1

Department of Biochemical Technology

Faculty of Chemical Technology

Slovak University of Technology

Bratislava

2

Department of Biochemical Technology, Faculty of Chemical Technology,

Slovak University of Technology, Bratislava

Research Institute of Rheumatic Diseases, Piešťany

Study of biological effects of the mud and thermal

water from Piešťany

Research report (HZ 137/96)

Principal investigators: prof. Ing. Ján Fuska, DrSc.

Prof. MUDr. Jozef Rovenský, DrSc.

Co-investigators: Ing. Bohumil Proksa, DrSc.

Ing. Mária Šturdíková. CSc.

Ing. Mária Stančíková, CSc.

Ing. Karol Švík, CSc.

Bratislava, 1997

3

Contents

Subject of contract .................................................................................................................. 4

1. Preparation of the major metabolite F-12............................................................................... 5

1.1. Metabolites of the fungus Neosartorya fischeri .............................................................. 5

1.2. Submerged cultivation of the strain of Neosartorya fischeri F-12 .................................. 7

1.3. Analytical assessment of the cultivation process ............................................................ 7

1.4. Isolation of the metabolites of Neosartorya fischeri F-12 ............................................. 11

1.5. Isolation of metabolites from the strain of Sporidiobolus salmonicolor B4 ................. 20

2. Preparation of the mud fractions and their in vitro biological assessment ........................... 21

3. In vivo assessment of elastolytic effects of the mud fractions ............................................. 22

4. Isolation of technologically significant microorganisms and the study of the impact of heat

on their survival ........................................................................................................................ 30

4.1. Isolation of microorganisms .......................................................................................... 30

4.2. Characterisation of isolated strains of microorganisms ................................................ 30

4.3. Study of the impact of temperature on survival of technologically significant

microorganisms .................................................................................................................... 31

4.4. Assessment of the growth of isolated bacteria at different temperature applying the

turbidimetric method ............................................................................................................ 32

5. Determination of Se content in samples provided................................................................ 35

6. Analysis of the sulphur content in the provided samples of water ....................................... 37

7. Discussion of obtained results .............................................................................................. 39

8. Annex – microscopic image of isolated strains of bacteria .................................................. 41

4

Subject of contract

1. Preparation of the major metabolite F-12. Assessment of biological effects,

determination of physical and chemical characteristics.

2. Preparation of fractions of the mud and the in vitro biological assessment thereof

3. In vivo assessment of elastolytic effects of the mud fractions:

- administration immediately after induction of arthritis

- administration of the preparation with a delay

4. Isolation of technologically significant microorganisms and the study of the impact of

heat on their survival

5

1. Preparation of the major metabolite F-12

1.1. Metabolites of the fungus Neosartorya fischeri

The fungi belonging to the genus Neosartorya are producers of several biologically active

compunds. E.g. Neosartorya fischeri var. glabra biosynthesizes the metabolites NK372135

A-D, which contain reactive isonitrile functions in their structure. These compounds work

antibacterially, antifungally and they inhibit the activity of 5-lipoxygenase, i.e. they

interfere into the biosynthesis of prostaglandins [1].

NK3722135 A: R1 = H R2 = OCH3

B: R1 = R2 = OCH3

C: R1 = OH R2 = OCH3

D: R1 = H R2 = OH

Fiscalines A, B, C consisting of chinazoline and indole skeleton isolated from the

cultivation medium N. fischeri inhibited the bond of peptides working as neuromodulators

(neurotransmitters) for the respective receptors. The isolated substances have analgesic

and anti-inflammatory effects [2].

Fiscalin A Fiscalin B Fiscalin C

If the culture was cultivated in a neutral environment (pH 7) at a temperature of around

25-30 °C in the presence of easily usable source of carbon, the production of

fumitremorgins A, B, or verruculogen, was observed in some strains of N. fischeri. These

substances are characterized by an efficient effect on the central nervous system [3,4].

6

In Japan a metabolite labelled NFA has been isolated from the cultivation medium of N.

fischeri [5]. Detailed data on biological activity of this substance are not available.

The thermotolerant strain of the fungus Neosartorya fischeri produced glucoamylase, the

optimum activity of which was observed at a temperature of 55-60 °C and pH 4.0 – 4.4

[6]. This strain released also the enzyme amino acid oxidase into the medium [7].

Verruculogen

7

References:

1. Nishimoto M., Masuda A., Fujita S., Morino T., Nishigori T.: Japanese patent 06,

135 979, CA 121, 155 918 (1994).

2. Wong S.M., Musza L.L., Kyda G.C., Kulling R., Gillum A.M., Cooper R.: J. Antibiot.

46, 543 (1993).

3. Nielsen P.V., Beuchat L.R., Frisvad J.C.: Appl. Environ. Microbiol. 54, 1504 (1988).

4. Beuchat L.R., Nielsen P.V.: Bioact. Mol. 10, 7 (1989).

5. Fujimoto H., Ikeda M., Yamaraki M.: Tennen Yuki Kagobutsu Toronkai Koen

Yoshishu 32, 229 (1990), CA 115, 49 190 (1990).

6. Hang Y.D., Woddams E.E.: Food Sci. Technol. 26, 483 (1993).

7. Toyo J.: Japanese patent 5, 141 289, CA 98, 67 941 (1982).

1.2. Submerged cultivation of the strain of Neosartorya fischeri F-12

We cultivated the micromycet Neosartorya fischeri F-12 on the Czapek-Dox medium

containing (g/L): sucrose 60, maize infusion 20, sodium nitrate 2, cryst. potassium dihydrogen

phosphate 1.0, potassium chloride 0.5, cryst. magnesium sulphate 0.5, cryst. iron disulphate

0.01. After adjusting the pH to 6.5 we filled the culture flasks of a volume 500 ml each with

100 ml of the medium for each flask and sterilized it for 20 at a temperature of 120 °C. We

used two-stage cultivation. We inoculated the above mentioned sterile medium with 10 ml of

vegetative inoculum. The cultivation in the production phase lasted 192 hours at 28 °C on the

rotation shaking machine (3.7 Hz). We monitored the course of the cultivation process in 24-

hous intervals. In the collected samples we determined the concentration of reducing sugars

(RS), the amount of biomass, pH levels of the fermentation medium and the production of

secondary metabolites applying thin layer chromatography (TLC).

1.3. Analytical assessment of the cultivation process

We extracted the sample of the fermentation medium together with the mycelium (4 ml) using

a mixture of solvents chloroform : isopropyl alcohol (5:1) continually stirred.

By centrifuging (10 min, 3000 rev/min) we separated a layer of solvent used for the extraction

and we spread the sample obtained in this way (100 μl) on the plates SILUFOL UV 254

(Kavalier, CR) and developed in the mixture of solvents chloroform : methanol : acetic acid

(9 : 1 : 0.5) and detected or on UV 254 nm using vanillin, iodine vapour or iron trichloride.

Determination of dry matter

After measuring the pH we centrifuged (10 min, 3000 rev/min) 5 ml of the cultivation

medium taken at 24-hour intervals. After separating the supernatant we washed the mycelium

(the solid sediment) 3 times with distilled water and centrifuged again. We dried the sediment

obtained in this way at a temperature of 65 °C to constant weight.

8

Determination of reducing sugars (RS)

In the supernatant of the fermentation medium we determined the reducing sugars

spectrophotometrically through the reaction with the 3.5-dinitrosalycil acid at 525 nm.

Results

The course of the cultivation of the strain F-12 is to be seen on the Picture 1. The increase in

biomass of N. fischeri is almost linear until the end of cultivation. There is a clear decrease in

the amount of reducing sugars, as well as in the pH levels of the fermentation medium since

the start of the cultivation until its end. In the 120 h of cultivation the carbon source is almost

exhausted. In the early hours of the fermentation (up to 48 hours) one major metabolite is

being formed. Its amount is increasing with prolongation of the fermentation. In 72 hours we

have already identified a total of 6 metabolites (Picture 2).

After terminating the cultivation in 192 hours we processed the fermented medium to isolate

the different secondary metabolites applying the procedure outlined in Scheme 1. Applying

multiple repeated cultivations (10-20 flasks) in two series of experiments labelled X and Y we

obtained the crude products. The extracts of the filtrates provided the following weight

proportions: 2 MEX 20 g, 12 MEY 5.09 g, or 12 FEX 0.8 g and FEY 1.18 g. The

classification of the secondary metabolites, the used frameworks and detection systems are to

be seen on the Picture 2.

The evaporation residues of the extracts of the cultivation media N. fischeri from the first

series of cultivation, 12 MEX and 12 MEY containing several metabolites, were divided by

separation methods into fractions NF 0106, NF 1124, NF 1024 and NF 2530. We assessed the

activity of the metabolites of the different fractions against the following models: antifungal

effect, inhibition of elastase and beta-glucosidase.

The antifungal activity of the secondary metabolites was determined by the diffusion disc

method. The following were used as models: Candida albicans, Candida parapsilosis,

Torulopsis glabrata, Cryptococcus neoformans and Trichosporon cutaneum. The results are

stated in the tables 1 and 2.

Table 1. Antifungal activity of the evaporation residues obtained in two cultivation series

Evaporation

residue

Inhibition zone (mm)

C. albicans C. parapsilosis T. glabrata T. cutaneum

12 MEX 20 24 13 50

12 FEX 0 0 0 48

Nystatin 0 0 20 15

MEX – extract of the mycelium, FEX – extract of the filtrate, nistatin 10 μg/disc

The highest activity on the models of dermatophytes was recorded for the fraction NF 0709,

followed by the fractions NF 1024 and NF 0106, whereas the fraction NF 2530 did not show

any activity. The second series of the filtrates from the cultivation of N. fischeri was

processed by Dr. Proksa who labelled the obtained fractions in the following manner: CR -1,

CR – 2, CR – 3, CR – 4, CR – 5, CR – 6 and CR – 7. The biggest fraction CR -2 in the form

of an already pure substance was not effective on the dermaphytes in our tests.

9

Picture 1. Cultivation of N. fischeri F-12

Production of biomass, consumption of glucose, pH levels during the

cultivation

Picture 2. TLC analysis of secondary metabolites of the strain F-12

The system chloroform : methanol : acetic acid (9 : 1 : 0.5)

Detection by UV 254 nm and vanillin.

10

Supernatant Mycelium

Extraction

Acetone

Extraction

Ethyl acetate

Acetone layer

condensed

Water layer Extract I

Extraction

Ethyl acetate

Extraction

Ethyl acetate

Water layer Extract II

Extract III Water layer

Extraction

Ethyl acetate

Extract IV Water layer

Evaporation

residue

Evaporation

residue

12ME 12FE

Scheme 1. The procedure in isolating metabolites of N. fischeri from the cultivation medium.

Fermentation medium

Mycelium

11

Table 2. Antifungal activity of some fractions obtained by separation of crude fractions

(evaporation residues)

Fraction Inhibition zone (mm)

C. albicans C. parapsilosis T. glabrata T. cutaneum

NF 0601 14 0 20 35

NF 1124 0 0 0 10

NF 0709 30 0 25 40

NF 1024 20 0 20 35

NF 2530 0 0 0 0

Nystatin 18 22 3 23

Samples tested in conc. 25 μl/disc, nystatin 10 μg/disc

1.4. Isolation of the metabolites of Neosartorya fischeri F-12

The dried extract of the mycelium was triturated by ether; the insoluble part was filtered out

(E1, 1.35 g). The part soluble in the ether (E2) was connected by the extract of the filtrate

after separating the mycelium (M), then condensed and again digested by the ether; the part

insoluble in the ether (E3 0.45 g) was filtered out, the soluble part (R1, 1.91 g) was

condensed. All prepared fractions were assessed by thin layer chromatography (Kieselgel

GF254 in the system: chloroform – methanol 9 : 1, detection by UV 254, UV 365, solution of

FeCl3 and solution of vanillin/sulphuric acid).

As according to TLC the fractions E1 and E3 contained the same spectrum of substances, they

were combined and processed using preparative column chromatography (distillation column

50 x 3 cm, Kieselgel 60, 0.063 – 0.200 mm, gradient elution by the mixture chloroform -

methanol). The eluates were monitored by thin layer chromatography under above mentioned

conditions. In this way fractions C-1 to C-4 were obtained:

Label Weight (g) TLC (Rf)

C-1 0.440 0.50

C-2 0.390 0.50 (0.48)

C-3 0.190 0.50, 0.48, 0.30, 0.25

C-4 0.100 (0.50), 0.48, 0.15

The fractions with Rf 0.50 and 0.25 gave the same colour with the detecting agents, which

suggests that with a high degree of probability they are compounds with the same basic

skeleton.

Fractions C-1 and C-2 were further purified by crystallization:

C-1 crystallized from the mixture acetone – ether 1 : 5 390 mg C-11

C-2 1 : 7 250 mg C-21

Crystallized mother liquors 95 mg C-12

12

The part of R1 was processed by chromatography in the same system as E1 and E3; by

processing the eluates the following 7 fractions were obtained:

Label Weight (g) TLC (Rf)

C-1 0.200 0.90, 0.85

C-2 0.221 0.50

C-3 0.341 0.50, 0.48, 0.35, 0.32, 0.28

C-4 0.185 0.48, 0.35, 0.32, 0.28, 0.18

C-5 0.126 0.32, 0.28, 0.18, 0.10

C-6 0.082 0.18, 0.10

C-7 0.111 0.00

A part of C-12 (95 mg) was acetylated in a mixture of 2 ml acetanhydride + 2 ml pyridine for

5 hours at a temperature of 60 °C. The solvents/dissolving agents were vacuum-distilled off,

the residue contains, according to TLC, two lipophilic compounds (TLC: silica gel in

benzene, Rf 0.90, 0.85) not containing phenolic group (inactive with iron trichloride).

For the studies focusing on investigation of the structure of the dominant compound C-1 the

NMR spectra (Varian VXR 300 MHz) were measured; the results can be seen in Pictures 3 –

8 (Picture 3. 1H-NMR, Picture 4.

13C-NMR, Picture 5. APT experiment, Picture 6. Selective

INEPT experiment optimized for 3-bond interactions, Picture 7. homonuclear COSY

experiment, Picture 8 HETCOR experiment) or in tables 3 – 5.

Table 3. 1H-NMR shifts of the compound C-1 (δ/ppm, CDCl3)

Signal/ppm Number of H Signal/ppm Number of H

13.92 s 1 3.65 s 3

13.79 s 1 2.8 m 2

11.55 s 1 2.35 m 5

11.38 s 1 2.05 m X

7.15 d 8.48 1 2.08 s 3

6.58 d 8.67 1 2.04 s 3

6.47 s 1 1.97 m X

5.28 d 1.38 1 1.92 s 3

4.37 m (q) 2.01 1 1.25 t 7.09 3

4.36 q 7.09 2 0.92 d 5.86 3

3.79 s 3

13

Table 4. 13

C-NMR shifts of the compound C-1 (δ/ppm, CDCl3)

No. δ/ppm No. δ/ppm

1 187.82 C 20 100.55 C

2 187.48 C 21 100.24 C

3 178.74 C 22 83.98 C

4 177.71 C 23 82.15 C

5 171.24 C 24 69.54 CH-1

6 170.62 C 25 67.06 CH-1

7 169.10 C 26 60. 39 CH-2

8 161.80 C 27 53.57 CH-3

9 159.89 C 28 53.30 CH-3

10 156.89 C 29 32.83 CH-2

11 155.51 C 30 27.88 CH-1

12 148.54 C 31 24.41 CH-2

13 140.00 CH-1 32 23.18 CH-2

14 118.54 C 33 21.08 CH-3

15 114.69 C 34 21.03 CH-3

16 110.30 CH-1 35 20.29 CH-3

17 108.98 CH-1 36 17.09 CH-3

18 106.88 C 37 14.21 CH-3

19 104.85 C

Table 5. Results of the HETCOR experiment

13C

1H

13C

1H

140.0 7.16 27.9 2.10

110.3 6.60 24.4 2.8 + 2.38

108.9 6.18 21.0 2.08

69.5 5.29 21.0 2.04

67.0 4.37 20.3 1.92

60.3 4.13 17.1 0.93

53.5 3.79 14.2 1.28

53.3 3.65

32.8 2.35

Conclusion: According to the results of NMR spectroscopy, the compound C-1, the dominant

metabolite of Neosartorya fischeri is not identical with any compound listed in the literature

review to this chapter and can be considered a new metabolite N. fischeri.

14

Picture 3. 1H-NMR of the compound C-1 (δ/ppm, CDCl3)

15

Picture 4. 13

C-NMR of the compound C-1 (δ/ppm, CDCl3)

16

Picture 5. 13

C-NMR of the compound C-1 (δ/ppm, CDCl3) APT experiment.

17

Picture 6. Selective INEPT experiment optimized for 3-bond interactions

18

Picture 7. COSY spectrum of the compound C-1

19

Picture 8. HETCOR spectrum of the compound C-1

20

1.5. Isolation of metabolites from the strain of Sporidiobolus salmonicolor

B4

Two strains of yeast were isolated from the healing mud of Piešťany: Rhodotola rubra 1B and

Sporidiobolus salmonicolor B4.

The strain S. salmonicolor was cultivated on petri dish (diameter 200 mm, 4 dishes in total)

on wort agar, which was covered by cellophane. To vaccinate the agar medium a suspension

of a 48-hours old culture was used. The cultivation lasted for 4 days at lab temperature. After

the cultivation, the biomass was mechanically removed from the surface of the cellophane,

suspended in distilled water and lyophilised. Altogether, 3.2 g of dry biomass was obtained.

The obtained biomass was extracted by methanol; the methanol solution was condensed and

precipitated by ether. The insoluble part was filtered off and the part soluble in ether was

condensed again. The part insoluble in ether was identified as trehalose. The part soluble in

ether had a moderate inhibitory effect on the activity of elastase.

21

2. Preparation of the mud fractions and their in vitro biological

assessment Fractions of the Piešťany thermal mud were prepared applying a gradual extraction by a series

of polar (water miscible) and non-polar, lipophilic solvents (water immiscible). The prepared

fractions were assessed by in vitro test on inhibition of activity of elastase. The results are to

be seen in Table 6.

Table 6. Influence of the fractions obtained from the thermal mud on the activity of the

pancreatic elastase

Mud fraction Elastase activity

Label μg/ml

A – 2 4 86

A – 2 40 83

A – 3 4 81

A – 3 40 0

A – 4 4 88

A – 4 40 87

A – 7 4 88

A – 7 40 88

A – 8 4 90

A – 8 40 89

22

3. In vivo assessment of elastolytic effects of the mud fractions The study with extracts of spa mud poses a continuation in the issue of assessment of effects

of the compounds present in the mud fractions in terms of their impact on inflammatory and

arthritic processes in animals.

Material and methods

For prevention and treatment of sewer rats with adjuvant-induced arthritis we used extracts

(mud fractions) labelled A 3/1, A 3/2 and dissolved in DMSO supplemented with olive oil

and fraction A4 in olive oil. The fractions were prepared applying a gradual extraction by a

series of polar (water miscible) and non-polar, lipophilic solvents (water immiscible). In the

experiments, adjuvant-induced arthritis was induced in males of the inbred strain Lewis

(Charles River Wiga GmbH, Federal Republic of Germany). We induced arthritis in the

animals by applying 0.1 ml of the suspension of thermally killed mycobacteria (12 mg/ml

Mycobacterium butyricum, company DIFCO) in incomplete Freund adjuvant.

The rats were divided into 7 groups with 7 individuals each in the following way: healthy

control group, untreated adjuvant arthritic control group, adjuvant arthritic group administered

with A3/1 in prevention (dosage of 2.0 mg per kg of live weight), adjuvant arthritic group in

prevention and treatment with A3/2 (dosage of 1.0 mg per kg of live weight), adjuvant

arthritic group in prevention and treatment administered with fraction A4 (dosage of 2.5 mg

per kg of live weight). In each group we used the standard compound feed MP (PD Dobrá

voda) as fodder and water was available ad libidum.

In treatment and work with the animals, the International guiding principles for Biomedical

Research Involving Animals, as well as the European Convention for the Protection of

Vertebrate Animals used for Experimental and Other Scientific Purposes were adhered to.

We applied the extracts in the amount of 0.1 ml per animal subcutaneously to the thigh. The

groups of untreated animals and healthy control were injected olive oil of the same volume

since the second day of the experiment 3 times a week (8 times in total).

In the blood samples from the 15th

, 22nd

and 29th

day of the experiment we studied the serum

albumin. We determined serum albumin applying the spectrophotometric method with

commercially available control serum (Q-PAK HYLAND Division, Travenol Laboratories,

Belgium).

We determined swellings of hind limbs using plethysmometer on the 14th

, 23rd

and 31st day

since the injection of the adjuvant.

We measured live weight on fixed day each week of the research.

We assessed the level of destruction of bone tissue of the hind limbs of the animals by X-Ray

images of the hind limb applying a 5-point scale following the Grim method on the 50th

day

of the experiment.

For the statistical evaluation of inflammatory and arthritic indicators we used basic statistical

values (arithmetic mean and standard deviation). We compared the individual studied groups

with each other following the Kolmogorov-Smirnovov non-parametric test.

Results

By studying the influence of the tested extracts on inflammatory and arthritic indicators in

diseased rats, we found a positive effect of the A3 fraction in comparison to the untreated

adjuvant arthritic group.

23

Live weight of arthritic rats increased until the 14th

day of the experiment. In the following

period we observed reduction in weight of the untreated group and the group preventively

treated with A4. The differences between these groups and the groups of preventively treated

with A3 were conclusive with both concentrations. On the 21st-28

th day between the groups

with A3/1 and arthritic untreated (p = 0.05) or with A3/2 and untreated, with A4 in prevention

(p = 0.01) with A4 in prevention (p = 0.05). (Picture 9, Table 7)

The amount of the serum albumin in the blood of sewer rats with adjuvant arthritis decreased

considerably. We detected significantly positive changes of this indicator in treated animals in

comparison to the untreated arthritic control group on the 22nd

day in groups with A3/1 (p =

0.05), resp. with A3/2 in prevention (p = 0.01) and on the 29th

day in the group with A3/2 (p =

0.05) (Picture 10, Table 8).

In the group preventively treated with A4 we found conclusively bigger swellings of the hind

limbs compared to other groups with different levels of significance already on the 14th

day of

the experiment. We observed the maximum swelling of the left and right hind limbs of the

rats on the 23rd

day of the experiment in all groups with adjuvant-induced arthritis. On the 23rd

day we found the conclusive changes in treated animals compared to the untreated ones in

these groups: the rats preventively treated with A3/1 (p = 0.05) and on the 31st day the rats

preventively treated with A3/1 (p = 0.05) or with A3/2 (p = 0.01) (Picture 11, Table 9).

Based on the assessment of the X-ray images on the 50th

day we found a lower level of

occurrence of bone erosions of joints of the hind limbs in animals treated with A3 compared

to the untreated arthritic control group. Assessed by the score, the most significant positive

effect was found in the group preventively treated with A3/1 (p = 0.01) and with A3/2 both in

prevention and treatment (p = 0.05) (Picture 12, Table 10).

The results above show that the extracts of the spa mud A3/1 and A3/2 positively influence

some indicators of the inflammatory and arthritic process. Their positive effect manifested

itself especially in preventive treatment of the rats with adjuvant arthritis.

Picture 9. Live weight of sewer rats after administration of fractions A3 and A4

Healthy

24

Table 7. Statistical evaluation of the results of the experiment with fractions A3 and A4 - live

weight

Live

weight

Healthy AA A3/1 P A3/2 P A3/2 L A4 P A4 L

1st day

17.10.96

Mean 175.57 163.30 66.86 173.00 172.14 171.86 172.86

STDV 20.94 19.64 10.17 10.79 5.87 9.44 8.45

9th

day

25.10.96

Mean 207.29 191.70 200.57 205.86 208.43 208.57 206.14

STDV 24.48 23.04 13.18 11.78 7.91 8.40 8.38

14th

day

30.10.96

Mean 221.86 195.90 200.57 205.86 208.43 208.57 206.14

STDV 25.54 22.53 19.33 12.96 13.25 14.15 14.53

21st day

6.11.96

Mean 239.14 187.40 210.57 214.43 197.86 184.57 201.86

STDV 27.41 10.86 28.98 21.11 20.41 13.99 24.32

28th

day

13.11.96

Mean 252.14 192.50 219.43 221.71 205.71 193.43 209.29

STDV 28.47 10.93 29.54 24.21 16.83 13.97 26.51

Live weight – statistically significant conclusive differences between the groups:

1st day: without any significant differences

9th

day: without any significant differences

14th

day: at p ≤ 0.05 healthy and AA, A4-P

21st day: at p ≤ 0.05 healthy and A4-L, AA and A3/1-P

at p ≤ 0.01 healthy and A3/2-L, A3/2-P , AA and A4-P

at p ≤ 0.001 healthy and AA, A4-P

28th

day: at p ≤ 0.05 healthy and A4-L, AA and A3/1-P , A3/2-P and A4-P

at p ≤ 0.01 healthy and A3/2-L, A3/2-P and A4-P

at p ≤ 0.001 healthy and AA, A4-P

25

Picture 10. The content of albumin in the blood serum of untreated animals and after

administration of fractions A3 and A4

Table 8. Statistical assessment of the results of the experiment with fractions A3 and A4 –

albumin in the blood serum of the sewer rats

Albumin Healthy AA A3/1 P A3/2 P A3/2 L A4 P A4 L

15th

day

31.10.96

Mean 55.73 29.47 29.30 31.21 29.56 29.94 30.71

STDV 1.36 2.08 0.93 2.05 1.23 0.50 1.38

22nd

day

7.11.96

Mean 37.36 30.14 32.04 34.11 30.65 30.13 31.14

STDV 1.28 1.92 1.37 2.57 1.90 1.01 0.90

29th

day

14.11.96

Mean 38.88 31.29 33.13 33.50 31.60 32.73 31.50

STDV 1.60 2.05 1.96 2.25 1.80 2.23 1.73

26

Albumin – statistically significant differences between the groups:

15th

day: at p ≤ 0.05 between group A3/1-P and the groups A3/2-P, A4-L

at p ≤ 0.001 between healthy and all other groups

22nd

day: at p ≤ 0.05 between healthy and A3/2-P

A3/1-P and AA, A3/2-P and A3/2-L, A4-L

at p ≤ 0.01 between A3/2-P and AA, A4-P, A3_ú-P and A4-P

at p ≤ 0.001 between healthy and all other groups except A3/2-P

29th

day: at p ≤ 0.05 between group A3/2-P and AA

at p ≤ 0.001 between healthy and all other groups

27

Picture 11. X-ray of the hind limbs of the sewer rats after administration of fractions A3 and

A4

Table 9. Statistical evaluation of the results of the experiments with fractions A3 and A4 – X-

ray of the hind limbs

Albumin Healthy AA A3/1 P A3/2 P A3/2 L A4 P A4 L

50th

day

4.12.96

Mean 0.00 2.33 0.71 1.00 1.00 2.14 1.57

STDV 0.00 0.71 0.95 1.29 1.15 1.07 0.79

X-ray of the hind limbs – statistically significant differences between the groups:

50th

day: at p ≤ 0.05 between AA and A3/2-P , A3/2-L , A3/1-P and A4-P

at p ≤ 0.01 between AA and A3/1-P

at p ≤ 0.001 between healthy and all groups with AA

28

Picture 12. Swellings of hind limbs of the sewer rats after administration of A3 and A4

Table 10. Statistical evaluation of the experiments with fractions A3 and A4 – swellings of

the hind limbs

Swellings

of hind

limbs

Healthy AA A3/1 P A3/2 P A3/2 L A4 P A4 L

14th

day

30.10.96

Mean 1.17 1.58 1.42 1.44 1.49 1.85 1.53

STDV 0.08 0.40 0.09 0.25 0.20 0.35 0.12

23nd

day

6.11.96

Mean 1.21 2.04 1.51 1.71 2.11 2.08 2.05

STDV 0.02 0.36 0.39 0.40 0.31 0.35 0.43

31th

day

16.11.96

Mean 1.21 1.84 1.52 1.49 1.64 1.73 1.76

STDV 0.02 0.20 0.31 0.25 0.18 0.28 0.29

29

Swellings of the hind limbs – statistically significant differences between the groups

14th

day: at p ≤ 0.05 between healthy and A3/2-P , A4-P and AA, A3/2-P, A3/2-L, A4-L

at p ≤ 0.01 between healthy and AA, A3/2-L, A3/1-P and A4-P

at p ≤ 0.001 between healthy and A3/2-L, A3/2-P , AA and A4-P

23rd

day: at p ≤ 0.05 between A3/1-P and AA and A3/1-P, A4-P , A4-L

at p ≤ 0.01 between healthy and A3/2-P, A3/1-P and A3/2-L

at p ≤ 0.001 between healthy and AA, A3/2-T, A4-P, A4-L

31st day: at p ≤ 0.05 between healthy and A3/1-P, A3/2-P, AA and A3/1-P, A3/2-L

at p ≤ 0.01 between A3/2-P and AA

at p ≤ 0.001 between healthy and AA, A3/2-L, A4-P, A4-L

30

4. Isolation of technologically significant microorganisms and the

study of the impact of heat on their survival

4.1. Isolation of microorganisms

Using impression method, the samples of mud from the mud kitchen of Pro Patria were

applied to the surface of a series of agar plates with culture medium API Broth, Thiobacillus

Broth and Thiobacillus thiooxidans Broth (HI MEDIA) and cultivated at temperatures of 28

°C, 40 °C and 60 °C in the course of 2 to 6 days. Applying the smearing method, the grown

up colonies of the cultures were inoculated to the agar plates with a medium of the same

composition and statically incubated at a temperature optimal for the growth of a chosen

colony. The procedure was repeated several times until we obtained a pure monoculture.

Applying the procedure described above we obtained 8 monocolonies, which differed both in

colour and morphology. Three from the cultures obtained had their optimal temperature at 28

°C and five at 60 °C.

4.2. Characterisation of isolated strains of microorganisms

The individual types of bacteria are to be seen in the Annex on the pictures 16 to 19. Three

isolated cultures of bacteria have been identified and so far stored in the Collection of

microorganisms CCM in Brno (strains S2, S3 and S7). So far, we have not succeeded in

identifying and classifying the strain S8.

Strain S3

The culture forms gram-labile rods and fibres in irregular clusters. The spores are oval, not

swelling the cell and placed subterminally. Morphology of the strain on MPA (meat-peptone

agar): the strain grows in the form of round colonies with a diameter of 2 to 4 mm, colonies

are matte, flat, fine grained, with a toothed edge. In terms of biochemistry, the strain is

characterised by the presence of catalase, ability to hydrolyse starch and DNA, to reduce

nitrates, to metabolise glucose, xylose, mannitol, cellobiose and fructose to acids. The strain

grows slightly at a temperature of 28 °C and 40 °C, good growth was observed at 56 °C, 60

°C and 65 °C.

Strain S2

This strain forms gram-labile rods – both individual and in clusters. Spores are oval, swelling

the cell and placed subterminally. Morphology of the strain on MPA: the strain grows well in

the form of round colonies with a diameter of 1 to 2 mm. These are smooth, glossy, flat with

an irregular edge. The strain has catalase activity, hydrolyses DNA, metabolises glucose,

mannitol, xylose, cellobiose, lactose, fructose and inositol. It grows at 40 °C, 56 °C and 60

°C.

The strains S3 and S2 have been identified and classified into the genus Bacillus.

31

Strain S7

It was identified as Pseudomonas putida. It forms gram-negative rods – two at a time in

irregular clusters. Morphology of the strain on MPA: grown up colonies with a diameter of 2

to 3 mm are round, glossy, smooth, slightly convex with a concentrated centre. Production of

fluoresceine, catalase, urease, oxidase, arginine dihydrolase has been observed. It metabolises

xylose and oxidatively glucose to acid. It doesn’t form acid from fructose, lactose, mannitol,

maltose and saccharose. It doesn’t form hydrogen sulphide. It grows on Mac Conkey agar.

Growth has been recorded at 28 °C and 37 °C. It doesn’t grow at 42 °C.

4.3. Study of the impact of temperature on survival of technologically

significant microorganisms

Another part of the work within the project was aimed at determining the optimum growth

temperature of the isolated bacterial strains utilizing sulphur. Sanitariness of the mud can be

achieved by heating it, e.g. by heating up the mud to 50 °C, which may, however, negatively

affect its biological activity. Our task was to try to study experimentally, whether the higher

temperature of the mud does not negatively affect the survival of the bacteria, which are

important for the biological activity of the mud.

Growth of the bacteria was assessed by plate dilution and turbidimetric method.

Determining the temperature optimum for the growth and reproduction of sulphur-

utilizing bacteria applying the plate method.

To study the effect of temperature on observed microorganisms we chose 4 strains of bacteria,

which we isolated from the mud on special culture mediums for sulphur-utilizing bacteria.

After inoculating the cultures on inclined agar culture media into liquid media and incubation

lasting 24 to 48 hours we prepared from them a suspension of bacteria, which we after

appropriate dilution (10 – 10) inoculated onto the surface of the agar plates (from each

dilution 3 parallel dishes).

The cultures on agar media were incubated for 6 days at three different temperatures: 28 °C,

40 °C and 60 °C. The results are shown in the Table 11.

32

Table 11. Growth of isolated chosen strains of bacteria on agar media API Broth and

Thiobacillus Broth

Strain of

bacteria

Temperature Number of KTJ

Suspension of bacteria

1 2 3 4 5 6

S2 28 47 5 1 0 0 0

S2 40 96 10 0 0 0 0

S2 60 360 95 32 18 10 2

S3 28 10 N 10 6 0 0

S3 40 68 14 6 0 0 0

S3 60 150 56 10 4 1 0

S7 28 N N N N N N

S7 40 - - - - - -

S7 60 0 0 0 0 0 0

4.4. Assessment of the growth of isolated bacteria at different temperature

applying the turbidimetric method

Determination of optimal temperature regime is preceded by experiments aimed at studying

the growth of the bacteria isolated from the samples of the mud coming from the mud kitchen

Pro Patria, at different temperatures (temperature at which the mud is modified from 40 °C to

50 °C). The cultures of isolated bacteria maintained on inclined agar were reproduced in

appropriate liquid media and inoculated into liquid culture media in flasks. During the static

cultivation samples of the media were collected and after adjusting them their growth was

assessed by measuring of absorbance of the sample at 450 nm.

The strain S2 grew fastest at 60 °C in API medium up to 72 hours of cultivation. The

maximum increment of bacteria was reached in 144th

hour of cultivation. Further prolongation

of the cultivation did not affect the growth of the culture at 60 °C. The bacteria grew also at

40 °C, however, less intensely. The almost linear growth of bacteria during the cultivation did

not reach the value of maximum increment of the strain at 60 °C on the 10th

day. At 28 °C the

culture almost didn’t grow (Picture 13). Similar is the course of the growth of the culture S3

on the medium API at 60 °C and 40 °C. The growth of the strain S3 is more intense at 28 °C

in comparison to the strain S2 (Picture 14). Bacteria S7 and S8 grew only at 28 °C in liquid

breeding ground (Thiobacillus Broth). The overall concentration of cells was low. Maximum

growth for the strain was achieved in 96th

hour of cultivation, for the strain S8 in 120th

hour of

cultivation (Picture 15). The reference strain Thiobacillus ferooxidans CCM 4253 requested

from the collection of microorganisms CCM didn’t grow on the media we chose for the

isolation of bacteria from the mud and for the observation of the influence of the temperature

on the growth of these microorganisms utilizing sulphur compounds. According to subjective

assessment the growth of Thiobacillus ferooxidans in liquid medium 66 (CCM) at 28 °C was

observed under microscope.

33

Picture 13. Growth of the bacteria of the strain S2 on API medium in flasks during static

cultivation at different temperatures

Picture 14. Influence of cultivation temperatures on the growth of bacteria of the strain S3 in

API medium under static conditions

34

Picture 15. Growth of bacteria S7 and S8 in liquid medium Thiobacillus Broth at different

temperatures

According to the subjective evaluation of the experiment, the growth of bacteria was clearly

different at different temperatures. For the strains S2 and S3 the increase in temperature

positively affected the growth of bacteria on agar medium. Both strains grew most intensely at

a temperature of 60 °C. They grew poorly at 28 °C. The strain S7 grows well at 28 °C, but at

60 °C the growth of these bacteria has not been observed.

35

5. Determination of Se content in samples provided

To determine trace amount of Se in the samples provided the analytical procedure described

in [1] was used. Electroanalytic method of differential pulse cathodic stripping voltammetry

(DPCSV) of determination of Se is based on the use of two reduction processes occurring at

the working hanging mercury drop electrode in the course of the analysis. The first one is the

formation of mercury II selenide during the deposition with potential -0.3 V vs. SCE in the

reaction:

Se(IV) + Hg + 4e- => HgSe

In the second step the selenide is reduced in the course of the stripping process with

polarisation of the working electrode into more negative potential following the equation:

HgSe + 2 H+ + 2e

- => H2Se + Hg

This reduction, which will manifest itself in the cathodic stripping peak at the potential Ep = -

0,56 V vs. SCE is used for quantitative analysis. The height of the stripping peak grows

linearly depending on the amount of Se present in the sample. The author states in his paper

the lowest limit of the method’s determinabilityy at 0.1 μg.dm-3

of Se and also that the

determination is not disturbed by even a 1000-times surplus of possibly interfering elements

such as Cu, Pb or Cd, which only confirms the suitability of the method chosen.

The analysis of the Se content in the samples provided was carried out using a polarographic

analyser PA 4 applying the above described method (DPCSV) with accumulation on the

hanging mercury electrode. The experimental conditions of the determination were set as

advised in the paper [1]:

- accumulation: potential of deposition -0.3 V vs. SCE, duration 840 s;

- stripping: polarisation speed 2 mV/s, modulation amplitude -25 mV, pulse

frequency 5 Hz.

Sample of water was analysed after adjustment of the environment to pH 1 with addition of

little HClO4. The Se content was determined by the multiple standard addition method by

comparing the magnitude of the peak signal at potential -0.56 V vs. SCE of the sample

without and with added standard additions.

The magnitude of the signal of the sample was comparable to the signal provided by the

standard addition of Se corresponding to 4ppB Se. The signal of the Se sample was clearly

distinguishable from the background signals. Given the curvature of the calibration curve

while testing the method using model samples with known amount of Se added to a solution

and a non-zero intersection of the y axis of the dependence of magnitude of signal on

concentration it is possible to give the result of the determination only with a relatively wide

confidence interval. The guaranteed amount of Selene in the water ranges around 4 ± 3 ppb

(i.e. 4 ± 3 μg.dm-3

) of Se.

As for the lyophilisate, the solution prepared from the amount of 20 mg dissolved in 10 ml of

acidified H2O (a volume necessary for the analysis) was analysed. There was no distinct

improvement of the accuracy of the analysis, as during the preparation of the solution of the

36

sample the sample is diluted substantially. In case of need of a more accurate result one has to

start with a larger amount, which was not available at the time of the analysis.

Solution of the sample of the evaporation residue for the analysis prepared from 40 mg of dry

matter dissolved in 10 ml acidified H2O also only confirmed the values obtained during the

analysis of water without substantially increasing the accuracy of the analysis. Higher

amounts of dry matter cause turbidity of the sample by insoluble solid particles (possibly

silicates).

The above mentioned result of the analysis of Se content can be considered certain within the

stated confidence interval.

References:

1. Adeloju B. B., Bond A. M., Analytica Chimica Acta 148, 59-69 (1983).

37

6. Analysis of the sulphur content in the provided samples of

water Determination of sulphur compounds by electroanalytic methods is reported already in earlier

papers, since these compounds give a clearly defined analytical signal within the electrode

reaction with the electrode material – mercury. One of such papers, which provides guidance

on the polarographic determination of S2O32-

, SO32-

, CNS- and S

2-, is paper [1]. The individual

sulphur compounds are determined by assessing the amplitude of the anodic polarographic

wave they give in the environment of 0.1 mol.dm-3

KNO3 at the following half-wave

potentials:

S2O32-

+ Hg – 2e- => HgS2O3 E1/2 = - 0.145 V vs. SCE

SO32-

+ Hg – 2e- => HgSO3 E1/2 = + 0.06 V vs. SCE

2CNS- + Hg – 2e

- => Hg(CNS)2 E1/2 = + 0.18 V vs. SCE

S2-

+ Hg – 2e- => HgS E1/2 = - 0.75 V vs. SCE

Determination of polysulphides by assessing the amplitude of their cathodic waves, which

they give in the environment of 2 mol.dm-3

NaOH, S22-

E1/2 = - 1.40 V vs. SCE is

described in paper [2], and S42-

E1/2 = - 1.42 V vs. SCE in the paper [3].

Determination of polytionates is addressed by the paper [4], in which the procedure to

determine S4O62-

in the environment of 0.1 mol.dm-3

KCl by assessing the polarographic wave

of its reduction with E1/2 = - 0.29 V vs. SCE is described.

From the newer papers, which use more modern electroanalytical methods enabling

determination of sulphur compounds on the level of trace concentrations, the paper [5] can be

mentioned, which describes the procedure of determination of sulphides also in the presence

of other interfering components applying the method of normal pulse polarography in the

environment of 0.1 mol.dm-3

Na2CO3 on concentration level ppm.

To verify the determination procedures described above model samples were used.

Polarographic analyser PA 4 applying the method of DC-polarography with classical mercury

drop electrode was used, since the expected content of sulphur compounds in the sample is

within the concentration range typical for the use of this reliable method.

Prepared model solutions of sulphur compounds (S2O32-

, SO32-

, CNS- and S

2-) in the

appropriate environments provided polarographic waves suitable for the analysis, as was

reported in the original cited papers. Within the investigated concentration range of 0.001

mol.dm-3

to 0.0001 mol.dm-3

, their amplitude depended linearly on the concentration.

It could be assumed that if the sample of Piešťany water was adjusted by addition of

appropriate environment the given sulphur compound in it would manifest itself through

a polarographic wave as in the case of model samples.

During the analysis of the real sample of Piešťany water we obtained a polarographic record

consisting of several polarographic waves based on a current beginning from the potential of

38

dissolving the material of the electrode Hg – 2e- => Hg

2+ . Such electrochemic behaviour is

common and determination is carried out using some of the methods of polarographic

quantitative analysis, most frequently the method of standard addition of one of the analysed

compounds (e.g. of S2O32-

and then of others) of known concentration. Standard addition

manifests itself as an increment of the one from the registered polarographic waves, which

corresponds to the added compound and from the amplitude of its increment the content of the

compound in the sample is calculated. By adding the standard addition a qualitative analysis

is carried out at the same time – matching of the polarographic wave with the determined

compound found approximately based on the value of the half-wave potential is confirmed.

However, during the analysis of the real sample of Piešťany water the standard additions of

the individual sulphur compounds used during the analysis of the model samples have always

led to the change of the whole polarographic record – to the change of ratio of the amplitudes

of all registered polarographic waves.

The result of the experiment leads to the conclusion that Piešťany water is a complex system

containing several sulphur compounds (and not only them), which are in equilibrium. Change

of environment or adding of one of the compounds leads to disturbance of this equilibrium

and stabilization of a new one, while the proportions of the individual sulphur compounds

also change. For the analysis of such a complex sample a deeper study is needed, the

understanding of the mechanisms taking place in the sample and of the equilibrium constants

of all chemical reactions taking place during the stabilisation of equilibrium in this complex

system. Under the given circumstances, this was beyond the possibilities of our analytical

laboratory.

References:

1. Kolthoff I. M., Miller C. S., J. Am. Chem. Soc. 63, 1405 (1941)

2. Werner E., Konopik N., Monatsh 83, 599 (1952)

3. Werner E., Monatsh 83, 1369 (1952)

4. Žežula I., Chem. Listy 47, 492 (1953)

5. Leung L. K., Bartak D. E., Anal. Chim. Acta 131, 167 (1981)

39

7. Discussion of obtained results

The research project carried out within HZ should be understood in two directions, which are

interlinked. The first direction is oriented to medical treatment – to the evidence of biological

effects of the mud or thermal water, but also to other potential components present in them.

The biological activity is given not only by the sulphur compounds, which can be considered

metabolites emerging during the biological or microbiological transformation. We also

assume that biological activity is also resulting from the organic compounds, which get into

the mud as metabolites of the microorganisms present in the mud. Both assumptions were

confirmed by our experiments. The extracts prepared from the healing mud have inhibitory

effects on the growth of a wide range of dermatophytes, but they also inhibit the activity of

elastase. We admit that a part of the biological activity against fungi, less so against yeasts,

can be attributed do sulphur compounds, but a part of the activity is formed by other organic

compounds. This is also confirmed by the results of the experiments, which show that the

metabolites effective against dermatophytes are also produced by microorganisms isolated

from Piešťany mud. The fungus Neosartorya fischeri produces compounds with such an

effect. Unfortunately the structure of the major metabolite produced by this culture has not

been resolved yet, however, according to the data available it is not identical with any of so

far isolated metabolites, which have been isolated from the above mentioned fungus. As can

be seen from the results, the culture F-12 produces several metabolites active against

dermatophytes. A similar picture was obtained also during the assessment of the extracts

prepared from the mud. Depending on their chemical and physical characteristics, the

metabolites dissolve and thus also are extracted into different solvents. As effective against

fungi were extracts prepared using different solvents, it may be assumed that different

metabolites (compounds) of different structure were concentrated in them. This finding is

important also because it confirms that the mud has a certain ability to autonomously inhibit

the growth of microorganisms, which can enter the mud in contact with human skin.

Equally important is the repeated finding that the fractions prepared from the mud inhibit the

activity of elastase. Although the used elastase is only a model, there is a finding that it

correlates very well with the results obtained with these fractions in vivo. It means that there

is a correlation between the results in vitro and in vivo. This finding is sufficient to be used to

evaluate the curative effects of mud in vivo in humans, but also to assess the quality of the

mud itself. The mud extracts should always exhibit in vitro activity against elastase, even

though their effect may range within one order. At the same time, the findings obtained so far

may be used to assess the effects of the mud and the thermal water on patients.

The second direction followed in the experimental section had a clearly technological focus.

We isolated several species of microorganisms from the mud and we prepared four of them in

clear form. The microorganisms obtained are not identical with Thiobacillus, however, we

have not succeeded yet in identifying them. Even the mere fact that they are so far

undescribed microorganisms is of importance. In some (3) of these strains we observed the

influence of temperature on their growth. We are convinced that interesting are particularly

those strains that grow at temperatures above 40 °C. Other strains are less involved in the

technological process. We have found out that the pure culture of Thiobacillus tiooxidans

grows well only at temperatures up to 30 °C, with increasing temperature its growth slows

down and stops at 40 °C. It is thus probable that Thiobacillus takes part in processes related to

the transformation reactions of sulphur compounds only in the maturing pools or in the dead

arm of the river. Since the mud enters the mud kitchen the activity of Thiobacilli is

substantially limited, even if we admit that strains-phenotypes might have get selected in the

mud, which maintain certain activity even at temperatures above 30 °C. Thermophilic bacteria

utilizing sulphur compounds we have isolated have different characteristics. Their

40

reproduction increases with growing temperature reaching their maximum at temperatures

around 65 °C. With decreasing temperature their growth decreases. Therefore it is probable

that these thermophilic bacteria are involved in the biological processes above 40 °C, i.e. also

in mud kitchens. We consider these findings important for further research, which should

focus on the improvement of the quality of preparation and regeneration of the healing mud.

As an addition to the objectives set in the HZ we focused on approximate analytical studying

of Selene and sulphur compounds in the thermal water and mud. Selene is present in the

thermal water, however, in concentrations which are by several orders lower than those

entering human body through food components we consume daily. We have neither found an

inhibitory effect of Selene on the activity of elastase in vitro. However, it is possible that its

effect may appear in synergy with other elements, for instance due to the fact that in some

compounds Selene may replace the atom of sulphur. However, it is improbable that the

biological activity of thermal water and mud would be dependent on the presence of Selene.

Studying transformations of sulphur and its compounds in thermal water is very complex.

Sulphur compounds are both in the water and the mud in a dynamic equilibrium, which

restores itself continuously. Any violation of the equilibrium results in a qualitative shift.

(Even during the mere device determination). Therefore, we based our experiments on the

model nutrient environment, i.e. we used the medium of defined composition. After

inoculating it with one of the microorganisms we had isolated from the mud, profound

changes in the medium composition occurred very quickly. The sulphate ion that seems to be

relatively stable changes over a short period of time, dynamic equilibrium is disturbed and

reinstated again. If we realize that both chemical and biological processes have their share on

the dynamic equilibrium, we also must realize that management of technological processes in

preparation and regeneration of the mud have to be regulated very carefully.

41

8. Annex – microscopic image of isolated strains of bacteria

Picture 16. Microscopic image of the strain S2 (enl. 2000x)

Picture 17. Microscopic image of the strain S3 (enl. 2000x)

42

Picture 18. Microscopic image of the strain S7 (enl. 2000x)

Picture 19. Microscopic image of the strain S8 (enl. 2000x)