Biotechnology Letters, Vol 19, No 1, January 1997, pp.

45–47

11
Fermentation parameters influencing
higher alcohol production in the
tequila process
Leticia Pinal1, Miguel Cedeño2, Humberto Gutiérrez3 and
11 Jaime Alvarez-Jacobs1*
1
División de Biotecnología, Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco
(CIATEJ), Av. Normalistas 800, Guadalajara Jal. 44270 MEXICO. 2Destiladora González González, Puerto Altata
1131, Guadalajara Jal. 44330 MEXICO. 3Departamento de Matemáticas, Centro Universitario de Ciencias Exactas e
Ingeniería, Universidad de Guadalajara, Blvd. Marcelino García Barragán y Calz. Olímpica, Guadalajara Jal. 44840
MEXICO.

In the fermentation process of tequila, the type of yeast strain, the temperature (30 or 35°C) and the C/N ratio
(62 or 188) had a significant influence in the level of higher alcohols produced. On the contrary, other parameters
11 like nitrogen source [urea or (NH4)2SO4] and inoculum amount (5% or 10%), have little or no influence on the produc-
tion of the compounds responsible for some of the organoleptic properties of tequila.

Introduction Yeast growth

Tequila is a distilled beverage derived from the fermen-
tation of the cooked core of the blue agave (Agave tequil-
ana Weber), in which the main source of fermentable
sugar is fructose. As in many other alcoholic fermenta-
11 tions, along with ethanol production several compounds
are produced, conferring to the final product particular
organoleptic characteristics (Romano and Suzzi, 1993).
Among these compounds, some of the most important,
are the higher alcohols, nevertheless having a high thres-
hold, they usually appear in relatively high amounts.
Several reports have been published, regarding the
production of higher alcohols, in beer (Engan, 1981),
in wine, as well as in distilled beverages, and it is
commonly accepted that the formed amounts of such
11 compounds depend on the nitrogen metabolism and
culture conditions (Berry and Watson, 1991). However,
very little is known about the tequila process (Cedeño,
1995). In this work we present results about the influ-
ence of process parameters, such as type of yeast strain,
temperature, inoculum size, C/N ratio and type of
nitrogen source in the final level of isobutyl alcohol,
isoamyl alcohol and ethanol in agave fermented wort.
Materials and methods
11 Strains used
Saccharomyces cerevisiae L-041 (isolated from wine broth)
and L-047 (from bakery), belong to the CIATEJ
1 Collection of Strains and Genes.
Both strains were grown in a tequila production
medium, in which 51% of the sugar came from cooked
agave juice and the rest from standard table sugar (96%
sucrose) plus 1g (NH4)2SO4/ l. The final sugar content
was 54 g/l. The growth time was 24 h for solid culture,
after which a loopful was taken to inoculate flasks with
180 ml of broth, for a 10-hour cultivation in an orbital
shaker at 30°C and 250 rpm. The final population using
a Neubauer chamber was 240–250 × 106 cells/ml with
100% viability.
Fermentation
Experiments were performed using a 25 factorial
arrangement. The variables and their levels were: Strain,
winery and bakery; temperature, 30 and 35°C; C/N,
62/1 and 188/1; nitrogen source, (NH4)2SO4 and urea,
inoculum amount, 5% and 10%. Fermentation was
carried out in 1 l erlenmeyer flasks with 800 ml of initial
volume, for 24 hours, after which time, the amount of
higher alcohols was determined. The culture medium
was similar to that used for growth, with the exception
of the final sugar concentration, 108 g/l.
Analytical procedures
Total reducing sugars were determined by the
phenol/sulfuric acid technique. Total alcohols were also
determined using the potassium dichromate procedure.

© 1997 Chapman & Hall Biotechnology Letters · Vol 19 · No 1 · 1997 45

 L. Pinal et al.
11 Ethanol and higher alcohols were determined by gas
chromatography with flame ionization detector.
Statistical analysis
An Analysis of Variance (ANOVA) was performed
according to Montgomery (1991), using the Statgraphics
software. The response variables were final concentra-
tions of isoamyl and isobutyl alcohol .
11 Results and discussion
Preliminary kinetic experiments
Kinetic experiments in fermentation conditions (35°C,
C/N 188/1 using ammonium sulfate, 10% v/v inoculum)
were performed, in order to determine growth, viability,
sugar consumption and ethanol and higher alcohol pro-
duction. Similar growth, viability, sugar consumption
and ethanol production was found for both strains.
However, different amounts of higher alcohols were
produced by strains L-041 and L-047 (figures 1 and 2),
11 consistent with several reports for alcoholic beverages,
in which different strains produced different amounts of
higher alcohols (Engan, 1981; Kunkee and Amerine,
11
Figure 1 Sugar consumption and ethanol and higher
alcohol production by strain L-041.
11
11
Figure 2 Sugar consumption and ethanol and higher
1 alcohol production by strain L-047.
46 Biotechnology Letters · Vol 19 · No 1 · 1997
1970; Ramsay and Berry, 1981). Ethanol production
started in the the first hours of the fermentation kinet-
ics, ending along with the growth. It was also found,
for both higher alcohols determined, that production
started when the sugar level substantially lowered, and
continued for several hours after the alcoholic fermen-
tation ended.
These results are consistent with the findings reported
for other beverages, in which the higher alcohols appear
at this stage of the alcoholic fermentation (Paterson and
Piggott, 1989). This suggests a change of the metabolic
status of the yeast, since the generation of these prod-
ucts is a consequence of the activation of the aminoacid
metabolism (Berry and Watson, 1991).
These findings confirm the empirical belief in the tequila
industry, that tequila produced with a slow fermenta-
tion process (24 hours or more) is richer in flavor and
aroma, in comparison with that produced in a fast
process (18 to 20 hours). It is also clear that there is
an important difference in higher alcohol production
Figure 3 Pareto diagrams for isoamyl alcohol production.
Figure 4 Pareto diagram for isobutyl alcohol production.
 Fermentation parameters influencing higher alcohol production in the tequila process
11 between the two strains used, which supports the idea
that the type of yeast employed plays an important role
in the organoleptic characteristics of tequila, perhaps
more important than the type and amount of agave used.
Evaluation of fermentation conditions
The influence of the different parameters tested was
evaluated by an ANOVA test. The results were summa-
rized in the Standardized Pareto diagrams depicted in
11 figures 3 and 4. The ‘effect level’ in the Pareto diagrams
is the difference in higher alcohol production between
the two levels of the variable tested (i.e. 30 and 35°C),
divided by the standard deviation.
It is clear that the most important factor influencing both
higher alcohol production is the strain used, followed by
C/N ratio and the combination of both factors. There are
several reports in distilled and in non- distilled beverages
that agree with these results, which are not surprising,
11 since higher alcohol production is an intrinsic property
of the metabolism of each strain. Five-fold differences in
higher alcohol production by brewery yeasts had been
reported (Engan, 1981; Ramsay and Berry, 1981).
Regarding C/N ratio, it was found, for both strains, that
low C/N levels result in low amounts of isoamyl alcohol:
19 mg/l in strain L-041 and 30 mg/l for L-047 vs. 27
and 64 mg/l respectively for high C/N level. These
trends are also valid for isobutyl alcohol production.
11 High C/N ratio mean a low nitrogen level, which might
provoke deamination reactions of aminoacids, leading
to the synthesis of higher alcohols, according to previous
reports (Ouchi et al., 1980; Ramsay and Berry, 1981).
Temperature was another factor with significant effect
on higher alcohol production, as well as the association
of this parameter with other variables, such as strain and
C/N ratio. For instance: in isoamyl alcohol production,
at 30°C the produced amounts were 21 and 39 mg/l for
strains L-041 and L-047 respectively; while at 35°C, the
11
values were 23 and 57 mg/l. It is well documented that
this variable has an clear role in the formation of volatile
compounds (Engan, 1981).
11
The type of nitrogen source, the amount of inoculum
and the combination of these factors, had little or no
significant effect on isoamyl or isobutyl alcohols, in
agreement with several reports (Berry and Watson,
1991).
The different fermentation parameters tested in this
study are perhaps five of the most important, however,
there are others of importance, as the carbohydrate
composition: in the 100% agave type of tequila the
sugar source is only fructose from inuline, in contrast
with the ‘mixed’ type having up to 49% of other kinds
of sugars, mainly sucrose. It is well known that the
100% agave has a higher amount of volatiles. The real
effect of the carbon source remains to be studied.
Acknowledgement
One of the authors (L.P.), held a scholarship from
CONACYT, México.
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