Effect of Fermentation Temperature - Wiley Online Library

Organic Acids in Cider, pp.229-232

Volume W5, No. 4,1999

Organic Acids in Cider with Simultaneous Inoculation

of Yeast and Malolactic Bacteria: Effect of Fermentation Temperature by Monica Herrero, Luis A. Garcia and Mario Diaz Department of Chemical Engineering and Environmental Technology, University of Oviedo, Spain

Received 1st December 1998

Analysis of major organic acids in cider was performed by HPLC after simultaneous inoculation

of selected yeast and malolactic bacteria in apple juice. Fermentations were carried out at two different temperatures, 15°C and 22°C, in order to compare the composition of the products obtained. Consumption of malic acid was affected by fermentation temperature, although in

both cases this acid was fully metabolised. At both temperatures tested, an important decrease in lactic acid was observed after completion of malic acid degradation, along with formation of high levels of acetic acid.

Key Words:

Organic acids,

yeast,

malolactic bacteria,

INTRODUCTION

apple juice,

temperature.

The specific identities of the organic acids found in

Alcoholic and malolactic fermentation (MLF) are the main microbiological processes in cider making. Malolactic

transformation (which converts stoichiometrically Lmalate into L-lactate and CO2) may occur spontaneously

due to the presence of malolactic bacteria (belonging to three genera: Leuconostoc, Lactobacillus and Pedioccus) in musts or in the cellar. Nevertheless, because cider is not

cider and their initial concentration depend mainly on

the initial composition of the raw material, but their concentrations change by the metabolic action of the micro-organisms conducting the process. Operating procedures, such as inoculation time and fermentation temperature, may considerably affect the organoleptic properties of the product.

a favourable media for lactic acid bacterial development,

In this work, major organic acids were analysed during

this process is unpredictable and may permit growth of

cider production after simultaneous inoculation of yeasts

undesirable microorganisms that cause spoilage of the

and malolactic bacteria, at two different fermentation

product. The use of starter cultures in cider would allow

temperatures. Results presented here were compared to

controlled fermentations and ensure a high-quality of

those obtained when the malolactic strain Leuconostoc oenos

uniformity in the final product.

Lc2 was inoculated in cider after completion of alcoholic

Malolactic bacteria can be inoculated simultaneously

with the yeast inoculum or after the alcoholic fermentation has been completed. The first method

might offer some advantages for bacterial development, due to the lower ethanol levels and the higher sugar content which exists in musts. On the other hand, simultaneous inoculation may produce a delay in

bacterial growth and malic acid degradation4 while sugar metabolism of lactic acid bacteria gives rise to

high levels of D-lactate and acetate. If bacterial inoculation is performed immediately after completion

of alcoholic fermentation, production of D-lactate and acetate from sugars is avoided, and essential nutrients

are available for bacteria as a result of yeast excretion and autolysis1. It seems appropriate to analyze in a

complete way both methods to determine the optimal procedure for each strain used under the operating conditions.

fermentation at 15CC, and MLF was performed under

the fermentation temperatures tested (15°C and 22°C)5. MATERIALS AND METHODS

Micro-organisms

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 an L oenos strain, which was selected amongst

others on the basis of its ability to perform malic acid degradation. The strain was maintained lyophilized and also frozen in 20% glycerol at -20°C. Experimental conditions Heat-concentrated

apple

juice,

supplied

by

an

industrial cider factory, was reconstituted with distilled This document is provided compliments of the Institute of Brewing and Distilling www.ibd.org.uk

Journal of The Institute of Brewing

Copyright - Journal of the Institute of Brewing

229

Organic Acids in Cider

Volume 105, No. 4,1999

water (1:6), yielding a final density of approx. 1060 g / litre. The juice was then sterilised in a tangential flow filtration device (Filtron Omegacell ISO71'1) connected to

a peristaltic pump, using polyethersulfone membranes (0.33um pore diameter).

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.

!"::rV^ i "^

^

1

0.5

Malolactic bacteria were grown in apple juice, prepared

as described above, supplemented with yeast extract

0.5% (w/v), incubated at 30°C, without shaking due to

0

0

5

10

15

the microaerophilic nature of this bacteria, for 6 days

20

25

30

35

40

Days

until stationary phase was reached. The apple must was inoculated with yeasts at a final

FIG. 2. Changes in lactic acid in fermentations at 15°C (■) and 22°C (•).

concentration of 106 CFU/ml and with a high bacterial inoculum (107 CFU/ml). Fermentations were carried out in pre-sterilised 250 ml Erlenmeyer flasks containing 100

ml of the culture medium placed in orbital shakers (New Brunswick, G25), at 100 rpm, at the assay temperatures (15°C or 22°C).

RESULTS

Malic acid (Fig. 1) was completely degraded at both fermentation temperatures, although it was observed that this process was faster at the higher temperature tested. At 22°C, malic acid was consumed in 9 days; at 15°C, 30

days were needed. In both cases, alcoholic fermentation

Sample preparation and analytical methods

Apple juice and cider samples were filtered through

and malic acid degradation began simultaneously.

0.45 um membranes. Organic acids in samples were

An increase in lactic acid concentration was detected

determined by HPLC (Waters, Alliance 2690), equipped

(Fig. 2), reaching a maximum level at 22°C earlier than at

with a photodiode array detector (Waters 996), as

15°C, in agreement with the consumption rate of malic

previously described2-7. A Spherisorb ODS2 (C18) analytical

acid. It should be kept in mind that part of the lactic acid

column (4.6 x 150mm, 3 |im, Waters) was used under the

measured by chromatographic techniques might correspond

following conditions: column temperature, 36°C; mobile

to D-lactate, as a result of sugar metabolism by L. oenos.

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

The malolactic bacteria population maintained

min, and 10 ul volume injection. Column effluents were

approximately the inoculation level in the fermentation

monitored at 210 nm. Solvents were HPLC grade.

media (data not shown), since growth was separated

Analytical

further

from the malolactic transformation (see Materials and

purification) were used as standards: quinic, pyruvic,

Methods). It is noteworthy that the amount of malic acid

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

degraded did not correspond to the expected amount of

were

lactic acid formed on the basis of the malolactic

grade

purchased

organic

from

acids

(without

Sigma-Aldrich

and

Merck.

Quantification was based on peak area measurements.

transformation, being much lower than expected,

Data treatment were performed with Millennium

specially if only low levels of D-lactate had been

software (v.2.15.01).

i

10

FIG. 1.

Malic acid degradation in fermentations at 15°C (|

and 22°C (•).

20

25

30

35

40

FIG. 3. Succinic acid production during fermentations at 15°C (■) and 22°C (•).

This document is provided compliments of the Institute of Brewing and Distilling www.ibd.org.uk

230

15



Volume 105, No. 4,1999


produced by the action of the heterolactic bacteria. In addition, an important decrease of lactic acid was

detected at both fermentation temperatures.

£

Production of succinic acid (Fig. 3) was observed,

reaching higher concentrations at 15°C than at 22°C. A slight decrease could be detected at the final stages of fermentation at 22°C. At both temperatures quinic acid (Fig. 4) was produced in the initial stages of fermentation,

1

FIG. 6. Changes in pyruvic acid in fermentations at both temperatures (the symbols represent the same temperatures).

u

'3

&

FIG. 4.

Quinic acid in apple juice fermented at 15°C (■) and

22°C (•).

0.004

I5

0.003

FIG. 7. Production of acetic acid during the course of fermentations (15°C (■) and 22°C (•)).

0.002

I

0.001

obtained at the temperatures assayed (Fig. 8): at 22°C,

during the first stage of fermentation, there was 0

5

10

15

20

25

30

35

40

production of this acid, followed by a degradation

phase; at 15°C, degradation took place from the beginning of the process, but at a slower rate than at

FIG. 5. Shikimic acid during fermentations at 15°C (■) and 22°C (•).

higher temperature.

0.012

but later, an important decrease was measured. Shikimic acid (Fig. 5), a minor component in apple juice, was

consumed earlier at higher temperature than at 15°C.

I

Production of pyruvic (Fig. 6) and acetic acid (Fig. 7) could be measured. The former is an important

intermediate in metabolic reactions, but it has been reported to be excreted by yeasts during alcoholic fermentation9. Higher levels of pyruvate were obtained

at 15°C than at 22°C. Acetic acid reached values near 1 g/ litre at both temperatures until malic acid was

0

almost degraded (days 9 and 20, respectively). From this

5

10

15

20

25

30

35

40

Days

moment, a second important production of acetic acid

took place, reaching very high levels that would spoil

FIG. 8.

the product. Different profiles for fumaric acid were

temperatures.

Fumaric acid. The symbols represent the same




231

Volume 105, No. 4,1999


malic acid was degraded could be attributed to the

DISCUSSION

Although sugars are the main carbon source for development of micro-organisms in cider, organic acids can be used as source of energy or they can be

synthesised by these micro-organisms during the

anaerobic lactate oxidation mechanisms mentioned, rather than sugar utilisation by lactic acid bacteria. CONCLUSIONS

fermentation process. In media that contain large

Consumption rate of malic acid was favoured when

amounts of organic acids, their metabolism by yeasts

fermentation was carried out at 22°C rather than at 15°C.

and lactic acid bacteria can be important, therefore

Malic add was completely degraded at both temperatures,

affecting the final product in either a favourable or

but based on the results obtained, other mechanisms

detrimental way. Apart from the specific characteristics

apart from malolactic transformation occurred. These

of the selected strains used as starter cultures, operating

transformations could include parallel malosucdnic

conditions at time of inoculation,

fermentation

fermentation and the utilisation of malic add by yeasts

temperature and duration of the process may

(giving rise to ethanol and CO2 by the action of the malic

considerably affect the organoleptic properties of the

enzyme, and/or formation of succinic add). An important

final product.

decrease in lactic add was detected, which can be related

As mentioned, two important events were observed at

both temperatures: the amount of lactic acid formed was lower than the theoretical amount expected from the malic acid consumption (considering only malolactic

to anaerobic lactate oxidation mechanisms. These two

features were also observed when fermentations were performed with sequential inoculation of yeast and malolactic bacteria Lc2, under the same temperatures5.

transformation), and secondly, an important decrease of

Production of high levels of acetate in the final stages

lactic acid with time took place. These features were also

of fermentation could be related to the mentioned lactate

observed in cider production when bacterial inoculation

oxidation mechanisms; maximum levels were reached

was performed after completion of alcoholic fermentation5.

when simultaneous inoculation was performed, although,

Regarding malic acid consumption, it should be taken

as the greatest production corresponded to the latest

into account that yeasts can either form or break down

phases of the fermentation process, it could not be

malic acid9 and as it was demonstrated in previous work,

attributed to sugar utilisation by the heterolactic

L. oenos Lc2 is able to degrade this acid. Yeasts are also

bacteria. Control of the duration of the process seems to

capable of converting malate to succinate9, but in

be critical to avoid further changes in organic acid

addition, some strains of Leuconostoc were found to

composition, espedally formation of high levels of acetic

perform this transformation3, known as malosuccinic

add that would lead to the spoilage of the product.

fermentation. Production of succinic acid was detected

at the temperatures assayed. When sequential inoculation was performed5, an important increase in succinic acid concentration occurred during alcoholic fermentation by

yeasts, although production was also observed once L. oenos Lc2 was inoculated. Fermentations carried out at

22°C showed lower levels of succinate formation, independent of the inoculation method (sequential or simultaneous).

The loss of lactic acid with time may be related to

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,

shikimate. Reduction of quiriate gives rise to dihydroshikimate, and simultaneously, lactate (both Dand L- isomers) is oxidised to acetate. These oxidoreductions proceed under anaerobic conditions.

Consumption of quinate and shikimate were monitored

at the temperatures tested, although differences were observed with the fermentation temperature. Similar results were obtained when experiments were carried out with sequential inoculation of yeast and the malolactic bacteria L. oenos Lc25.

It was also reported6 that lactic acid may be oxidised to acetate and CO2 by lactic acid bacteria, with pyruvate as intermediate. The high levels of acetate obtained once

1

FICYT

(Foundation for

Beelman, R.B., Keen, R.M., Banner, MJ. and King, S.W., Developments in industrial Microbiology, 1982,23, 107.

2

Blanco, D., Moran, M.J., Gutierrez, M.D. and Mangas, J.J., Chromatographia, 1988, 25, 1054.

3

Carr, J.G. and Whiting, G.C., Reports of Long Ashton

4

Research Station for 1955,1956,163. Gallander, J.F., American Journal of Enology and Viticulture, 1979, 30,157.

5

Herrero, M., Cuesta, I., Garda, L.A. and Diaz, M., journal of the Institute of Brewing, 1999,105,191.

6

Kandler, O., Antonie van Leeuwenhoek, 1983,49, 209.

7

Mangas, J.J., PhD thesis. 1992. Department of Analytical Chemistry, University of Oviedo. 8 Whiting, G.C., Lactic acid bacteria in beverages and food, Ed. J.G. Carr, C.V. Cutting and G.C. Whiting, London, Academic Press, 1975,69.

9

Whiting, G.C., Journal of the Institute of Brewing, 1976, 82, 84.


232

and by

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