Further studies on inhibitory effects

of juniper berry oils on

ethanol fermentation
Viada B. Veljkovi~,* Miodrag L. Lazi~,t Dobrila J. Ruti~t
and Mihajio Z. Stankovi~*
* Faculty o f Technology, University o f N i # , Leskovac, Yugoslavia
Pharmaceutical and Chemical Works "Zdravlje," Leskovac, Yugoslavia

The inhibitory effect of juniper berry oil on ethanol fermentation by Bakers' yeast is shown. The higher
the amount of the juniper berry oil, the more intensive is the inhibition. The lag time and the duration
of the ethanol fermentation are considerably extended, sugar utilization and ethanol production are
retarded, while organic acid formation is increased in the presence of the juniper berry oil.

Keywords:Ethanol fermentation, inhibitionof; Juniper communis; juniper berry oil, inhibitory effects of; Bakers'
yeast

Introduction concluded that the duration of the ethanol fermentation

is much longer if the juniper berries, regardless of
The fruit of Juniper communis, the juniper berry, has
whether or not the essential oil has been previously
a wide use not only for the recovery of the essential oil
removed, are present in the fermentation medium.
but also for the production of juniper-flavored alcoholic
Also, the ethanol yield is considerably higher if the
beverages. 1 Of special commercial interest are the dis-
water extract of juniper berries is used as the ferment-
tilled spirits from fermented juniper berries, often
ation substrate. 7 Thus, the inhibitor(s) might not be
mixed or redistilled with grain or fruit spirits.
eliminated by removal of the essential oil or, moreover,
Up to now, juniper berries have mainly been used
after separation of the residual plant material. The neg-
in ethanol fermentation either as entirely dried, crushed
ative effects of juniper berries on ethanol fermentation
berries to which water is added 2-4 or as a mash of
are attributed to the inhibitory effects of tannin, terpe-
residual juniper material obtained after removal of the
nic constituents of the essential oil, and resinous sub-
essential oil by water-vapor distillation. ~-6 Recently, it
stances. 2'5 The inhibitory effect of juniper berry oil is
has been shown that the water extract obtained from especially emphasized. 7
crushed juniper berries by removal of both the essential In this paper, the direct evidence for the inhibitory
oil and the residual plant material can also be used
effect of juniper berry oil on ethanol fermentation by
successfully as the fermentation substrate. 7 Table 1
Bakers' yeast are presented.
summarizes the conditions (microorganism, fermenta-
tion medium, operating conditions) applied in studies Materials and methods
on ethanol fermentation of juniper berries (without add-
ing mineral salts) found in the literature; the duration Microorganism
of the fermentation under different working conditions
The same Bakers' yeast (Saccharomyces cerevisiae)
is also presented. From the presented data it can be
as before 7 was used.

Media

Address reprint requests to Dr. Veljkovi6at the Faculty of Technol- The compositon of the fermentation medium was (g
ogy, Universityof Ni~, Durmitorska 19, 16000Leskovac, Yugoslavia d m - 3): glucose, 80, or an equimolar mixture of glucose,
Received 6 September 1988; revised 31 March 1989 40, and fructose, 40; peptone, 10; yeast extract, 5, at

706 Enzyme Microb. Technol., 1990, vol. 12, September © 1990 Butterworth-Heinemann
 Effects o f j u n i p e r berry oils on ethanol f e r m e n t a t i o n : V. B. Veljkovid e t al.
Table 1 Survey of working conditions employed in studies of ethanol fermentation of juniper berries (without addition of mineral
salts)

Ratio of Duration of
Fermentation berries Inoculum Temperature fermentation
Reference Yeast substrate to water size (°C) pH (days)

Matzik a2 Bakers'or Crushed berries 1:2 2.5-7.0 g d m -3 25 Not regulated 7-14

Brewers' of mash
Stankovid and Baker's Crushed berries 1 "4 9 g kg 1 of 30 3.5-4.0 4
Randjelovidb4 berries Not regulated
Mash after removal 1 : 2c 5
of the essential oil
Gro.~s Baker's Crushed residual plant 1:4 9 g kg -1 of plant 32 3.8-4.5 5
material after removal materials Not regulated
Wine of the essential oil d 8
Veljkovid Baker's Crushed berries 1:2 2 g dm-3 of 30 4.0 3
et al.7 substrate Regulated
Mash after removal 2
of the essential oil
Water extract 1

a Conditions are cited from the other literature sources

b Optimum conditions reported are presented
c Ratio of the berries to water for water-vapor distillation
d Residual plant material after removal of the essential oil was dried at 40-45°C for the purpose of long-term storage

pH 4.0. The media were sterilized in situ at 120°C for 15
min. The commercial juniper berry oil (Pharmaceutical
and Chemical Works "Zdravlje," Leskovac, Yugosla-
via) was added (0.1 or 5 cm 3 dm -3 of medium) to the
sterilized medium.
The juniper berry oil used was produced from
crushed juniper berries of Yugoslav origin (West Mace-
donia) by water-vapor distillation and met the require-
ments of DAB 7 (density, d 2° = 872.7 kg m-3; index
of refraction, n 2° = 1.477; specific rotation, a2°(l = l
dm) = -5.8°). By using the GLC analysis (Varian
Aerograph series 1400; column: Carbowax 20M, 1 = 2
m, ~ = 3.2 mm; column temperature: initial, 50°C, final
220°C; program rate: 2°C min-l, linear; carrier gas flow
rate: 10 ml min-~, nitrogen; detector: FID, hydrogen/
air) 38 components were detected, the main compo-
nents being identified as c~-pinene (15.6%), /3-pinene
(1.0%), myrcene (1.0%), limonene (9.3%), o-cymene
(15.1%), p-cineole (15.7%), terpineol-4 (16.9%), caryo-
phyllene (7.4%), borneol (12.1%), and geraniol (5.1%).
Operating conditions
The inoculum (2 g of the yeast per liter of medium) was
prepared by homogenizing the yeast with the medium.
All incubations were carried out in a 14-1 laboratory
fermenter with a working volume of 5 1 under strictly
defined conditions of agitation (100 rev min- 1), temper-
ature (30°C), and pH (4.0). The pH of the fermentation
broth was automatically regulated with 5 M NaOH so-
lution.
Measurements and analytical methods
Yeast growth. The growth of the yeast was measured
spectrophotometrically at 660 nm.
Sugar concentration. The concentration of glucose
when present alone in the fermentation medium and
the concentration of total carbohydrates (glucose +
fructose) were measured spectrophotometrically using
the colored reaction with picric acid. s The concentra-
tion of fructose in the mixture with glucose was mea-
sured spectrophotometrically using the colored reac-
tion with pentavalent vanadium in sulfuric acid. 9 The
concentration of glucose was calculated from the con-
centration of total sugars and the concentration of
fructose.
Ethanol concentration. The ethanol concentration was
determined from the density of the alcoholic redistillate
obtained from the fermentation broth.
Results and discussion
The influences of 5.0 and 0.1 cm 3 of the juniper berry
oil per liter of the medium compared to the control
without the oil during batch ethanol fermentation by
the Bakers' yeast can be seen in Figure 1. The volume
of the juniper berry oil of 5.0 cm 3 dm -3 approximately
corresponds to the amount of the essential oil which
would be introduced by the crushed juniper berries
containing 1% of the oil if the ratio of berries to water
is 1 : 2, and 0.1 cm 3 of the juniper berry oil per liter
of medium is assumed to correspond to the residual
amount of the juniper oil remaining after removal of its
main part by water-vapor distillation. It can clearly be
seen that the juniper berry oil strongly inhibits the
ethanol fermentation, the inhibition being increased by
increasing the amount of the juniper berry oil. The
maximum values of the specific growth rate, the glu-
cose uptake rate, the ethanol production rate, as well

E n z y m e M i c r o b . T e c h n o l . , 1990, v o l . 12, S e p t e m b e r 707

Papers
strates. In this way, ethanol fermentation of juniper
berry sugars is simulated, as the mixture of glucose and
1.5I
fructose approximately in the ratio of 1 : 1 is present in
.J
juniper berries and is the main part of the carbohydrates
present. The inhibition of ethanol fermentation by the
juniper berry oil (added in the amount of 5.0 cm 3 dm -3
of medium) compared to the control fermentation can
~0 Lu
13 be seen in Figure 2. Some important variables of the
0 ethanol fermentation are presented in Table 3 and illus-
.~0~ 13
3 trate the inhibitory effect of juniper oil quite clearly.
In the presence of the juniper berry oil, the growth
of the yeast is fourfold slower than in the control fer-
mentation. The yeast requires a prolonged adaptation
1o 20 3o &o 50
Time, h to the medium containing the juniper berry oil, and
the lag time and the duration of the fermentation are
Figure 1 Effects of juniper berry oil on yeast growth, glucose extended. About fivefold the time is needed to produce
utilization, and ethanol production the same amount of ethanol (30 g dm -3) as the control
fermentation. Both the glucose uptake rate and the
Volume of juniper fructose uptake rate are greatly decreased by the juni-
Parameter of berry oil (cm 3 dm -3) per berry oil. As a result, ethanol is produced at a much
fermentation 0 0.1 5.0 slower rate. Part of the sugars is dissipated through
other metabolic pathways, and the ethanol yield is de-
Optical density © /x creased in the presence of the juniper berry oil. Also,
Glucose concentration • • •
Ethanol concentration @ A []

4o~,1s
as the lag time and the duration of the fermentation are
a
summarized in Table 2. The yeast growth is greatly
retarded, and the lag time and the duration of the fer-
mentation are significantly extended by the presence
of the juniper berry oil; the rates of glucose utilization ~02 / z
13
and ethanol formation are decreased compared with :2
the control fermentation. 1^ dlS 3E
Further experiments were carried out with the me- °8! C
dium containing an equimolar mixture of glucose and
fructose (each sugar, 40 g dm -3) as the limiting sub- 0 J0
Time,h

Table 2 Effects of juniper berry oil on ethanol fermentation in 1

a glucose medium 1.5
0

Volume of juniper berry oil b 3o~ g

Parameter of (cm3 dm-3) ~, ,o'5
I) 0
fermentation 0 0.1 5.0 "I[:)
zoo -r
-6
13 u o
Specific growth =.s
ratea (h 1) 0.14 0.11 0.043
Maximum glucose I/3
.J
uptake rate " E
(g dm -3 h -1) 7.4 4.9 2.8 3
Maximum ethanol * 0 -D
10 2o 3o 40 5o eo
production rate Time, h
(g dm -3 h -1) 3.3 1.9 1.5
Lag time b (h) 4-5 7-8 16-17
Figure 2 Effects of juniper berry oil on yeast growth, glucose
End of fermentation ~ (h) 15 20-25 40 and fructose utilization, ethanol production, and acid formation
in a glucose-fructose medium. (a) Control fermentation, without
a Calculated from the change of optical density of the fermenta- juniper berry oil. (b) Fermentation with juniper berry oil (5 cm 3
tion broth dm-3 (©) Optical density; (~) glucose concentration, (A) fructose
b Estimated on the basis of the beginning and the end of ethanol concentration; (El) ethanol concentration; ( x ) volume of 5 M
production NaOH solution added to maintain the pH at 4.0

708 Enzyme Microb. Technol., 1990, vol. 12, September

 Effects of juniper berry oils on ethanol fermentation: V. B. Veljkovi6 et al.
Table 3 Effects of juniper berry oil on ethanol fermentation in a glucose-fructose medium

Medium without Medium with

juniper berry juniper berry
Parameter of fermentation oil oil

Specific growth rate (h 1) 0.13 0.038

Sugar uptake rate
(maximum value) (g dm -3 h -1)
Glucose 7.3 1.9
Fructose 6.5 1.9
Maximum ethanol production
rate (g dm -3 h -1) 3.8 1.0
Rate of NaOH addition
(maximum value) (mmol dm -3 h 1) 1.58 0.62
Total amount of NaOH added (mmol) 53 63.5
Moles of NaOH per mole of ethanol 0.019 0.029
Lag time (h) 4 20
End of fermentation (h) 12 50-55
Maximum ethanol yield (g dm 3) 33.5 30.0
With respect to the
theoretical ethanol yield (%) 82 74

the production of the organic acid is increased due to
the effect of juniper berry oil.
An interesting conclusion can be drawn from the
kinetic studies of sugar utilization by Bakers' yeast
grown in the glucose-fructose medium with or without
the juniper berry oil. In both cases, the uptake of glu-
cose is preferred to that of fructose while both sugars
are present. However, this preference of the yeast for
glucose appears to be more emphasized in the presence
of the juniper berry oil. The utilization of fructose is
delayed, practically begins when the glucose is ex-
hausted, and is much more slowly increased if the juni-
per berry oil is present. Thus it might be assumed that
the juniper berry oil inhibits the ethanol fermentation
by affecting the sugar transport system(s), not only the
yeast growth.
Conclusion
The inhibitory effect of juniper berry oil on ethanol
fermentation by Bakers' yeast is clearly shown. The
intensity of the inhibition is dependent on the amount
of the juniper berry oil present in the fermentation
medium; the higher the amount of the juniper berry oil,
the stronger is the inhibition. The fermentation medium
containing the juniper berry oil is unfavorable for the
growth of the Bakers' yeast, and the lag time and the
duration of the fermentation are greatly extended in the
presence of the juniper berry oil. The sugar uptake
rates and the ethanol production rate are decreased,
while the organic acid production rate is increased due
to the inhibitory effect of the juniper berry oil. The
ethanol yield is smaller due to sugar dissipation as
compared to the control fermentation in the absence of
the juniper berry oil.
The results of the study on the inhibitory effect of
juniper berry oil on ethanol fermentation by Bakers'
yeast completely confirm the effects of juniper berries
before and after removal of the essential oil, as already
reported by the authors. 7 Furthermore, it can be ex-
pected that a certain amount of the inhibitor(s) shall
remain in the fermentation medium even after removal
of the residual juniper material. The addition of some
yeast nutrients, such as ammonium and phosphate
ions, at optimum levels to the water extract of juniper
berry can overcome the inhibitory effects of juniper
berries and cause further shortening of the duration
of ethanol fermentation and increase in the ethanol
yield. ~0
Acknowledgments
The authors are grateful to Mrs. Mirjana Stankovi~,
Mrs. Ljubica Milenkovi6, and Mr. Miiovan Peri~ for
their expert technical assistance.
References
1 Simpson, A. C. in Economic Microbiology, Vol. 1: Alcoholic
Beverages (Rose, A. H., ed.) Academic Press, London, 1977,
pp. 537-593
2 Matzik, B. Z. LebensmitteI-Unters-Forsch. 1966, 130, 345-348
3 Kagdfik, J., Mosteck~, J., Hfila, S. and Vanura, J. Czech. Pat.
CS 213,044, 1984
4 Slankovi6, M. and Randjelovi6, M. Hem. Ind. 1980, 34,
355-377
5 Dyr, J. and Uher, J. Czech. Pat. 134,003, 1970
6 Grog, I. Kern. Ind. 1983, 32, 575-578
7 Veljkovi6, V. B., Lazi6, M.L., Ruti6, D. J. and Stankovi6,
M. Z. Enzyme Microb. Technol. 1988, 10, 440-441
8 Manski, W., Skurska, Z. and Nawrocki, J. Archiwum lmmuno-
logii i Terapii Doswiadczalnej 1953, 1, 321-334
9 Haldorsen, K. M. J. Chromatogr. 1978, 150, 485-490
10 Veljkovi6, V. B., Lazi6, M. L., Ruti6, D. J. and Stankovi6,
M. Z. Acta Bioteehnol. 1989, 9, 397-400

Enzyme Microb. Technol., 1990, vol. 12, September 709