Changes in Organic Acids During Malolactic Fermentation at Different Temperatures in Yeast-Fermented Apple Juice

Changes in Organic Acids During Malolactic Fermentation pp.191-195 Volume 105 No. 3,1999

Changes in Organic Acids During Malolactic Fermentation

at Different Temperatures in Yeast-Fermented Apple Juice

by Monica Herrero, Isabel Cuesta, Luis A. Garcia and Mario Diaz

Department of Chemical Engineering and Environmental Technology, University o/Oviedo, Spain

Received 23rd October 1998

Organic acids were analyzed during controlled malolacticfermentation conducted by a selected

Leuconostoc oenos strain, performed at different temperatures (15 °C, 22 °C and 27 °CX The apple

juice had been previously fermented by a Saccharomyces cerevisiae strain, at 15'C and major

organic acids were also monitored during this phase. The aim of this work was to study the

effect offermentation temperature on organic acid composition and on the main reactions that

take place during malolacticfermentation, applicable to improving the cider making process at

the industrial level.

Key Words: Apple juice, organic acids, high performance liquid chromatography,

fermentation.

malolactic

INTRODUCTION

Organic acid composition in alcoholic beverages, and in

cider in particular, is a very important feature that

affects the organoleptic properties of the product. Being

a constituent of the sourness group, each acid in

alcoholic beverages conveys a characteristic flavour,

aroma or taste. During malolactic fermentation, lactic

acid bacteria transform malic acid into lactic acid, thus

reducing the excessively high acidity found in apple

juice. Malolactic conversion is also accompanied by

formation of products other than lactic acid, modifying

both flavour and texture.3-8-4 Malolactic fermentation can

remove strong vegetative/herbaceous aromas, enhance

fruity and floral aromas, improve the mouthfeel and

extend the duration of the aftertaste. Not only

composition, but also concentration, of each acid is

essential in the quality of the product. Concentration

threshold values and the relative sourness of the most

important non-volatile acids in cider have been

previously reported15.

When malolactic fermentation occurs spontaneously,

the flavour characteristics are unpredictable due to

the different microorganisms which may be present in

the must or in the cellar. The use of starter cultures in

wine and cider making ensures achievement of

malolactic fermentation in a more rapid and

predictable manner, and also provides uniformity to

the final product.

Technological parameters also affect the odour and

taste of cider. For example, fermentation temperature

strongly affects the development of malolactic

fermentation and lactic acid bacteria metabolism10. The

aim of this work was to determine the main changes in

organic acids that take place during malolactic

fermentation, conducted at different temperatures. A

selected malolactic indigenous strain was used as starter

culture, in diluted concentrated apple juice previously

fermented by a selected yeast strain. This study, at the

laboratory level, was carried out with the goal of

determining the optimal conditions to perform a

controlled malolactic fermentation in cider production.

MATERIALS AND METHODS

Microorganisms

A commercial active-dried yeast strain of

Saccharomyces cerevisiae was used. The malolactic

bacteria (strain Lc2) was previously isolated in the cellar

of the cider industry Escanciador, S.A. (Villaviciosa,

Asturias, Spain), and was identified as Leuconostoc oenos.

It was selected on the basis of its ability to perform malic

acid degradation.

Experimental conditions

Concentrated apple juice, supplied by an industrial

cider factory, was reconstituted with distilled water

(1:6), yielding a final density of aprox. 1060 g/litre. The

Journal of The Institute of Brewing 291

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Volume 105, No. 3,1999 Changes in Organic Acids During Malolactic Fermentation

juice was sterilized in a tangential flow filtration device

(Filtron Omegacell 150™) connected to a peristaltic

pump, using polyethersulfone membranes (0.33/<m pore

diameter).

Fermentations were carried out in pre-sterilized 250

ml Erlenmeyer flasks containing 100 ml of the culture

medium which were placed in an orbital shaker (New

Brunswick, G25) at 100 r.p.m, at the assay temperatures.

Yeast active-dried preparation was rehydrated in

sterile apple juice and grown under aerobic conditions

at 250 r.p.m., 28°C, for 18 h. The apple juice was then

inoculated with yeast at a final concentration of 106

cfu/ml. Alcoholic fermentation by yeasts was carried

out at 15"C.

Once the alcoholic fermentation was completed and the

density reached approximately 1005 g/litre, a high bacterial

inoculum (107 cfu/ml) was added to the flasks to start

malolactic transformation. The samples were incubated at

the assay temperatures, under the same conditions.

Malolactic bacteria was previously grown in apple juice,

prepared as described above, supplemented with yeast

extract 0.5% (w/v) and statically incubated at 30°C, due to

the microaerophilic nature of this bacteria. Incubation was

for 6 days until the stationary phase was reached.

Sample preparation and analytical methods

Apple juice and cider samples were filtered through

0.45 /*m membranes. Medium density was mesured by

picnometry. L-malic, D- and L-lactic acid were

determined by enzymatic assays (Boehringer

Mannheim). Organic acids in samples were determined

by HPLC (Waters, Alliance 2690), equipped with a

photodiode array detector (Waters 996), as previously

described7-'. A Spherisorb ODS2 (C18) analytical column

(4.6 x 150mm, 3/<m, Waters) was used under the

following conditions: column temperature, 36°C; mobile

phase, 10-2 m KH2PO4/H3PO4 pH 2.65; flow rate, 0.5

ml/min, and 10/<l volume injection. Column effluents

were monitored at 210 nm. Solvents were HPLC grade.

Analytical grade organic acids (without further

purification) were used as standards: quinic, pyruvic,

malic, shikimic, lactic, acetic, fumaric and succinic acids

were purchased from Sigma-Aldrich and Merck.

Quantification was based on peak area measurements.

Data treatment was performed with Millenium software

(v.2.15.01).

RESULTS AND DISCUSSION

Apple juice was initially fermented by the selected

yeast strain at 15°C. Once the alcoholic fermentation was

completed, the inoculum of malolactic bacteria was

added to the flasks (107 cfu/ml) and further incubation

was performed at 15*C, 22°C and 27°C under the same

conditions.

Some aspects may be highlighted about the alcoholic

fermentation by yeast at 15°C. Pyruvic acid reached a

maximum when approximately half the sugar has been

fermented (Fig. 1). This acid is an intermediate in the

Embden-Meyerhof-Parnas pathway and a precursor to

many other substances. In spite of its importance as a

MLF

FIG. 1. Pyruvic acid evolution during alcoholic

fermentation (AF) by S. cerevisiae at 15°C, and during

malolactic fermentation (MLF) by L. oenos at 15°C (■),

22°C (•) and 27'C (A).

metabolic intermediate, it is excreted by yeast during

fermentation, sometimes in concentrations ranging in

cider from 0.08 to 0.6 g/litre.14 As it is shown in Figure 2,

the strain of S. cerevisiae used in this work was able to

consume malic acid. Yeasts may either break down or

form malate during fermentations. They convert malate

to ethanol and CO2 anaerobically via the malic enzyme.

In a survey of 300 strains of Saccharomyces using

synthetic medium, the proportion of malate degraded

MLF

FIG. 2. L-Malic acid during AF (15°C) and MLF (15°C

(■), 22°C (•) and 27'C (A)).

192 Journal of The Institute of Brewing


Changes in Organic Acids During Malolactic Fermentation Volume 105 No. 3,1999

MLF MLF

35 40

10 15 20 25 30 35

FIG. 3. D-lactic acid (open symbols) and L-lactic acid

(solid symbols) during AF (15°C) and MLF at the three FIG. 6. Fumaric acid during AF (15°C) and MLF at 15°C

temperatures tested: 15°C (■), 22°C (•) and 27°C (A). (■), 22°C (•) and 27°C (A).

MLF

15 20 25 30 35 40

FIG. 4. Production of acetic acid during alcoholic

fermentation (AF) at 15'C and malolactic fermentation

(MLF) at 15°C (■), 22°C (•) and 27"C (A).

MLF

0 15 20 25 30 35 40

CO

FIG. 5. Succinic acid production at 15°C (■), 22'C (•)

and 27°C (A) during MLF and AF at 15°C.

for each strain varied from 5 to 40%.u Production of L-

lactate was detected (Fig. 3). Reduction of pyruvate may

result in formation of either D(-)- or L(+)- lactate.14

In addition, formation of acetic (Fig. 4), succinic (Fig. 5)

and fumaric (Fig.6) acids were observed. The later

showed a maximum in the first stages of alcoholic

fermentation.

Once malolactic bacteria were inoculated in the

fermentation media, L-malic acid was rapidly consumed

and as shown in Figure 2, this process was significantly

affected by temperature. At 15°C, the rate of

consumption of malic acid was slower than at higher

temperatures (22°C and 27°C). Based on this result, 22°C

should be selected as the optimal temperature for malic

acid degradation, since at 27*C residual malic acid was

detected until 32 days. Previously published results

have determined that the optimal temperature for

malolactic fermentation in wine production was 20-

25°C.6 It should be pointed out that the amount of L-

lactic acid formed (Fig. 3) did not correspond to the

amount of L-malic acid consumed under all

temperatures assayed. This fact could be explained by

taking into account the fact that yeast metabolism could

be responsible for part of malic acid degradation

occurring during this phase. However, two additional

features were detected at the three fermentation

temperatures tested. An interesting decrease in L-lactic

acid was observed, being higher at 27°C than at 15°C. It

has been reported in the literature that lactic acid

bacteria have the ability to oxidize lactic acid to acetate

anaerobically.13 Both D- and L-lactate are equally good

hydrogen donors for the reductive steps and

simultaneously lactate is oxidized to acetate. In

addition, some strains of Leuconostoc isolated from

ciders and perries produced appreciable amounts of

succinate from malate,2 the proportion varying with the

particulate isolate and increasing with the pH of the

medium. This reaction is known as "malosuccinic



Volume 105, No. 3,1999 Changes in Organic Acids During Malolactic Fermentation

fermentation". Succinic acid production was observed at

the three fermentation temperatures (Fig. 5) with the

greatest concentration being reached at 27*C and 15°C

The concentration of sucdnic add at these temperatures

was inversely related to the levels of L-lactic add

measured. Some yeasts are also capable of converting

some malate to succinate and smaller amounts of

lactate,14 which was demonstrated using 14C-labelled

malate. Previous work9 regarding Spanish cider

manufacture (from the Asturian region) reported similar

results: an L-lactic add decrease with time during

malolactic fermentation and the amount of L-lactic add

formed was less than the theoretical value expected

from the malic acid conversion.

A second major organic add present in apple juice,

together with malic add, is quinic add. Some Leuconostoc

strains reduce quinate and the related shikimate,13 which

is a minor component in apple juice. Quinic add is

reduced to dihydroshikimic add after the completion of

malolactic fermentation. Its esters, chlorogenic add and

p-coumarylquinic acid, are usually reduced at a later

stage of fermentation. Shikimic add, is coupled to a

major redox system (shikimate /dihydroshikimate) in

the metabolic action of lactic add bacteria, involving the

oxidation of substrates such as fructose and lactic

acid.11-12 The concentrations of quinate (Fig. 7) showed

significant differences between 15°C and the higher

fermentation temperatures of 22°C and 27°C, where this

add was not detected from days 25 and 19, respectively.

4

3.5

3

2.5

2

1.5

1

0.5

n

AF

Ti—

•

■

MLF

%

\%«

\V

>•

10 15 20 25

Days

30 35 40

FIG. 7. Quinic add. The symbols represent the same

conditions.

Quinic acid metabolism is related to malic acid

consumption and is involved in anaerobic lactate

oxidation mechanisms. Consumption of shikimic add

was also monitored and was metabolized earlier at

higher fermentation temperatures (Fig. 8).

The formation of pyruvic add was also affected by the

fermentation temperature. At higher temperatures,

0.004

0.0035

0.003

*& 0.0025

I 0.002

| 0.0015

0.001

0.0005

0

AF MLF

■■ ■

MM MM *

0 5 10 15 20 25 30 35 40

Days

FIG. 8. Shikimic add. The symbols represent the same

temperatures.

increasing maximum levels were reached in succesive

later stages of fermentation (Fig. 1). It has been

postulated that lactic add may be oxidized to acetate

and CO2 by lactic add bacteria, with pyruvate as an

intermediate product.5 These maximum levels of

pyruvate during malolactic fermentation could be

related to this reaction.

Fumaric acid formation reached a maximum at the

beginning of the malolactic transformation, and then

decreased in a similar way as malic add degradation at

each fermentation temperature. Once malic add was

fully metabolized, a second increase was detected, at

15'C and 22*C.

The rate of acetic add formation during the first

stages of malolactic fermentation (Fig. 4) was greatest at

the highest temperature tested. No significant

differences were observed for acetic add formation

between 15°C and 22*C, both reaching a maximum level

of 0.8 g/litre. From day 20 until the end of the

fermentation process, an important increase in the rate

of production of acetic acid was observed at all

temperatures assayed, reaching concentrations of

lg/litre at 15°C, and higher concentrations at 22°C and

27°C. These concentrations of acetic acid would

negatively affect the organoleptic properties of the

product. In all experiments, fermentation conditions

were mantained for 40 days, although malic acid was

consumed earlier in all cases. Acetic acid production at

the later stages can be directly related to the oxidation

mechanisms of lactate metabolism (involving quinate

and dihydroshikimate, or pyruvate as intermediate)

both giving rise to acetate as the final product. In cider

production, malic acid consumption usually indicates

the end of the fermentation process, avoiding further

acetic acid formation.



Changes in Organic Acids During Malolactic Fermentation Volume 105 No. 3,1999

CONCLUSIONS

Fermentation temperatures during malolactic

transformation significantly affect the concentration

levels of the major organic acids in cider. Regarding the

malic acid consumption rate, 22°C turns out to be the

most favourable temperature. Based on these results , it

has been postulated that in all experiments carried out,

a parallel malosuccinic fermentation may be occurring

in addition to the malolactic fermentation. An important

decrease in L-lactic acid was observed at each

temperature tested, once malic acid had been degraded.

This can be explained by anaerobic lactate oxidation

mechanisms from the action of malolactic bacteria.

Consumption of quinate and shikimate and formation of

pyruvate as an intermediate product have been

measured, producing acetate as a final product at the

later stages of the fermentation process. To avoid

elevated concentrations of acetic acid in the product,

fermentation temperatures between 15°C and 22°C

seems to be more suitable than 27°C.

Acknowledgements. This work was financially

supported by the following Asturian cider industries:

Sidra Escanciador, S.A., Valle, Ballina y Fernandez, S.A.,

Sidra Mayador, S.A. and Industrias Zarracina, S.A.

(Asturias, Spain) and by FICYT (Foundation for

Scientific and Technical Research, Asturias, Spain).

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10. Vaillant, H., Formisyn, P. and Gerbaux, V. Journal of Applied

Bacteriology, 1995, 79, 640-650.

11. Whiting, G.C. and Coggins, R.A. Biochemical Journal, 1969,115,60-61.

12. Whiting, G.C. and Coggins, R.A. Antonie van Leeuwenhoek. 1971, 37,

33-49.

13. Whiting, G.C. Lactic acid bacteria in beverages andfood, Ed. J.G. Carr,

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Changes in Organic Acids During Malolactic Fermentation at Different Temperatures in Yeast-Fermented Apple Juice