Testing of Some Enzymatic Levels in Ocna Sibiului Lakes Therapeutic Mud

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1 Acta Universitatis Cibiniensis Seria F Chemia 9(2006-1):21-28 Testing of Some Enzymatic Levels in Ocna Sibiului Lakes Therapeutic Mud Abstract Neli Darie 1*, Letitia Oprean 1, Claudia Felicia Ognean 1 and M. Ognean 1 The Ocna Sibiului lakes therapeutic mud, produced in time by geological and biochemical transforming, have significant enzymatic activities. The work s aim was to study the correlation between the mud s three enzymatic activities and the therapeutic effect in various seasons of the year by estimating the phosphatase, dehydrogenase and catalase activities in the eight lakes mud. The samples have been collected in winter 2005 (in February) and in summer 2005 (in June). The experimental results show a season influence on dehydrogenase and catalase activities (great activities in summer) and phosphatase (great activity in winter). Keyword: therapeutic mud, Ocna Sibiului lakes, dehydrogenase, catalase, phosphatase. I. Introduction Ocna Sibiului lakes muds have appeared after geological and biochemical long time transforming, in which a wide range of microorganisms were involved. Their activity have continued after the mud s formation and the activity consequences were the constituents changes and also black and dispersed mud s genesis, which have therapeutic properties [1,2]. 1 Lucian Blaga University, Faculty of Agricultural Sciences, Food Industry and Environmental Protection, 7-9 Dr.I.Ratiu St., Sibiu, Romania * To whom the correspondence should be addressed. 21

2 Neli Darie, Letitia Oprean, Claudia Felicia Ognean and M. Ognean The mud s basic compounds are very fine clay and clayey marl particles. The mud s black bluish color is due to the considerable content of iron sulphide, which has therapeutic effects by easy decomposition. Mud looses his black color in few hours and turning into grey, in presence of the air, because of the iron sulphide oxidation into iron oxide or colloidal iron hydrate. After the dense mud formation, in the most deeply layers have begun a slow destroy consisting of mud gradual content in the marl layer from the bottom of the lake [3]. The mud destroy is the outcome of colloids degradation, sediments gradually mineralization and water gradually release. In the profound layer, there was proceeded stratification and the clay quantity has risen as consequences of the feldspars transformation. It has pointed out a decrease of the enzyme activity. Some scientific papers quote certain enzymes (as saccharase, protease, urease, phosphatase, dehydrogenase and catalase) which play a major role in establishing the mud s therapeutic properties. Carried out studies on the Sovata lakes mud emphasizes a greater enzymatic activity in the unctuous, black and grey mud than that in the sandy mud [4, 5]. In the study there were investigated the relationship between the enzymatic activities of the mud s phosphatase, dehydrogenase, catalase and the mud s therapeutic effect in the various seasons of the year. II. Experimental Mud samples The enzymatic studies have been carried out on the mud s samples from eight various Ocna Sibiului lakes (table 1). Sampling activities have taken place in two different batches, one collected in winter 2005 and one in summer Table 1 Mud s samples analyzed by enzymatic methods Crt.nr. Source (lakes name) Mud s features 1. Visa soft consistency, dark grey color 2. Lacul mâńelor soft consistency, dark grey color 3. Gura minei sandy consistency, grey yellowish color 4. Crişan soft and unctuous consistency, dark grey color 5. Lacul negru unctuous consistency, black grey color 6. Ocna pustie soft and clayey consistency, grey color 7. Lacul cu nămol unctuous consistency, dark grey color 8. OcniŃa clayey consistency, grey yellowish color Enzymatic activities assays [6,7] a. Dehydrogenase activity Organic substances, preexistent in the mud, can donate hydrogen, under dehydrogenase s action, to the 2, 3, 5-triphenyltetrazolium which color has modified from colorless to red (formasane). After extraction by acetone, the formasane has been spectraphoto-metrically measured at 485 nm. The formed formasane quantity, as a straight 22

3 Acta Universitatis Cibiniensis Seria F Chemia 9(2006-1):21-28 proportion of the dehydrogenase s activity, is expressed as µg formasane / mud g. Parallel, a control sample and a standard one have been done and in this case, the 2,3,5- triphenyltetrazolium reduction to formasane has been done by zinc powder. b. Phosphatase activity Phosphatase rich mud sample has been treated with a specific substratum, disodium phenyl phosphate, which after the enzymatic hydrolysis at ph 9, 4 has broken into phenol and disodium phosphate. Cibba reactive (2, 6-dibromoquinone chloramines) has fitted the produced phenol and a specific indophenol blue color has developed. The outcome indophenol has photoed colorimetric assayed and there has been found a positive correlation between the outcome indophenol s concentration and the phosphatase activity, expressed as µg phenol/mud g. A standard curve has been created using phenol solutions well-known concentrations as standard sample. c. Catalase activity Mud catalase decomposes the hydrogen peroxide substratum at ph 6, 8. After the incubation time, sulphuric acid has inhibited the enzymatic reaction and the undesired compounds have removed by filtration technique. The excess hydrogen peroxide was permanganometrically assayed and the catalase activity has been expressed as mg H 2 O 2 / mud g. As standard sample, there has been used a without mud sample but containing hydrogen peroxide substratum. III. Results and discussions The experimental data performed on the mud s samples collected from the eight Ocna Sibiului lakes have been given in tables 2-4 and the enzymatic activities variation in accordance with seasons in figures 1-3. All summer collected mud samples have significant phosphatase, dehydrogenase and catalase activities. The dehydrogenase activity is due to the capable to proliferate lively micro flora and the experimental data revealed high values for summer period in mud samples collected from Crişan lake (3.59 µg), OcniŃa lake (2.64 µg), Lacul cu nămol (2.08 µg) and Lacul mâńelor (2.00 µg). The other mud lakes do not exhibit a high dehydrogenase activity. An important phosphatase activity has been revealed and acquired values have been represented. As the phosphatase is accumulated by the organic mineral bio-components there cannot be establish a good correlation between dehydrogenase and phosphatase activities. The greatest phosphatase activity was found in OcniŃa lake ( µg) followed by Crişan lake ( µg) and Lacul cu nămol (92.64 µg). Table 2 The enzymatic activities * in the collected mud from the Ocna Sibiului salt lakes in summer 2005 (June) Source Dehydrogenase activity Phosphatase activity Catalase activity (lakes name) (µg formasane/g mud) (µg phenol/g mud) (mg H 2 O 2 /g mud) Visa Lacul

4 Neli Darie, Letitia Oprean, Claudia Felicia Ognean and M. Ognean mâńelor Gura minei Crişan Lacul negru Ocna pustie Lacul cu nămol OcniŃa * Average values (n = 5) The catalase activity shows relatively high oxidation-reduction potential of the collected mud from the salt lakes. In the eight analyzed lakes, meaningfully catalase activities have been found out in the mud from OcniŃa Lake (3.64 µg), Crişan lake (2.55 µg) Lacul cu nămol (1.98 µg) and Lacul negru (1.23 µg). The data analysis of the collected samples in winter period (February 2005) exhibit a dehydrogenase activity decreasing for mud samples of the referred lakes having a significant enzymatic activities. The values obtained for this period of the year was Crişan Lake (1.32 µg), OcniŃa lake (0.61 µg), Lacul cu nămol (0.42 µg) and Lacul mâńelor (0.33 µg). A relatively high phosphatase activity has been registered in the cold season, slightly higher than that obtained in the summer season. In winter season the mud lakes having greater phosphatase activity are the Lacul cu nămol ( µg), OcniŃa Lake ( µg), Crişan lake ( µg) and Lacul negru ( µg). Concerning catalase activity detected in winter season it is slightly decreasing comparatively with summer catalase activity. From the eight lakes subjected in this study, the greatest catalase activity has been found in the OcniŃa lake (0.98 µg) followed by Crişan lake (0.69µg), Lacul cu nămol (0.47µg) and Lacul negru (0.31µg) mud. The other studied lakes mud show lower catalase values, about 0.24 µg average. Table 3 The enzymatic activities * of the collected mud from the Ocna Sibiului salt lakes in winter 2005 (February). Source (lakes name) Dehydrogenase activity (µg formasane/g mud) Phosphatase activity (µg phenol/g mud) Catalase activity (mg H 2 O 2 /g mud) Visa Lacul mâńelor Gura minei Crişan Lacul negru Ocna pustie Lacul cu nămol OcniŃa * Average values (n = 5) 24

5 Acta Universitatis Cibiniensis Seria F Chemia 9(2006-1):21-28 Analyzing the average values, for each season, there was found out that dehydrogenase, phosphatase and catalase enzymatic activities depend upon the seasons, as shown in figures 1, 2 and 3. dehydrogenase activity (µg formasane/g mud) 4 3,5 3 2,5 2 winter 2005 summer ,5 1 0,5 0 Visa Lacul mâńelor Gura minei Crişan Lacul negru Ocna pustie Lacul cu nămol OcniŃa Fig. 1. Seasonal dehydrogenase activity evolution in saline s mud. In winter months the dehydrogenase activity is much lower than in the summer ones, while the phosphatase activity average is greater in winter ( µg) than in summer (84.05 µg). 25

6 Neli Darie, Letitia Oprean, Claudia Felicia Ognean and M. Ognean 250 autumn 2005 phosphatase activity (µg phenol/g mud) Visa Gura minei Pânzelor Lacul negru Poporului Brâncoveanu Baza de tratament Rândunica summer 2005 lakes muds Fig. 2. Mud s phosphatase activity modification in the seasons of the year. The average catalase activity is about four times lower in winter than in the summer period. Table 4. The average values for the seasonal enzymatic activities in the different salt lakes mud from Ocna Sibiului. Type of enzymatic activity Season winter summer Dehydrogenase (µg formasane/g mud) Phosphatase (µg phenol/g mud) Catalase (mg H 2 O 2 /g mud)

7 Acta Universitatis Cibiniensis Seria F Chemia 9(2006-1):21-28 catalase activity (mg H2O2/g mud 5,0 4,5 4,0 3,5 3,0 2,5 2,0 1,5 1,0 0,5 0,0 Visa Lacul mâńelor Gura minei Crişan Lacul negru Ocna pustie Lacul cu nămol OcniŃa summer 2005 winter 2005 lakes muds Fig. 3. Seasonal catalase activity variation of lakes mud from Ocna Sibiului. IV. Conclusions The enzymatic activity study, in eight saline mud lakes from Ocna Sibiului, collected both in winter and summer seasons, has shown that mainly three enzymatic activities were highlighted: dehydrogenase, phosphatase and catalase activities. The experimental data have concerned the seasonal influences on enzymes activities, sustaining that season influences are great enough to modify the enzymatic activity. Thus the dehydrogenase and the catalase have the maximum activities detected in summer period while the phosphatase has the maximum activity in the cold season. There was no positive correlation noticed between the phosphatase activity and the dehydrogenase one. The therapeutic mud from the lakes OcniŃa, Lacul cu nămol, Crişan and Lacul negru have dehydrogenase, phosphatase and catalase activities over the average seasonal values. On the basis of the experimental data it can be considered that the eight studied saline lakes mud are enzymatic ally active and are also rich in microorganisms, making them interesting for therapy applications. V. References 1. Panzaru, T.G., Complexul lacustru de pe masivul de sare de la Ocna Sibiului, Instit. Pedag., Oradea, Por, F.D., Marine Ecolog., 1980, 1, Griffiths, R.P., Mar. Pollut Bull., 1983, 14,

8 Neli Darie, Letitia Oprean, Claudia Felicia Ognean and M. Ognean 4. Kiss, S., Radulescu, D., Dragan-Bularda, M., Bulgareanu, C.V.A., Studia Univ. Babes Bolyai. Biol., 1979, XXIV/2, Kreps, E., Organic catalysts or enzymes in sea water, 3 rd edn.univ.liverpool Press, Darie, N., Biochimie. Aplicatii practice, Ed.Univ.din Sibiu, Devlin, T.M. (ed.), Biochemistry and clinical correlations, 3 rd edn.,wiley Press, New York,

ADDVALUE LINE CELLULOSE DERIVATIVES ON OBTAINING LOW CALORIE SPREADING FROM HIGH FAT RAW MATERIAL. Abstract

V. Jascanu, et all. Journal of Agroalimentary Processes and Technologies, Volume XIII, No.1 (2007), 157-162 Full Paper Natural Food Extracts and Additives ADDVALUE LINE CELLULOSE DERIVATIVES ON OBTAINING