The Ability of the Rumen Ciliate Protozoan Diploplastron Affine to Digest and Ferment Starch

teria, the ciliates were incubated overnight with ampicil-lin, streptomycin and chloramphenicol prior to the homogenization. The amylolytic activity of the ciliates was examined by quantification of products released during incubation of the cell extract with appropriate substrates. Fractionation of the crude enzyme preparation was performed by molecular filtration using a glass column packed with Sephadex G-150. Enzymes were identified by a zymography technique following separation of Abstract The ciliate Diploplastron affine is known as a common species of the rumen fauna in cattle and sheep. This protozoon is able to digest cellulose, whereas its amylolytic activity is not well known. The objective of the reported studies was to examine the ability of D. affine to digest starch and to use this polysaccharide to cover the requirement for energy. The enzymatic studies showed that the protozoal cell extract degraded starch to reducing products with the rate being equivalent to 2.4± 0.47 μmol/L glucose per mg protein per min. Maltose, maltotriose and a small quantity of glucose were the end products of starch degradation. The degradation rate of maltose was only 0.05 μmol/L glucose per mg protein per min. Two peaks in α-amylase and a single peak in maltase activity were found following molecular filtration of ciliate cell extract, whereas three starch-degrading enzymes were identified by a zymographic technique. Incubation of the bacteria-free ciliates with starch in the presence of antibiotics resulted in a release of volatile fatty acids with the net rate of 25 pmol per protozoan per h. Acetic acid followed by butyric acid was the main product of starch fermentation. The results confirmed the ability of D. affine to utilize starch in energy-yielding processes.

Abstract The ciliate Diploplastron affine is known as a common species of the rumen fauna in cattle and sheep. This protozoon is able to digest cellulose, whereas its amylolytic activity is not well known. The objective of the reported studies was to examine the ability of D. affine to digest starch and to use this polysaccharide to cover the requirement for energy. The enzymatic studies showed that the protozoal cell extract degraded starch to reducing products with the rate being equivalent to 2.4± 0.47 μmol/L glucose per mg protein per min. Maltose, maltotriose and a small quantity of glucose were the end products of starch degradation. The degradation rate of maltose was only 0.05 μmol/L glucose per mg protein per min. Two peaks in α-amylase and a single peak in maltase activity were found following molecular filtration of ciliate cell extract, whereas three starchdegrading enzymes were identified by a zymographic technique. Incubation of the bacteria-free ciliates with starch in the presence of antibiotics resulted in a release of volatile fatty acids with the net rate of 25 pmol per protozoan per h. Acetic acid followed by butyric acid was the main product of starch fermentation. The results confirmed the ability of D. affine to utilize starch in energy-yielding processes.

Introduction
The ciliate Diploplastron affine is a common species of rumen fauna in the domesticated ruminants (Dogiel 1927). To date, the fibrolytic properties of this protozoan were studied by Coleman (1985) whereas its ability to digest and utilize starch is not well known. The objectives of the reported studies were to examine the ability of D. affine to digest starch and to use this polysaccharide to cover the requirement for energy.

Material and methods
The ciliates were isolated from the rumen fluid of sheep. The cell extract for enzymatic studies was obtained by homogenization of purified protozoa and removal of the particulate matter by centrifugation. To restrict the bac-    protein on polyacrylamide gel (Gabriel and Wang 1969). Volatile fatty acids (VFA) were measured chromatographically during the incubation of the protozoa with starch and antibiotics.

Results and discussion
Ciliate D. affine possesses enzymes degrading starch and its derivatives. Protozoal cell extract prepared from ciliates incubated with antibiotics released reducing sugars from starch, dextrin, maltose and isomaltose (Fig. 1,  Table 1). On the other hand, pullulanase activity was not found (Table 1). Similar results were obtained earlier when amylolytic activity of Eudiplodinium maggii was studied (Bełżecki et al. 2007 (Table 2). This indicates that the products of starch degradation were utilized to cover the requirement of ciliates for energy. The determination of amylase activity after the separation of protozoal protein by molecular filtration revealed that this activity formed two distinct peaks (Fig. 2). The single peak of maltase activity was also present there. Conversely, Bełżecki et al. (2007) found only a single peak of amylolytic activity when the crude enzyme preparation of E. maggii was fractionated by ion exchange chromatography. On the other hand, three starch-degrading enzymes were identified by a zymographic technique following the separation of D. affine protein by native polyacrylamide gel electrophoresis (Fig. 3). For comparison, four such enzymes were found in E. maggii crude enzyme preparation (Bełżecki et al. 2007). Ciliate D. affine belongs to the rumen microorganisms which are able to digest starch and to utilize the obtained products as a carbon source in energyyielding processes.
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