Appl Microbiol Biotechnol (1988) 29:208-210 Applied Microbiology

Biotechnology © Springer-Verlag 1988

Nucleic acid reduction in yeast

Rosa Alvarez and Antonio Enriquez

Centro Nacional de Investigaciones Cientificas, Depar tamento 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.

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.

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

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 NH40H

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 -

70-

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~ 50-

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z 30-

20-

10-

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I I I I ~ 0 40 50 . 60 70 80

Temperature {°C }

13%

5% 2,5%

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

i , / / 1 ' ~ _ 4 1 5 %

.= 6.5°,° ; 5 %

i i [ i i m 40 50 60 70 80

Temperature (°C)

Fig. 1. Influence of incuba- tion temperature on reduction of nucleic acid levels and pro- tein and biomass losses in Saccharomyces cerevisiae at different concentrations of NHnOH

Analytical determinations

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).

Results and discussion

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-

~ 60- P

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Fig. 2. Reduction of nucleic acid levels and protein and bio- mass losses in Kluyveromyces fragilis at different concentra- tions of NHnOH. ( O - 0 )

65°C; ( 0 - - 0 ) 70°C

210 R. Alvarez and A. Enriquez: Nucleic acid reduction

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-

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a. 10-

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Z NH~OH (°/0] NH 4 OH (%)

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~ 20

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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

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

Alvarez R, Aguila B (1983) Cin6tica de liberaci6n de ~cidos nucleicos y proteinas durante el tratamiento t6rmico con hidr6xido de amonio en levadura panadera. Rev Cien Biol 14:345--354

Contreras R, Vald6s I (1984) Yeast selection for human con- sumption. Biol6gia (Bratislava) 39: 707--715

GAP (1975) Informe de la Reuni6n del Grupo Especial de tra- bajo del GAP sobre evaluaci6n clinica y concentraciones aceptables del hcido nucleico en las PUC destinadas al consumo de los seres humanos. Bol GAP 5:20--30

GAP (1976) Informe de la Reuni6n del Grupo Especial de tra- bajo del GAP sobre proteinas unicelulares. Sol GAP 6:1-- 6

Gierhart DL, Potter NN (1979) Effects of ribonucleic acid re- moval methods on proteolytic activity and protein solubil- ity in Candida utilis. Biotechnol Bioeng 21:1963--1980

G6mez J, Viniegra G (1977) Extracci6n del hcido ribonucleico (ARN) de Saccharomyces cerevisiae en condiciones alcali- nas suaves. Rev Soc Quim M6x 21:97--102

Lindblom M, Mogren H (1974) The influence of alcali and heat treatment on yeast protein. Biotechnol Bioeng 16:1123--1130

Lovland J, Harper JM, Frey AL (1976) Single cell protein for human food. Lebensm Wiss Technol 9:131--142

Lowry OH, Rosebrough NJ, Farr AL (1951) Protein measure- ment with the folin reagent. J Biol Chem 193:265--275

Maul SB, Sinskey A J, Tannenbaum SR (1970) A new process for reducing the nucleic acid content of yeast. Nature 228:181

Otero MA, Cabello A (1980) Single cell protein low in nucleic acids by alkaline treatment. Biotechnol Lett 4:149--152

Rut M (1973) Determination of nucleic acids in yeast. Kvasn~r Prymysl 19:131--133

Vananuvant P, Kinsella JE (1975) Extraction of protein low in nucleic acid, from Saccharomyces fragilis grown contin- uously on crude lactose. J Agric Food Chem 23:216--221

Vfikari L, Linko M (1977) Reduction of nucleic acid content of SCP. Proc Biochem 16:17-- 19

Received March 19, 1987/Accepted October 5, 1987