Appl Microbiol Biotechnol (1988) 29:208-210
Microbiology
Nucleic acid reduction in yeast
Rosa Alvarez and Antonio Enriquez
Centro Nacional de Investigaciones Cientificas, Departamento Microbiologia Industrial, Apartado 6880, C. Havana, Cuba
Summary. A method for reduction of nucleic acid
levels in preparations of the yeasts Saccharomyces
cerevisiae and Kluyveromycesfragilis by means of
alkaline treatment has been developed. Under
similar conditions (4.5% NH4OH, 65 ° C, 30 min) a
low nucleic acid content (less than 2%) was ob-
tained for both strains. Higher losses of proteins
and biomass were obtained with K. fragilis than
with S. cerevisiae.
Introduction
High nucleic acid content and low cell wall diges-
tibility are two of the most important factors lim-
iting the nutritional and toxicological value of
yeast for animal and/or human consumption
(GAP 1975, 1976; Lovland et al. 1976).
By means of suitable treatment, a proteina-
ceous product with a higher quality can be ob-
tained, not only due to nucleic acid removal, but
also because the extraction methods may possibly
increase protein digestibility and the functional
properties of yeast-supplemented foods (G6mez
and Viniegra 1977; Gierhart and Potter 1979).
Different physicochemical treatments have
been used to obtain the partial disintegration of
cell wall structure, the main factor in digestibility
of yeasts, as well as reduction in nucleic acid lev-
els (Lindblom and Mogren 1974; Vananuvant and
Kinsella 1975). Some workers have employed
acids and alkalis plus high temperatures for this
purpose (Maul et al. 1970; Otero and Cabello
1980; Alvarez and Aguila 1983; Viikari and Linko
Offprint requests to: R. Alvarez
Applied
Biotechnology
© Springer-Verlag 1988
1977). The purpose of the present work was to ob-
tain the reduction of nucleic acid levels in two
yeast strains by a moderate treatment with
NHaOH. Saccharomyces cerevisiae (baker's yeast),
widely used in several foods, and Kluyveromyces
fragilis, proposed as one of the more suitable
yeasts for human consumption (Contreras and
Vald6s 1984), were employed.
Materials and methods
Microorganisms used
S. cerevisiae (baker's yeast). Fresh cake yeast (25% dry matter)
obtained from a factory was employed. The material was kept
at 4 ° C for no more than 7 days.
K. fragilis L/1930. Growth of the culture was carried out in a
Biolafitte stirred fermentor (1 m 3 capacity). Sugarcane mo-
lasses containing 2% reducing sugars as a carbon source was
fed to the fermentor, supplemented with (NH4)2SO,, 1.6 g/l,
(NH4)zHPO4 1.08 g/1 and urea 0.73 g/1. The fermentation was
carried out at 34°C, pH 4.0, air 0.93 vvm and 200 rpm. Yeast
growth was monitored by cell counts at timed intervals. Under
the described conditions a specific growth rate (It) of 0.71 h - 1
and a final cell concentration of 1.5 x 109 celis/ml were ob-
tained.
At the end of the exponential growth phase, the biomass
was centrifuged and washed once with tap water, before ex-
perimental use.
Thermal treatment with N H 4 0 H
Yeast suspensions containing 14% dry matter, were put in a
reactor of 2-1 capacity with mechanical stirring. Treatments
with 0.33--13% of NHaOH (related to yeast dry matter) were
assayed for 30 rain at 45 °, 50 °, 60 °, 65 °, 70 °, 75 ° and 80 ° C. In
all cases, the suspension needed 15 min to reach the chosen
temperature. Samples were taken at the end of the process and
analysed.
R. Alvarez and A. Enriquez: Nucleic acid reduction 209

90-

80-

30- 13%

70- 5%
o
20- 2,5%

c 60- E
o
~6 o 10-
~5
~ 50-

'
40 5'o '
60 7'o ''
80
o 40- Temperature (°C)
ilJ

-- 30-
z 30-

to
20- 20-
o
i,//1'~_415 %
Fig. 1. Influence of incuba-
10-
.= 6.5°,°
;5% tion temperature on reduction
2 ~o-
13- of nucleic acid levels and pro-
tein and biomass losses in
I I I I ~ 0 i i [ i i m Saccharomyces cerevisiae at
40 50 . 60 70 80 40 50 60 70 80 different concentrations of
Temperature {°C } Temperature (°C) NHnOH

Analytical determinations Results and discussion

Gravimetric dry matter. Yeast suspension (10 ml) was centri-
fuged at 6000 rpm for 15 min, washed twice with distilled wa-
ter and dried at 80°C to constant weight.
Nucleic acid content. The nucleic acid content was determined
by a simplified spectrophotometric method, based on removal
of nucleic acid with 0.5 N HC104 at 90°C for 20 rain and
measuring the supernatants at 270 and 290 nm (Rut 1973).
Protein content. Lowry's method was used to determine the
protein content in cells and supernatants (Lowry et al. 1951).
As can be observed in Fig. 1, removal of nucleic
acids and biomass losses increased with tempera-
ture. It is significant that the highest protein
losses were obtained between 50 ° and 55°C,
probably indicating that proteases reached maxi-
mal activity in this temperature range. This effect
was enhanced at higher ammonia concentrations,
suggesting that the temperature should increase to

~ 80- 40-

J 40-
J
~ 60-
P o
30-
o
30-

rT
Y
~ ~O- ,c 20- oE 20-
o
/3- m

z 20- 10- 10-

Fig. 2. Reduction of nucleic
acid levels and protein and bio-
mass losses in Kluyveromyces
O- I I - 0 I I ii. 0 I I fragilis at different concentra-
4 8 4 8 4 8 tions of NHnOH. ( O - 0)
% NH4OH referred to yeast dry matter) 65°C; ( 0 - - 0 ) 70°C
210
65 °C as quickly as possible, in order to minimize
the activity of these enzymes.
In the experiments with K. fragilis at 65°C
and 4.5% NH4OH (Fig. 2), a reduction in nucleic
acid content close to 90% was observed. This rep-
resents a final nucleic acid content of nearly 1.2%
of the biomass. A higher incubation temperature
(70 ° C) did not notably increase the reduction of
nucleic acid levels, but protein and biomass losses
increased considerably, showing that the treat-
ment was severe and that cellular integrity was de-
stroyed. Therefore, incubation temperatures
above 65°C seem unsuitable, since the protein
and biomass losses were higher, while nucleic
acid reduction was negligible.
In Fig. 3, the two strains of yeast are com-
pared under the same conditions (65°C, 4.5%
NHaOH). K. fragilis, with an initial nucleic acid
content of 12.08%, reached a lower final nucleic
acid content (1.2%), compared with S. eerevisiae,
which had a final content of 1.4%, despite its
lower initial content (7%). Regarding protein and
biomass during the treatment, K. fragilis showed
higher percentage losses. This effect increased
with temperature and ammonia concentration.
~ 80-
g GAP (1976) Informe de la Reuni6n del Grupo Especial de tra-
6o-
"13
~ Z.O-
g c
o
~ 20-
-5
Z
o i --'-
o Z
NH~OH (°/0]
30
~ 20
Ol r i
0 t.,
NHt.ON (%)
Fig. 3. Comparison of nucleic acid levels and protein and bio-
mass losses between the strains of S. cerevisiae (© -- O) and K.
fragilis ( 0 - - 0 ) after 30 min treatment at 65 °C
R. Alvarez and A. Enriquez: Nucleic acid reduction
The strain of S. cerevisiae showed higher resist-
ance to ammonia attack, probably due to more re-
sistant cell wails, as indicated by the lower pro-
tein, biomass and nucleic acid losses. This aspect
is very important, because it shows the possibility
of obtaining similar results in K. fragilis with less
energetic treatments. This also suggests the neces-
sity of a proper balance in the variables, in order
to obtain .adequate reduction in nucleic acid lev-
els with minimal losses of protein and biomass.
The treatment with NH4OH shows several ad-
vantages in relation to other chemical agents.
With relatively low concentrations (4.5%), a final
nucleic acid content of less than 2% for both
strains can be obtained. In addition, the use of
ammonia is more suitable from the point of view
of its elimination or/and utilization of residues
after the treatment.
References
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nucleicos y proteinas durante el tratamiento t6rmico con
hidr6xido de amonio en levadura panadera. Rev Cien Biol
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6
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Received March 19, 1987/Accepted October 5, 1987