A bioreactor model system specifically designed for Tetrahymena growth and cholesterol removal from milk.

This work describes the configuration and operation of a bioreactor system especially designed for Tetrahymena cultivation and its use for milk improvement, particularly cholesterol elimination by the action of this cell. An advantage of the proposed method is the re-use of the growth medium; thus, the medium is used twice to provide two batches of Tetrahymena biomass without the need of further inoculation. This makes the procedure of producing the cell biomass faster and more economical. Cells are concentrated in the culture vessels by sedimentation at room temperature and then transferred to milk suspensions, where they can further grow for at least one generation with the benefit of reducing steeply cholesterol level. Milk treated according to this process is separated from the biomass by centrifugation. Under these conditions, less than 5% of the cells remain in the milk, and cholesterol elimination amounts to 75 +/- 10% of that initially present. No changes in sensorial properties of the milk, such as clotting or butyric odor, were observed as a result of this treatment. In addition, the bioreactor allows the aseptic recovery of the spent growth medium, which contains diverse enzymes of interest, and the cell pellets, to exploit particular lipids like phosphonolipids, abundant poly-unsaturated fatty acids and co-enzyme Q(8).

The use of Tetrahymena thermophila mutant cell line for removal of cholesterol from milk.

The nonpathogenic ciliate Tetrahymena thermophila converts cholesterol from foodstuffs into provitamin D compounds in high yields. However, prolonged incubation with wild-type strain CU-399 at high densities results in a final deterioration of milk properties, possibly as a result of secreted hydrolases. Here we attempted to solve this problem using MS-1 Tetrahymena strain, a stable mutant with a low rate of hydrolase secretion. Densities of to 2 x 10( 6 ) cells/ml can be incubated for up to 5 h in milk, without any clotting or change in appearance. Moreover, centrifugation of this suspension eliminates most of the cells, and results in an about 75% +/- 10 (n = 10) decrease of the initial cholesterol. Sterols are recovered in the cell pellets, which show that Tetrahymena is able to avidly capture them from the medium. Therefore, this mutant strain is optimal for milk cholesterol depletion, avoiding unfavorable sensory alterations.

A method for the preparation of Tetrahymena thermophila phospholipase A1 suitable for large-scale production.

A rapid and economical method for the purification of phospholipase A1 (PLA1) from the extracellular medium of the ciliate Tetrahymena thermophila is presented. Essentially, the procedure, here designated as purification by selective interaction (PSI), entails the incubation of media containing PLA1 with liposomes made of soy bean phospholipids. The PLA1-lipid complexes are precipitated by the addition of CaCl2 and collected by centrifugation. Elution of the PLA1 is effected by treating the complexes with 40% dimethylformamide, a reversible inhibitor of this enzyme, which is easily removed by dialysis. In combination with DEAE cellulose ion exchange chromatography, PSI yielded homogeneous PLA1 preparations with a 14% recovery and a 416-fold increase in specific activity. This procedure, which can be completed within 1 day, may prove useful for the isolation of phospholipases from other sources. This practical method for the purification of a microbial PLA1 opens the way to large-scale production of these types of enzyme, which are not as yet commercially available.

The role of secreted acid hydrolases in the utilization of complex nutrients byTetrahymena.

In an attempt to extend our knowledge of the biology of feeding of the ciliateTetrahymena thermophila, this organism was grown axenically on complex organic material. The nutrient substrate was based on autoclaved wheat grains and adjusted to either pH 5.5 or 7.5. In wild type cultures the cells grew and multiplied only under acidic conditions. In cultures of a mutant cell line blocked in the secretion of acid hydrolases the cells did not grow at either pH value. Thus released acid hydrolases may play a key role in the utilization of complex nutrients in combination with uptake of small organic molecules. Mechanisms in the feeding biology ofTetrahymena thermophila andParamecium tetraurelia are compared.