatinization, the beads were maintained

in the CaCl2 solution for 30 min. The beads obtained had anaverage diameter of 2.9 mm.

2.4. Microbiological counting

Lactic acid bacteria were followed throughout fermenta-tion experiments by counting viable cells in Petri plates.Serial dilutions were performed in saline solution, platingduplicates in statistically significant dilutions on MRS (Bio-kar) supplemented with antibiotics to inhibit yeast growth(100 ppm cycloheximide and 25 ppm 8-OH-quinoline).Dishes were incubated at 30°C for 5 days.

2.5. Sample preparation and analytical methods

Analyses were performed on apple juice or cider afterfiltration through 0.45mm membranes (Whatman). Samples(10 ml) were taken only once from each flask. Mediumdensity (without cells) was measured using a picnometer.Organic acids in samples were determined by HPLC (Wa-ters, Alliance 2690), equipped with a photodiode array de-tector (Waters 996), as previously described [12]. A Spheri-sorb ODS2 (C18) analytical column (4.63 150 mm, 3mm,Waters) was used under the following conditions: columntemperature, 36°C; mobile phase, 1022 M KH2PO4/H3PO4

pH 2.65; flow rate, 0.5 ml/min, and 10ml volume injection.Column effluents were monitored at 210 nm. Solvents andreagents were HPLC grade. Analytical grade organic acids(without further purification) were used as standards:quinic, pyruvic, malic, shikimic, lactic, ethanoic, fumaricand succinic acids (Sigma-Aldrich and Merck). Quantifica-tion based on peak area was performed using Millenniumsoftware (v.2.15.01; Waters).L-malic acid was also deter-mined by enzymatic assays (Boehringer Mannheim).

Volatile compounds with boiling points lower than145°C were analyzed using a gas chromatograph (GC-14B,Shimadzu) equipped with a FID detector and an auto injec-tor (AOC-20i, Shimadzu), fitted with a Supelcowax 10(Supelco) column (60 m3 0.25 mm i.d., phase thickness0.25 mm). Chromatographic conditions were as follows:initial temperature 40°C for 10 min; programme rate 4°C/min to 80°C; 80°C for 10 min; programme rate 35°C/min;final temperature 200°C for 15 min. Finally, column tem-perature was equilibrated at the initial temperature for 20min until the next injection. Injector and detector tempera-ture were 200°C and 230°C, respectively; the carrier gaswas He at 150 kPa; volume injected, 5ml. Samples weredirectly injected after membrane filtration. Analytical gradecompounds (without further purification) were used as stan-dards: ethyl ethanoate, methanol, ethanol, propan-1-ol,2-methylpropan-1-ol, butan-1-ol, 2-methylbutan-1-ol,3-methylbutan-1-ol, purchased from Sigma and Merck.Data obtained were registered and processed in a Chro-matopac C-R6A (Shimadzu), based on peak area measure-ments.

36 M. Herrero et al. / Enzyme and Microbial Technology 28 (2001) 35–41

2.6. Microscopic techniques

Cells entrapped in the calcium alginate beads were fixedwith 1% osmium tetroxide [13], dehydrated with acetone,immersed in Epon 812 resin (Taab Laboratories, England)and stored at 60°C to promote polymerization. Gel beadswere cut into 1mm slices using an LKBV ultramicrotome.After staining with toluidine blue, micrographs were ob-tained using a LEICA DMR-KA optical microscope.

3. Results and discussion

The immobilized malolactic bacteria showed good fer-mentation performance. The appearance of alginate beadsdid not alter during 17 days of fermentation (see Fig. 1) andfewer than 100 CFU/mlO. oeni were released into the

medium. The low numbers of bacteria released into themedium would have been insufficient to performed malo-lactic fermentation [1]. This result contrasts with previouswork in our laboratory [11,14,15], carried out with othermicroorganism and different culture medium, when thebeads ruptured. In the present work, cell growth has beenuncoupled from the malolactic transformation in the fer-mentation media (see Material and methods).

3.1. Evolution of organic acids

As shown in Table 1, malic acid was metabolized over asimilar period using either immobilized or free cells; in both

Fig. 1. Optical microscopic observation of immobilizedO. oeni.Edge of a section. Fermentation time: (a) day 0; (b) 17 days. (LEIKA DMR-XA microscope,JVC TV camera and a MITSUBISHI video printer).

Fig. 2. Consumption of malic acid (D) and evolution of lactic acid (L)during the malolactic fermentation using inmobilized (open symbols) andfree (solid symbols) cells. Day 0 shows the concentration at the end ofyeast fermentation.

Table 1Consumption of malic acid during the malolactic fermentation usingimmobilized (IM) and free cells (FC). ND5 not detected. Day 0 showsthe concentration at the end of yeast fermentation.

Days IM (mg/I) FC (mg/I)

0 3.2 3.22 1.5 1.54 0.9 1.27 ND ND9 ND ND

14 ND ND16 ND ND18 ND ND21 ND ND

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cases this acid could not be detected in the fermentationmedia from day 7 after bacterial inoculation with the ana-lytical conditions used. Higher levels of lactic acid (Fig. 2)were reached with free cells. Thereafter, lactic acid concen-trations fell possible because of the metabolism of this acid[16] .

Immobilized cells synthesized less ethanoic acid (Fig. 3)and reduced the concentration of quinic acid less than freecells (Fig. 4).

Formation of fumaric acid was obtained when usingimmobilized malolactic bacteria (Fig. 4), and similar pro-files in the evolution of pyruvic, shikimic and succinic acidswere observed (Fig. 5).

3.2. Volatile compounds

This type of substances are mainly produced during yeastmetabolism, having an important influence on cider flavor,but during malolactic fermentation several volatile com-pounds may be formed or their concentrations altered.

Although higher concentrations of ethanol were obtainedusing immobilized cells (Fig. 6), achieving a maximum

level 4 days after malolactic bacteria inoculation, later on animportant decrease was detected in both cases, probably dueto esters and/or acetaldehyde formation during maturation.Apart from chemical conversions, a physic phenomenonsuch as evaporation might have taken place.

Ethyl ethanoate levels (Fig. 7) were higher using freecells, although in both cases concentrations are in the rangepreviously described for strains ofSaccharomycesin cider[17].

Higher concentrations of propan-1-ol, 2-methylpropan-1-ol and butan-1-ol (Fig. 8) were obtained when fermenta-tion was performed with immobilized cells; similar levelsfor methanol, 2-methylbutan-1-ol and 3-methylbutan-1-ol(Fig. 9) were found for free and immobilized bacteria.

3.3. Comparison of the substrate consumption in free andimmobilized cells

The immobilized cells have a different environment thanfree cells, thus affecting their physiological conditions.Therefore, in order to contribute to the knowledge of themetabolism and the consumption efficiency of the cells inthe internal bead conditions, kinetic parameters must bedetermined, and for this aim a model of substrate consump-

Fig. 3. Ethanoic acid measured during fermentations with immobilized (e)and free (f) cells.

Fig. 4. Evolution of quinic acid (V) and fumaric acid (D) with immobilized(open symbols) and free cells (solid symbols).

Fig. 5. Levels of other organic acids. With immobilized cells: pyruvic ({),succinic (D) and shikimic (V); and free cells: pyruvic (l), succinic (Œ)and shikimic (x).

Fig. 6. Profiles of the ethanol content during fermentations with immobi-lized (V) and free (F) cells.

38 M. Herrero et al. / Enzyme and Microbial Technology 28 (2001) 35–41

tion was developed. Malic acid was selected for the study asa key substrate since it is present in the medium in a highconcentration and it is a major variable in the malolactictransformation.

Firstly, the kinetic study has been carried out in thefermentation with free cells. Numbers of bacteria remainedconstant during malolactic fermentation and the rate ofutilization of malic acid may be related to substrate concen-tration and bacterial biomass. The equation

dS

dt5

kS

k9 1 SX

(that presents the same symbolic form as the Michaelis-Menten equation) was used to fit the experimental data [S5malic acid concentration;X 5 biomass concentration; andkandk9 are constants]. Fig. 10 shows experimental values incomparison with the theoretical model for values ofk 572.98 day21 andk9 5 2.77 g/liter (the software MicroMathScientist for Windows. Version 2.0 was used to fit theexperimental data). These data can also be fitted by a firstorder equation with quite good results.

To analyze the system with cells immobilized in alginate

an homogeneous diffusion model [14] was used with valuesof 0.88 for the porosity of the bed and 0.00145 m for theradius of the beads. For the malic acid diffusivity into thebeads, the value of 73 10210 m2/s given by Øyaas et al.[18] for the effective diffusivity of lactic acid through algi-nate, has been assumed. In the same way the externalresistance discussed in previous works [14,19] has beencalculated from the external resistance obtained for BSAdiffusing to the alginate beads. We have taken into accountthat the mass transfer coefficients increase the diffusivity tothe power 2/3, obtaining a value of 83 1027 m/s. Anywaywe have observed that variations in a 50% range in thevalues of the diffusivity and the external resistance almostdo not affect the obtained results because the control is byreaction not by diffusion. The same kinetic equation as infree cells has been considered for the modelization of thissystem. Initially thek andk9 values introduced in the modelhave been the same as those obtained for free cells.

The simulation results obtained in the previous condi-tions have shown that thek value for free cells does not fitthe experimental data in the case of immobilized cells and itis a parameter that could be reasonably optimized. Different

Fig. 7. Ethyl ethanoate formed using free cells (f) and immobilized (M).

Fig. 8. Evolution of propan-1-ol (V), 2-methylpropan-1-ol (M) and butan-1-ol (D) when using immobilized (open symbols) and free cells (solidsymbols).

Fig. 9. Evolution of methanol (V), 2-methylbutan-1-ol (M) and 3-methyl-butan-1-ol (D) using immobilized (open symbols) and free cells (solidsymbols).

Fig. 10. Theoretical results (line) for malic acid consumption using freecells, when a Monod kinetic model is considered. Experimental data:obtained by enzymatic assays (f) and HPLC (Œ).

39M. Herrero et al. / Enzyme and Microbial Technology 28 (2001) 35–41

values fork have been tested in the simulation using theproposed model and the best value obtained for this constantfitting the experimental results is in this case 13 day21

(thek9 value has been maintained). In Fig. 11 experimen-tal and theoretical data are shown. It can be observed thatthe value fork is approximately 5.6 times higher for freethan for immobilized cells. This difference, as indicatedabove, might be explained by the different environmentthat surrounds the bacteria in both cases. It must be bornein mind that in the model the possible effect that yeastscan present in the malic acid consumption is not takeninto account.

4. Conclusions

The use of the malolactic bacteriaO. oeniimmobilized inalginate beads as starter culture to conduct the malolacticfermentation in cider has proven to be efficient. It is impor-tant to say that similar values of malic acid consumption inboth fermentations with free and immobilized cells can beobtained, although the bacteria concentration was differentin both cases, 5 times higher for the immobilized system onthe basis of total volume of the fermenter. Based on thecomparison of the kinetics obtained by means of a diffusionmodel for malic acid consumption in immobilized and freecells, it has been found that both systems can be modeled bythe same kinetic equation with a value for the kinetic con-stantk approximately 5.6 higher in free than in immobilizedcells. Lower levels of ethanoic acid were reached whenfermentation was carried out with immobilized bacteria.Although production of major volatile compounds dependsmainly on the yeast strain used to conduct the alcoholicfermentation, some differences could be observed in theirconcentrations during the malolactic fermentation depend-ing on the type of inoculation used. Only ethyl ethanoatereached higher levels when fermentation was performedwith free cells, but concentrations of alcohols were higher or

at least similar using immobilized bacteria. The lower con-centration of ethanoic acid and higher concentrations ofalcohols resulting from the use of immobilized cells mightlead to production of ciders with better flavors [20,21].Then, this cell immobilization technique is worthy of fur-ther consideration in cider production, facilitating cell/liq-uid separation processes or major versatility in the selectionof suitable reactors.

Acknowledgments

This work was financially supported by the followingAsturian cider industries: Sidra Escanciador, S.A., Valle,Ballina y Ferna´ndez, S.A., Sidra Mayador, S.A. and Indus-trial Zarracina, S.A. (Asturias, Spain) and by FICYT (Foun-dation for Scientific and Technical Research, Asturias,Spain).

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