J. Inst. Brew., November-December, 1991, Vol. 97, pp.

453-456

453

ANALYTICAL CONTROL OF CIDER PRODUCTION BY TWO TECHNOLOGICAL METHODS

By D. B. Gomis*. M. D. Gutierrez, M. J. Moran

Deparlamento de Quimica Fisica y Analllica, Universidad de Oviedo, 33006 Oviedo, Spain

AND

J. Moreno, E. Dapena, C. Cabranes, J. M. Alonso

Centro de Experimentation Agraria, Conscjeria de Agriculture! y Pesca del Prinipado de Asturias, Villaviciosa, Spain
Received 15 November 1990

Two cider-production procedures involving direct fermentation of apple juice and a prefermentative
clarification step or defecation were controlled microbiologically, while chemical monitoring of the major

sugars, polyalcohols and organic acids produced during the fermentation was carried out by HPLC. The
defecation process markedly slows down alcoholic fermentation and the malolactic conversion. Good
correlations between the contents of D(-) lactate and volatile acidity were obtained for the ciders
studied, which indicates that the increased volatile acidity of the ciders was basically a result of
metabolism mediated by lactic-acid bacteria, the yield being higher in barrels where enzymatic defecation
was not applied. Sucrose was rapidly hydrolysed in the first step of the fermentation process, while
glucose is the preferential carbon source for the yeasts. Citric acid was produced by the yeasts in the first
step of the fermentation process and was later taken up by these micro-organisms. Conversion of quinic
and shikimic acids seemed to point to a signicant activity of heterofermentative lactic-acid bacteria in
storage and maturation of ciders.

Key Words: Cider, high performance liquid chromatography,

sugars, organic acids, polyalcohols.
Introduction
The normal development of production processes of different
apple derivatives, particularly cider, depends heavily on the
transformations that the different components of the fruit (e.g.
organic acids and sugars) undergo throughout the process as
they determine the sensory features of the above mentioned
derivatives.
Sugars are the main source of carbon available to micro
organisms (yeasts, lactic acid and acetic acid bacteria) for
development during the production of cider.
Organic acids can be used as a source of energy or
synthesized by these microorganisms during the biochemical
transformations involved in the cider making. Thus, the
L(- )malic acid, the principal acid of the apple, is converted to
L(+)lactic acid by lactic acid bacteria (malolactic conversion)
This deacidification action increases the pH and improves the

sensory quality of the product (Tracey & Van Rooger21).

Quinic acid, together with malic acid, is one of the major

acids occurring in apples; in fact, it is present at high concen
trations in some varieties of cider apples. It is reduced to
dihydroshikimic acid by lactic acid bacteria; the latter case,
can be subsequently enolized, dehydrated and decarboxylated
to catcchol, which has an adverse effect on cider quality (Carr
& Whiting9). Shikimic acid, a minor component of apples,
is coupled to a major redox system in the metabolic action

of lactic acid bacteria (shikimate/dihydroshikimate), which

involves the oxidation of substrates such as fructose and lactic

acid to acetate and carbon dioxide (Whiting & Coggins25-26).

Citric acid can be initially synthesized by yeasts during the

fermentation and subsequently assimilated and catabolized
within the cells. Lactic acid bacteria are liable to convert citric

acid to acetate, diacetyl and acetoin (Shimazu20). D(-) lactate

and L( + ) lactate are produced by homo and heterofermen
tative lactic acid bacteria during the fermentation of sugars

(Randier14). They can also be produced, though in smaller

amounts, by yeasts through reduction of the pyruvate formed
in the fermentation process (Ribereau-Gayon17). Succinic acid

has a strong salty-bitter flavour (Kaneuchi15) which may

adversely affect the sensory features of cider. It is produced by

yeasts in the course of the alcoholic fermentation. Its final
concentration depends on the nitrogen source present in the
growth medium. Its yield is increased by glutamate and proline

(Heerde and Radler13).

Regarding lactic acid bacteria, we should note that several

lactobacillus species can produce succinic acid from malic acid

(Whiting23).

Normally, in the first stage of apple juice fermentation a

spontaneous defecation takes place. The nitrogen compounds
decreases and as a consequence, the fermentation rate also
decreases. The cider thus obtained was more aromatic and
microbiologically stable with less acetification activity. The
spontaneous defecation control was difficult because the pectin
contents and pectin methyl esterase activity depends on apple
maturity. In addition, apple maceration was smaller every day
for economic reasons. In order to control the defecation process
an enzymatic defecation was carried out by addition of calcium
and pectin methyl esterase.
All this, prompted us to study cider production through
different processing techniques (with and without enzymatic
defecation) with the aim not only of controlling the various
parameters of general interest (density, volatile acidity) but also
of monitoring the contents of sugars, polyalcohols and organic
acids, the transformations of which determine the quality of the
end product.
Organic acids, polyalcohols and sugars can be analysed by

various chromatographic techniques (Goiffon", Mentasti16).

High performance liquid chromatography is a rapid, straight

forward alternative to the analysis for carbohydrates and
organic acids in foods.
This paper deals with the control and determination of
carbohydrates, polyalcohols (glyccrol and sorbitol), cthanol
and organic acids in the course of the fermentation of cider by
using chromatographic separation methods described in earlier

papers (Blanco57).

We also studied the changes in volatile acidity and the

density and determined the L( +) and D( -) lactic acid contents
enzymatically on two apple juices obtained from the same
batch of apples but through different processing techniques at
different fermentation stages.

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454

[J. Inst. Brew.

CIDER PRODUCTION CONTROL

Materials and Methods

juice (barrel I), and another, in which the juice was subjected

Instrumental set-up and operating conditions

A Liquid Chromatograph equipped with a Waters 510
pump, a Wisp 712 automatic injector, a Waters 410 RI detector
and a Digital 380 data station was used to determine carbo
hydrates and polyalcohols.
The Sugar Pak I column used (300x6.5 mm i.d.) from
Waters Associates was operated under the following

and Drilleau2). This involved adding 1,200 units/Hi of pectin

conditions:

mobile

phase,

water

containing

50

ppm

Ca(Na)2EDTA; flow-rate, 0.5 ml/min; column temperature,

80C; detector temperature, 37C; detector sensitivity, 32;
injected volume, 10 ul.
Major organic acids occurring in apple juice and cider were
determined by using a Spherisorb ODS-2 column (5 urn, 250 *
4 mm i.d.) and a LK.B high performance liquid chromatograph,
equipped with a LKB 2150 pump, a rheodyne 7125 injection
valve, a LKB 214 rapid spectral detector and an IBM data
station. Column effluents were monitored at a wavelength of
206 nm.
The operating conditions were as follows: mobile phase
0.01 M KH2PO4/H3PO4 buffer, pH 2.25; flow-rate, 0.5 ml/min;

column temperature, 25C; ionic strength, 10~2 M; injected

volume, 20 ul.

to a defecation process prior to its fermentation (Baron

esterase (rapidase CPE-Gist-Biocades) and 9 mM of Ca2+ as

dihydrate calcium chloride to the apple juice (barrel II). The

experiments were carried out in duplicate barrels for each
juice treatment during two consecutive harvests (1987-1989).
Changes in the different parameters throughout the fermen
tation process by the two production procedures were then
monitored.

The results obtained confirmed the effect of prefermentive

clarification on the time and rate of the two main biochemical
transformations involved in the manufacturing process, i.e.
alcoholic fermentation and malolactic conversion. As can be
seen in Figure 1, from the gradual changes in density, malic and
lactic acid concentrations, it is apparent that both the alcoholic
fermentation and malolactic conversion take place at a slower
rate in the juice subjected to defecation as a consequence of
yeast and lactic acid bacteria diminution as can be seen in
Figure 2.

TABLE 1. Chemical and microbiological composition of apple juice

procedure developed by Boehringer and Mannhcin*.

Density
Total acidity
Total nitrogen
Sugars
Polyphenols
Pectin

(10% HC1) at 75'C for 20 min.

Total nitrogen was determined by the modified Hach method

pH
Brix

Other analytical methods

L( + ) and D(-)lactic acid were analysed by the enzymatic

The total sugar content was determined by the Bertrand

method. Sucrose was previously hydrolized in an acid medium

(Hach12, Watkins22), while ammonia nitrogen was assayed by

distillation over boric acid and subsequent titration with HCI.
The density and volatile acidity were determined according
to standard methods (AOAC1).

1,043 g/litre
44.90 meq/litre
97.80 mg/litre
98.50 g/litre
0.96 g/litre
0.66 g/litre
4.00
10.90

4.4xl05cfu/ml
9.7xlO5du/inl
7.5xl05cfu/ml

Yeast
Lactic bacteria

Acetic bacteria

Reagent and sample preparation

All reagents were of analytical grade and were used without

further purification. Organic acids, sugars and polyalcohols
were purchased from Sigma Chemical (St Louis, MO, USA)
and Merck (Darmstadt, FRG). HPLC-grade solvents were

BARREL I
1045

employed as mobile phases.

Apple juices and ciders were filtered through a Sep Pak Qg
cartridge (Waters Associates) prewetted with methanol to

retain colour pigments for sugar analyses. Prior to injection, all

samples were filtered through a 0.45-um Millex membrane

(Millipore). Carbonated ciders were previously degassed for

5 min in an ultrasonic bath.
60

Recovery studies
Acids and sugars were quantified by the external-standard
method from peak areas. Ciders were analysed in triplicate
before and after adding known amounts of sugars and acids.
Three different concentration levels of spiked sugars and acids
were assayed. The average recoveries were between 91 and
105% for sugars and over 100% for all acids, thereby showing
the method used to be accurate enough for the analysis of these
solutes. The coefficients of variation for the three replicates of
each sample were generally less than 6%.

80

100

Time (days)

BARREL II

Results and Discussion

A mixture of apples of different sensory propertiesCollaos
(mild sharp) 30%, Raxao (sharp) 40%, Duron Arroes (Sweet)

10%, Coloradona (bittersweet) 15%, Meana (bitter) 5%

endowing the resulting cider with an overall acidic nature was
chosen. Such a mixture was processed in a hammer mill and a
batch mechanical press (three day pressing time). The chemical
and microbiological composition of apple juice are listed in
Table I. The juice thus obtained was then processed in two
different ways, viz. by the traditional technique used in

Northern Spain, which involves the direct fermentation of the

20

40

60

80

100

Time (days)
Fig. I. Evolution of the density (O) and the L-lactic (D) and L-malic
acid (A) contents throughout the cider fermentative process.

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Vol. 97, 1991]

455

CIDER PRODUCTION CONTROL

BARREL
log (cfu/ml)

10

15
Minutes

40

60

80

100

Time (days)
Fig. 2. Evolution or yeast (O) and lactic acid bacteria (D) throughout
the cider fermentative process.

Regarding the sugars yielded along the alcoholic fermen

tation pathway, and as can be seen in Figure 3, sucrose was
hydrolysed in the first step of the fermentative process, and
the microorganisms used glucose first and then fructose as
their carbon source. Sorbitol cannot be fermented by yeasts,
although it can indeed be used by acetic acid bacteria, which
oxidize it to sorbose and, possibly, 5-ketofructose (Beech and

Carr3-4). On the other hand, the percentage of sugars employed

in the formation of glycerol was 8.7% and 9% for barrels I and

II, which is consistent with the polyalcohol yields found in

other fermented beverages (Ribereau-Gayon17).

Succinic acid, produced during the fermentation of apple

juice in the two barrels, could be considered a major acid. The
yield of this acid correlated well with that of glycerol (Table II),
which was probably a result of the metabolism of the pyruvic
acid formed in the course of the glycerin-pyruvic fermentation.
Hetcrofermentative lactic acid bacteria (Leuconostoc oenos)
have been shown shown to metabolize sugars via heterolactic
fermentation to form lactate, carbon dioxide and ethanol or
acetate mainly. The concentration of acetate yielded probably
depends on the redox properties of the inedium and on the
assimilated substrate.
Leuconostoc oenos, found in the two barrels, has been
reported to occur in the production of cider particularly in the

malolactic conversion (Salih19); this heterofermentalive micro

organism produces D( ) lactate (Garvi10, Salih18).

As can be seen from Table HI the volatile acidity and D(-)

lactate yields are correlated, the latter was lesser in barrel II as

Fio. 3. Typical chromatograms of sugars and alcohols in apple juice in

fourth stages or fermentation. Column, Sugar Pak 1,300 * 6.5 mm I.D.
Mobile phase: water containing 50 ppm Ca(Na),EDTA. Flow rate:
0.5 ml/min. Temperature column: 80C. (1) organic acids, (2) sucrose,
(3) glucose, (4) fructose, (5) glycerol, (6) ethanol, (7) sorbitol.

TABLE 2. Correlation between succinic acid and glycerol

Barrel II

Barrell
Succinic acid
(g/litre)

Glycerol
(g/litre)

Succinic acid

Glycerol

(g/litre)

(g/litre)

0.5108
0.6970
0.7359
0.7721

2.80
3.40
3.83
4.14

0.4350
0.4405
0.6035
0.6209

1.68
1.40
3.39
4.31

m=0.1921
b/a =0.0017
r=0.9598

m=0.0707

b/a=0.3343
r=0.9738

m=slope; b/a=intercept; r=correlation coefficient.

consequence of smaller heterofermentative lactic acid bacteria

activity (Fig. 2). This fact appears to indicate that the increased
volatile acidity of the two ciders was basically a result of the
metabolism mediated by lactic acid bacteria.
As can be seen in Figure 4, the citric acid concentration
reaches a maximum during the alcoholic fermentation and then
decreases with time throughout the production process. This

shows that citric acid, as claimed by Whiting24, is produced by

the yeasts in the first step of the fermentative process and is

later taken up by the microorganisms.

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456

CIDER PRODUCTION CONTROL

TABLE 3. Correlation between D-laclic acid and volatile acidity

Barrel II

Barrel I

Volatile acidity
(g/litre)

D(-)lactate

(g/litre)

Volatile acidity
(g/litre)

D( - )lactate
(g/litre)

0.19

0.07
0.10
0.14
0.16
1.36

0.22
0.27
0.27
0.40
0.61

0.09

0.21
0.27
0.34
0.75

0.10
0.13
0.27

0.40
m= 1.1608
b/a=0.1241
r=0.9855

m=0.4051
b/a=0.2037
r = 0.9805

m=slope; b/a = intercept; r=correlation coefficient.

0.025

40

60

80

Time (days)

BARREL II

20

40

60

80
Time (days)

lactobacillus genus (Beech and Carr34). After that, there is a

decrease in the content of this acid which seems to be related to
the formation of shikimic acid by heterofermentative lactic acid

bacteria as reported by Whiting and Coggins26.

The shikimic acid concentration decreases during the cider

maturation step, especially in barrel II (Fig. 4), probably
because of its metabolization by lactic acid bacteria to dihydro-

shikimic acid. Therefore according to Carr and Whiting9, one

should also bear in mind that quinic and shikimic acid and
dihydroshikimic acid, involved in the redox system employed
by lactic acid bacteria in anaerobic oxidations, can be oxidized
by acetic acid bacteria to 3,4-dihydroxy-5-oxo-cyclohexane-lcarboxylic acid.

Acknowledgement: This work was financially supported by the

Comision Asesora para la Investigation Cientifica y Tccnica
(CAICYT, PA 310/85).

BARREL I

20

[J. Inst. Brew.

100

Fig. 4. Evolution of the citric (D), quinic (A) and shikimic acid (O)
contents throughout the cider fermentative process.

Regarding the quinic acid transformation (Fig. 4), it is

maximum in the final step of the malolactic conversion. This
can be explained on the basis of the hydrolysis of chlorogenic
acid, which is generally produced by lactic acid bacteria of the

References
1. AOAC. 1984. Official Methods of Analysis of the Association of
Analytical Chemists. Virginia: Sidney Williams, 1984, 220.
2. Baron, A. & Drilleau, J. F. Use of Enzymes in Food Technology.
Paris: P. Dupuy, 1982, 471.
3. Beech, F. W. & Carr, J. G. Alcoholic Beverages. London: A. H.
Rose, 1977, 185.
4. Beech, F. W. & Carr, J. G. Alcoholic Beverages. London: A. H.
Rose, 1977. 188.
5. Blanco, D., Moran, M. J., Gutierrez, M. D. & Sanz, A.
Chromatographia, 1987, 24,347.
6. Blanco, D., Gutierrez, M. D., Mangas, J. J. & Noval, A.
Chromatographia, 1988, 25,701.
7. Blanco. D., Moran, M. J., Gutierrez, M. D. & Mangas, J. J.
Chromatographia, 1988, 25, 1054.
8. Boeringer, G. H. & Mannhein, F. R. Methods of Biochemical
Analysis and Good Analysis Using Test Combinations. Boehringer

Manheim S.A., 1975.

9. Carr, J.G.& Whiting, C.C. Journal of Applied Bacteriology. 1971,
34,81.
10. Garvi, E. I. In Bergey's Manual of Systematic Bacteriology.
Baltimore: Williams & Wiekims, 1986, Volume 2,1071.
11. Goiffon, J. P., Blachere, A. & Reminiac, C. Analusis, 1985,13,218.
12. Hach, C. C, Bowden, B. K., Kopelove, A. B. & Brayton, S. V.
Journal of the Association of Analytical Chemists. 1987,70,783.
13. Heerde, E. & Radler, F. Archives of Microbiology, 1978,117,269.
14. Kandler, O. Antonie van Leeuwenhoek. 1983,49,209.
15. Kaneuchi, C, Seki, M. & Komagata, K. Applied and Environ
mental Microbiology. 1988,54,3053.
16. Mentasti, E., Genaro, M. C, Sarzanini, C, Baiochi, C. &
Savigliano. M. Journal of Chromatography. 1985,322, 177.
17. Ribercau-Gayon, J., Peynaud, E., Ribereau-Gayon, P. & Sudraud,
P. Sciences et Techniques du vin. Paris: Dunod, 1975, Tome II, 522.
18. Salih, A. G., Drilleau, J. F., Cavin, F., Divies, F. & Bourgeois,
C. M. Journal Institute of Brewing, 1988,94, 5.
19. Salih, A. G., Le Quere, J. M. & Drilleau, J. F. in press.
20. Shimazu, Y., Uehara, M. & Watanabe, M. Agricultural and
Biological Chemistry, 1985,12,423.
21. Tracey, R. P. & van Rooyen, T. J. Journal of Applied Bacteriology.
1988,65,113.
22. Watkings, K. L., Trygve, L. V. & Krause, G. F. Journal of the
Association of Official Analytical Chemists. 1987,70, 6073.
23. Whiting, G. C. Lactic Acid Bacteria in Beverages and Food.
London: Academic Press, 1975, 69.
24. Whiting, G. C. Journal Institute of Brewing. 1976,82, 84.
25. Whiting, G. C. & Coggins, R. A. Biochemical Journal. 1969, 115,
60.
26. Whiting, G. C. & Coggins, R. A. Antonie van Leeuwenhoek, 1971,
37,33.

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