37 Journal homepage: www.fia.usv.ro/fiajournal Journal of Faculty of Food Engineering, Ştefan cel Mare University of Suceava, Romania Volume XIV, Issue 1 - 2015, pag. 37 - 44 BIOCH E MICA L A ND M OL EC ULAR-GE NE T IC CHAR AC T ERIZ ATI ON OF A S TRA IN ISO LA T ED FROM A THER MAL HE ALI NG S PR ING I N STARO ZAGORSKI M INE RA LN I BANI, STA RA ZAG ORA RE GION, BU LGA RIA *Nedyalka VYLCHEVA-ZHEKOVA1, Zapryana DENKOVA2, Radosveta NIKOLOVA2 1 PHT “Aleksander Paskalev”, Haskovo, 2University of Food Technology, Department of Microbiology zdenkova@abv.bg *Corresponding author Received 5th February 2015, accepted 30th March 2015 Abstract: Mineral waters of springs near the village of Starozagorski Mineralni Bani, Stara Zagora region, are well known for their healing effect in diseases of bones and joints, peripheral nervous system, gynecological, kidney and urological, gastro-intestinal and hepatic and biliary diseases. The physico-chemical and microbiological parameters of healing spring water in the village of Starozagorski Mineralni Bani, Stara Zagora region were determined. According to its physico- chemical and microbiological parameter, spring water meets the standard requirements for quality of drinking water. A strain was isolated from the examined healing spring as a pure culture and its colonial and morphological characteristics were examined. The isolated strain SMB was rod-shaped, Gram - positive, motile, sporeforming. It formed a precipitate on the surface of the liquid culture medium, did not turbidify the liquid culture medium and formed a precipitate on the bottom of the tube. Strain SMB was a facultative aerobe. It was identified by biochemical (API 50 CHB) and molecular - genetic methods (sequencing of the gene for the 16S rRNA) as a representative of the species Bacillus thuringiensis. After performing further examinations of its antimicrobial activity against phytopathogenic molds strain SMB would be incorporated in the composition of biological preparations against plant diseases. Keywords: mineral water, physico-chemical, microbiological, identification, sequencing, 16S rRNA 1. Introduction Mineral water from springs near Starozagorski Mineralni Bani, Stara Zagora region, come to the surface with a temperature of about 40°C from about 1600 m in depth. They are known to have healing effect in diseases of bones and joints, peripheral nervous system, gynecological, kidney and urological, gastro-intestinal and hepatic and biliary diseases [1 - 8]. Mineral water is slightly mineralized, hyperthermal, with neutral pH (pH=6.9) due to the presence of 60 mg of carbon dioxide in it. It contains hydrocarbons, sulfates, calcium, magnesium, silicon, fluorine, and other trace elements; the mineral content is 0.498 g/l to 2 g/l; the content of metasilicic acid in colloidal state is 34 mg/l, which makes it effective in a number of diseases. Potable water used in enterprises of the food and microbiological industry must meet the requirements for potable water. http://www.fia.usv.ro/fiajournal mailto:zdenkova@abv.bg Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XIV, Issue 1 – 2015 N e d yal k a VY L C HE VA- Z HEK O VA , Z a pr ya na D E N KO VA, R ad o s ve t a N I K OL O VA B i oc h e m i c al a nd m o l e c u l ar - g e n et i c c ha r ac t e ri z a t i o n of a st r ai n i s ol a t ed f r o m a t h e rm al he a li n g s pr i n g i n S t a ro z a go r s k i M i n e r al ni Ba n i , S t a r a Z a go r a r e gi o n , B ul g a r i a , Foo d a nd E nv ir o nm e nt Sa f e t y , V o l ume X IV , Is s ue 1 – 2 0 15 , pa g . 3 7 - 4 4 38 Thus the total bacterial abundance (TBA), the number of coliform bacteria and pathogenic bacteria should be determined and monitored periodically. Pathogenic bacteria are controlled by hygienic - epidemiology laboratories. In Bulgaria there are a number of mineral springs with unexamined microflora. Studies have shown that microorganisms with valuable properties can be isolated from healing and spring waters. The thermal healing spring with water temperature of 40°C in Starozagorski Mineralni Bani, Stara Zagora region has not been examined so far. The purpose of the present research was the physico-chemical and microbiological analysis of the spring water from the thermal healing spring with water temperature of 40°C in Starozagorski Mineralni Bani, Stara Zagora region and the physiological, biochemical and molecular-genetic identification of a strain isolated from the spring. 2. Materials and Methods 2.1. Physico-chemical methods The colour was determined according to the Rublyovska Scale - method BS 8451: 1977; The odor was determined according to the the method for determination of the odor at 20°C - method BS 8451: 1977, technical means – Mercury thermometer, conditions № 21; The turbidity was determined according to EN ISO 7027, technical means - turbidity meter type TURB 355 IR ID № 200807088; The pH was determined according to BS 3424: 1981, technical means - pH meter type UB10 ID №UB10128148; The oxidation was determined according to BS 3413: 1981; Method for determination of chlorides - BS 3414: 1980; The nitrates were determined according to VLM - NO3 - №2, technical means – photometer “NOVA 60 A” ID № 08450505; The nitrites were determined according to VLM - NO2 - №3, technical means – photometer “NOVA 60 A” ID № 08450505; The ammonium ions were determined according to VLM - NH4 - №1, technical means – photometer “NOVA 60 A” ID № 08450505; The total hardness was determined according to ISO 6058; The sulphates were determined according to VLM - SO4 - №4, technical means – photometer “NOVA 60 A” ID № 08450505; The calcium was determined according to ISO 6058; The magnesium was determined according to BS 7211: 1982; The phosphates were determined according to VLM - PO4 - №5, technical means – photometer “NOVA 60 A” ID № 08450505; The manganese was determined according to VLM - Mn - №7, technical means - photometer “NOVA 60 A” ID № 08450505; The iron was determined according to VLM - Fe - № 6, technical means - photometer “NOVA 60 A” ID № 08450505; The fluoride was determined according to VLM - F - № 8, technical means - photometer “NOVA 60 A” ID № 08450505; The electrical conductivity was determined according to BS EN 27888, technical means - conductivity inoLab cond № 720 ID 11081137. 2.2. Microbiological methods The applied methods for microbiological indicators were in accordance with Ordinance № 9/2001 Darjaven vestnik, Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XIV, Issue 1 – 2015 N e d yal k a VY L C HE VA- Z HEK O VA , Z a pr ya na D E N KO VA, R ad o s ve t a N I K OL O VA B i oc h e m i c al a nd m o l e c u l ar - g e n et i c c ha r ac t e ri z a t i o n of a st r ai n i s ol a t ed f r o m a t h e rm al he a li n g s pr i n g i n S t a ro z a go r s k i M i n e r al ni Ba n i , S t a r a Z a go r a r e gi o n , B ul g a r i a , Foo d a nd E nv ir o nm e nt Sa f e t y , V o l ume X IV , Is s ue 1 – 2 0 15 , pa g . 3 7 - 4 4 39 issue 30 and Decree № 178 / 23.07.2004 on the quality of water intended for drinking purposes. The presence of Escherichia coli and coliform bacteria were determined according to BS EN ISO 9308-1: 2004; The presence of enterococci were determined according to BS EN ISO 7899- 2; The presence of spores of sulfite reducing anaerobes was determined according to BS EN 26461-2: 2004; The total number of aerobic and facultative anaerobic bacteria was determined according to BS EN ISO 6222: 2002; The presence of Pseudomonas aeruginosa was determined according to BS EN ISO 16266: 2008; 2.3. Determination of the sulphytereducing anaerobic bacteria (Clostridium perfringens) in water The samples were heated in a water bath at 80°C for 15 min and pour plated in tubes. The inoculated tubes were incubated at 37°C for 24 h until the appearance of black colonies and cavities in the medium. The amount of sulphytreducing sporeforming anaerobes (Clostridium perfringens) was determined by their titer (the smallest volume of water in which they were established) using standard tables. 2.4. Identification methods Determination of the biochemical profile The system API 50 CHB (BioMerieux SA, France) was used for the identification of the species of the genus Bacillus based on their ability to utilize 49 carbon sources. Fresh 24-hour culture of the studied strain was suspended in API 50 CHB medium, an integral part of the used kit. The API strips were placed in the incubation boxes, the microtubules were inoculated with the prepared cell suspension and sealed with sterile liquid paraffin. The results were reported on the 24th and the 48th hour of incubation at 37±1°C. Reporting was done, based on the colour change of each microtubule, compared to the colour of the control microtubule (microtubule 0). Positive results were recorded in the cases of color change from red to orange or bright yellow. The obtained results were processed with apiweb® identification software. Molecular-genetic methods Isolation of total DNA [1] 16S rDNA amplification All PCR reactions were performed using the PCR kit – PCR VWR, in a volume of 25 µl in a Progene cycler (Techne, UK) according to the manufacturer’s instructions. 50 ng total DNA of the studied strain and 10 pmol primers were mixed in each reaction. DNA of the studied strain was amplified using universal primers for the 16S rDNA gene - 27f (5’AGAGTTTGATCMTGGCTCAG3’) [2] and 1492r (5’ACCTTGTTACGACTT3’) [2]. The amplification program included: denaturation - 95°C for 3 minutes, 40 cycles - 93°C for 30 s, 55°C for 60 s, 72°C for 2 min, final elongation - 72°C for 7 min. The resulting PCR product from the 16S rDNA amplification of the tested strain was visualized on a 2% agarose gel, stained with ethidium bromide solution (0.5 µg/ml), using an UVP Documentation System (UK) [3]. Purification of the product of the PCR- reaction – 16S rDNA – from TAEagarose Gel The purification of 16S rDNA was conducted using DNA-purification kit (GFX MicrospinTM) according to the manufacturer’s instructions. Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XIV, Issue 1 – 2015 N e d yal k a VY L C HE VA- Z HEK O VA , Z a pr ya na D E N KO VA, R ad o s ve t a N I K OL O VA B i oc h e m i c al a nd m o l e c u l ar - g e n et i c c ha r ac t e ri z a t i o n of a st r ai n i s ol a t ed f r o m a t h e rm al he a li n g s pr i n g i n S t a ro z a go r s k i M i n e r al ni Ba n i , S t a r a Z a go r a r e gi o n , B ul g a r i a , Foo d a nd E nv ir o nm e nt Sa f e t y , V o l ume X IV , Is s ue 1 – 2 0 15 , pa g . 3 7 - 4 4 40 DNA-sequencing Sequencing of the gene encoding the 16S rRNA was conducted by „Macrogen Europe Laboratory”, the Netherlands using the Sanger method for DNA-sequencing. The obtained forward and reverse partial sequences of the two ends of the 16S rRNA gene were assembled using the software CLC Sequence Viewer. The total gene sequence of the 16S rRNA gene was compared with the sequences available in the online GenBank database through the online software BLASTn and the species identification was determined by the rate of correspondence between the squence of the studied strain and the reference strain from the online database [4]. 3. Results and Discussion Determination of the physicochemical characteristics of thermal healing spring in the village of Starozagorski Mineralni Bani, Stara Zagora region with water temperature of 40°C The values of the main physico-chemical parameters of the healing spring in the village of Starozagorski Mineralni Bani, Stara Zagora region were compared with the requirements set by the standards (Table 1). Table 1 Physico-chemical analysis of the healing spring water in the village of Starozagorski Mineralni Bani, Stara Zagora region Controlled parameter Unit Maximum value Result Colour according to the Rublyovska scale Colour degrees Acceptable for the consumer Acceptable Odor at 20 °С Total result Acceptable for the consumer Acceptable Turbidity NTU Acceptable for the consumer Acceptable рН pH units ≥ 6,5 and ≤ 9,5 6,9 Oxydation mgO2/dm 3 5,0 0,6 Chlorides mg/dm3 250 26 Nitrates mg/dm3 50 7 Nitrites mg /dm3 0,50 0,00 Ammonium ions mg/ dm3 0,50 0,00 Total hardness mgekv/dm3 12 7 Sulphates mg/dm3 250 14 Calcium mg/dm3 150 72 Magnesium mg /dm3 80 30 Phosphates mg/dm3 0,5 0,0 Manganese mg/dm3 50 0 Iron µg/dm3 200 16 Fluorides mg/dm3 1,5 0.0 Conductivity µS/dm3 2000 750 Experimental data showed that the thermal healing spring waters met the required values for all controlled parameters of Ordinance № 9/2001 Dyrjaven vestnik, issue 30 and Decree № 178/23.07.2004 for the quality of potable water. Determination of the microbiological safety of thermal healing spring in the village of Starozagorski Mineralni Bani, Stara Zagora region with water temperature of 40°C The experimental data from the microbiological examination of the thermal healing spring water indicated that the water met the standard criteria for microbiological quality of mineral and potable water (Table 2). Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XIV, Issue 1 – 2015 N e d yal k a VY L C HE VA- Z HEK O VA , Z a pr ya na D E N KO VA, R ad o s ve t a N I K OL O VA B i oc h e m i c al a nd m o l e c u l ar - g e n et i c c ha r ac t e ri z a t i o n of a st r ai n i s ol a t ed f r o m a t h e rm al he a li n g s pr i n g i n S t a ro z a go r s k i M i n e r al ni Ba n i , S t a r a Z a go r a r e gi o n , B ul g a r i a , Foo d a nd E nv ir o nm e nt Sa f e t y , V o l ume X IV , Is s ue 1 – 2 0 15 , pa g . 3 7 - 4 4 41 Microflora of the thermal healing spring water from the spring in the village of Starozagorski Mineralni Bani, Stara Zagora region with water temperature of 40°C One strain was isolated from the spring water. The colonial and morphological characteristics of the isolated bacterial strain were determined (Table 3). The isolated strain was rod-shaped, Gram - positive, motile, sporeforming. The strain formed a precipitate on the surface of the liquid culture medium, did not turbidify the liquid culture medium and formed a precipitate on the bottom of the tube. The strain was a facultative aerobe (Table 4). The ability of the isolated strain to absorb the 49 carbon sources included in the kit system for rapid identification of bacilli API 50 CHB/E was examined. After processing of the test results with apiweb® the species identification of strains with the corresponding percentage of reliability was obtained (Table 5). The strain Bacillus SMB belongs to the species Bacillus thuringiensis with percentage of reliability of 99%. Table 2 Microbiological analysis of healing spring water from the spring in the village of Starozagorski Mineralni Bani, Stara Zagora region with water temperature of 40°C Controlled parameter Norm, cfu/cm3 Result, cfu/cm3 Coliforms 0/100 0/100 Escherichia coli 0/100 0/100 Enterococci 0/100 0/100 Sulphyte reducing anaerobic bacteria (Clostridium perfringens) 0/100 0/100 TBA at 22 °С 100 0 TBA at 37 °С 20 0 Pseudomonas aeruginosa 0/250 0/250 Table 3 Colonial characteristics of the isolated strain Strain Description of the colonies Visualization Description of the cells Visualization SMB Round, soft, smooth and shiny, whitish, drop-like colonies with wave-like edges, size – 2 – 3 mm Gram-positive short, thick rods with round edges, motile, sporeforming, arranged singly, in pairs and in long chains Table 4 Growth of the isolate from the thermal healing spring water in the village of Starozagorski Mineralni Bani, Stara Zagora region in liquid medium for 24-48 h, at temperatures 3°C - 45°C (P=precipitate; T=turbidification; S=sludge) Strain 3 °С 25 °С 30°С 37 °С 45 °С Attitude to oxygen P T S P T S P T S P T S P T S SMB ─ ─ ─ + ─ + + ─ + + ─ + ─ ─ ─ Facultative aerobe + - Occurance of Precipitate, Turbidity, Sludge; ─ - Absence of Precipitate, Turbidity, Sludge. Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XIV, Issue 1 – 2015 N e d yal k a VY L C HE VA- Z HEK O VA , Z a pr ya na D E N KO VA, R ad o s ve t a N I K OL O VA B i oc h e m i c al a nd m o l e c u l ar - g e n et i c c ha r ac t e ri z a t i o n of a st r ai n i s ol a t ed f r o m a t h e rm al he a li n g s pr i n g i n S t a ro z a go r s k i M i n e r al ni Ba n i , S t a r a Z a go r a r e gi o n , B ul g a r i a , Foo d a nd E nv ir o nm e nt Sa f e t y , V o l ume X IV , Is s ue 1 – 2 0 15 , pa g . 3 7 - 4 4 42 Table 5 API 50 CHB/E of the strain Bacillus SMB # Carbohydrates SMB 1 Glycerol - 2 Erythriol - 3 D-arabinose - 4 L-arabinose - 5 Ribose + (90-100%) 6 D-xylose - 7 L-xylose - 8 Adonitol - 9 -metil-D-xyloside - 10 Galactose - 11 D-glucose + (50%) 12 D-fructose + (50%) 13 D-mannose - 14 L-sorbose - 15 Rhamnose - 16 Dulcitol - 17 Inositol - 18 Manitol - 19 Sorbitol - 20 -methyl-D-mannoside - 21 -methyl-D-glucoside - 22 N-acetyl-glucosamine + (90-100%) 23 Amigdalin - 24 Arbutin + (90-100%) 25 Esculin + (90-100%) 26 Salicin + (90-100%) 27 Cellobiose - 28 Maltose + (90-100%) 29 Lactose - 30 Melibiose - 31 Saccharose + (90-100%) 32 Trehalose + (90-100%) 33 Inulin - 34 Melezitose - 35 D-raffinose - 36 Amidon + (50%) 37 Glycogen + (90-100%) 38 Xylitol - 39 -gentiobiose - 40 D-turanose - 41 D-lyxose - 42 D-tagarose - 43 D-fuccose - 44 L-fuccose - 45 D-arabitol - 46 L-arabitol - 47 Gluconate - 48 2-keto-gluconate - 49 5-keto-gluconate - Identification Bacillus thuringiensis % of reliability 99 Bacillus thuringiensis B62, 16S ribosomal RNA gene, partial sequence; Sequence ID: gb|JX010983.1| Length: 1455 Number of Matches: 1 Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XIV, Issue 1 – 2015 N e d yal k a VY L C HE VA- Z HEK O VA , Z a pr ya na D E N KO VA, R ad o s ve t a N I K OL O VA B i oc h e m i c al a nd m o l e c u l ar - g e n et i c c ha r ac t e ri z a t i o n of a st r ai n i s ol a t ed f r o m a t h e rm al he a li n g s pr i n g i n S t a ro z a go r s k i M i n e r al ni Ba n i , S t a r a Z a go r a r e gi o n , B ul g a r i a , Foo d a nd E nv ir o nm e nt Sa f e t y , V o l ume X IV , Is s ue 1 – 2 0 15 , pa g . 3 7 - 4 4 43 Score Expect Identities Gaps Strand 2773 bits(1442) 0.0 1444/1445(99%) 0/1445(0%) Plus/Plus * Query 10 CGGCGTGCCTATACATGCAGTCGAGCGAATGGATTAAGAGCTTGCTCTTATGAAGTTAGC 69 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 7 CGGCGTGCCTATACATGCAGTCGAGCGAATGGATTAAGAGCTTGCTCTTATGAAGTTAGC 66 Query 70 GGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCATAAGACTGGGATAACTCCGGGAAA 129 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 67 GGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCATAAGACTGGGATAACTCCGGGAAA 126 Query 130 CCGGGGCTAATACCGGATAATATTTTGAACTGCATGGTTCGAAATTGAAAGGCGGCTTCG 189 |||||||||||||||||||| ||||||||||||||||||||||||||||||||||||||| Sbjct 127 CCGGGGCTAATACCGGATAACATTTTGAACTGCATGGTTCGAAATTGAAAGGCGGCTTCG 186 Query 190 GCTGTCACTTATGGATGGACCCGCGTCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCA 249 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 187 GCTGTCACTTATGGATGGACCCGCGTCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCA 246 Query 250 AGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGC 309 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 247 AGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGC 306 Query 310 CCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGA 369 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 307 CCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGA 366 Query 370 GCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAAC 429 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 367 GCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAAC 426 Query 430 AAGTGCTAGTTGAATAAGCTGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACT 489 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 427 AAGTGCTAGTTGAATAAGCTGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACT 486 Query 490 ACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTA 549 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 487 ACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTA 546 Query 550 AAGCGCGCGCAGGTGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGG 609 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 547 AAGCGCGCGCAGGTGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGG 606 Query 610 TCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAAAGTGGAATTCCATGTGTAGCGGT 669 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 607 TCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAAAGTGGAATTCCATGTGTAGCGGT 666 Query 670 GAAATGCGTAGAGATATGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTG 729 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 667 GAAATGCGTAGAGATATGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTG 726 Query 730 ACACTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCG 789 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 727 ACACTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCG 786 Query 790 TAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGAAGTTAACGCATT 849 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 787 TAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGAAGTTAACGCATT 846 Query 850 AAGCACTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCC 909 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 847 AAGCACTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCC 906 Query 910 CGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCT 969 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 907 CGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCT 966 Query 970 TGACATCCTCTGAAAACCCTAGAGATAGGGCTTCTCCTTCGGGAGCAGAGTGACAGGTGG 1029 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 967 TGACATCCTCTGAAAACCCTAGAGATAGGGCTTCTCCTTCGGGAGCAGAGTGACAGGTGG 1026 Query 1030 TGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAA 1089 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 1027 TGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAA 1086 Query 1090 CCCTTGATCTTAGTTGCCATCATTAAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAAC 1149 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 1087 CCCTTGATCTTAGTTGCCATCATTAAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAAC 1146 Query 1150 CGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGT 1209 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 1147 CGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGT 1206 Query 1210 GCTACAATGGACGGTACAAAGAGCTGCAAGACCGCGAGGTGGAGCTAATCTCATAAAACC 1269 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 1207 GCTACAATGGACGGTACAAAGAGCTGCAAGACCGCGAGGTGGAGCTAATCTCATAAAACC 1266 Query 1270 GTTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGCTGGAATCGCTAGTAATC 1329 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 1267 GTTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGCTGGAATCGCTAGTAATC 1326 Query 1330 GCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACC 1389 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 1327 GCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACC 1386 Query 1390 ACGAGAGTTTGTAACACCCGAAGTCGGTGGGGTAACCTTTTGGAGCCAGCCGCCTAAGGT 1449 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 1387 ACGAGAGTTTGTAACACCCGAAGTCGGTGGGGTAACCTTTTGGAGCCAGCCGCCTAAGGT 1446 Query 1450 GGACC 1454 ||||| Sbjct 1447 GGACC 1451 Fig. 1. Comparison of the nucleotide sequence of the 16S rRNA gene of the strain Bacillus SMB and the partial sequence of the 16S rRNA gene of Bacillus thuringiensis B62. Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XIV, Issue 1 – 2015 N e d yal k a VY L C HE VA- Z HEK O VA , Z a pr ya na D E N KO VA, R ad o s ve t a N I K OL O VA B i oc h e m i c al a nd m o l e c u l ar - g e n et i c c ha r ac t e ri z a t i o n of a st r ai n i s ol a t ed f r o m a t h e rm al he a li n g s pr i n g i n S t a ro z a go r s k i M i n e r al ni Ba n i , S t a r a Z a go r a r e gi o n , B ul g a r i a , Foo d a nd E nv ir o nm e nt Sa f e t y , V o l ume X IV , Is s ue 1 – 2 0 15 , pa g . 3 7 - 4 4 44 The 16S rRNA gene was sequenced and the resulting sequence was processed using the software BLASTn and it was confirmed that the studied strain belongs to the species Bacillus thuringiensis (Fig. 1). 4. Conclusion The physicochemical and microbiological parameters of thermal healing spring water in the village of Starozagorski Mineralni Bani, Stara Zagora region were examined. The spring water met the requirements laid down in the Ordinance № 9/2001 Dyrjaven vestnik, issue 30 and Decree № 178/23.07.2004 for the quality of potable water when it comes to its physicochemical parameters. From a microbiological point of view the spring water was safe to use. The strain SMB isolated from the spring was identified using physiochemical, biochemical (API 50 CHB) and molecular-genetic (sequencing of the gene for the 16S rRNA) methods as a representative of the species Bacillus thuringiensis. After further examinations of its antimicrobial activity against phytopathogenic molds strain SMB would be incorporated in the composition of biological preparations against plant diseases. 5. References [1]. BENDER T, BÁLINT P, BÁLINT G, A brief history of spa therapy. Ann. Rheum. Dis. 61,949 (2002). [2]. BENDER T, KARAGÜLLE Z, BÁLINT G, GUTENBRUNNER C, BÁLINT P, SUKENIK S, Hydrotherapy, balneotherapy and spa treatment in pain management. Rheumatol. Int. 4:220–224 (2005). [3]. GEYTENBEEK J, Evidence for effective hydrotherapy. Physiotherapy 9:514–529 (2002). [4]. RIYAZ N, ARAKKAL F,Spa therapy in dermatology. Indian J. Dermatol. Venereol. Leprol. 77:128–134 (2011). [5]. SEITE S, Thermal waters as cosmeceuticals: La Roche-Posay thermal spring water example. Clin. Cosmet. Investig. Dermatol. 6:23–28, (2013). [6]. SUKENIK S, FLUSSER D, ABU-SHAKRA M, The role of spa therapy in various rheumatic diseases. Rheum. Dis. Clin. N. Am. 25:883–897 (1999). [7]. TISHLER M, ROSENBERG O, LEVY O, ELIAS I, AMIT-VAZINA M, The effect of balneotherapy on osteoarthritis. Is an intermittent regimen effective? Eur. J. Intern. Med. 15:93–96 (2004). [8]. TONKO C, KIRÁLY A, MIZSEY P, PATZAY G, CSEFALVAY E, Limitation of hardness from thermal water by means of nanofiltration. Water Sci. Technol. 67:2025–2032 (2013). [9]. DELLEY M, MOLLET B, HOTTINGER H, DNA probe for Lactobacillus delbrueckii. Appl. Environ. Microbiol., 56,1967–1970 (1990). [10]. LANE DJ, 16S/23S rRNA sequencing. In: Nucleic acid techniques in bacterial systematics. Stackebrandt, E., and Goodfellow, M., eds., John Wiley and Sons, New York, NY, pp. 115-175 (1991). [11]. DENKOVA R, GEORGIEVA L, DENKOVA Z, URSHEV Z, YANAKIEVA V, Biochemical and technological properties of Lactobacillus plantarum X2 from naturally fermented sourdough. Journal of Food and Packaging Science Technique and Technologies 1(1):59 - 64 (2012a). [12]. DENKOVA R, YANAKIEVA V, DENKOVA Z, URSHEV Z, GORANOV B, SOTIROVA E, Identification and examination of some probiotic properties of Lactobacillus plantarum F3. Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University – Suceava 9 (4):22 – 29 (2012b).