Microsoft Word - 4 Denkova_corectat.doc Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XI, Issue 4 – 2012 22 IDENTIFICATION AND EXAMINATION OF SOME PROBIOTIC PROPERTIES OF LACTOBACILLUS PLANTARUM F3 Rositsa DENKOVA1, Velichka YANAKIEVA2, *Zapryana DENKOVA2, Zoltan URSHEV3, Bogdan GORANOV2, Elena SOTIROVA2 1Department of Biotechnology, Sofia University „St. Kliment Ohridski”, rositsa_denkova@mail.bg 2Department of Microbiology, University of Food Technologies, zdenkova@abv.bg 3DNA Analyses Lab, LB Bulgaricum *Corresponding author Received 16 November 2012, accepted 10 December 2012 Abstract: In order to be included in the composition of probiotic preparations each strain has to meet a number of requirements. The strain Lactobacillus F3 from naturally fermented sourdough is identified as a Lactobacillus plantarum strain using molecular-genetic methods (ARDRA and 16S rDNA sequencing). Some of its probiotic properties are examined: ability for industrial cultivation and survival in the model conditions of the gastro-intestinal tract. High concentrations of active cells are retained during cultivation at pH=2 + pepsin, pH=4,5 +pancreatin and pH=7 + pancreatin as well as at different concentrations of bile salts – 0,15%, 0,3%, 0,6%, 1%. The strain allows industrial cultivation with accumulation of high concentrations of viable cells. The results of the studies on some probiotic properties of Lactobacillus plantarum F3 make the strain a potentially probiotic one. Keywords: ARDRA, sequencing, probiotic, batch cultivation, pepsin, pancreatin, bile salts 1. Introduction A number of factors influence negatively the interaction between intestinal microorganisms, such as stress and diet. Unfortunately they lead to detrimental effects on human health. There is increasing evidence indicating that consumption of ‘probiotic’ microorganisms helps maintaining a favourable microbial profile as results of which several therapeutic benefits are observed [7]. Probiotics are live microorganisms that confer a beneficial effect on the host when administered in proper amounts [4]. The beneficial effects of probiotic preparations on gastrointestinal infections, the protection of the immune system, the reduction of serum cholesterol, the improvement in inflammatory bowel disease and suppression of Helicobacter pylori infection, Crohn's disease, restoration of the microflora in the stomach and the intestines after antibiotic treatment; they are also characterized by anti-cancer properties, antimutagenic action, anti- diarrheal properties are well known [8]. Lactobacilli and bifidobacteria are a natural part of the intestinal microflora of the healthy human. They are included in the composition of probiotics and probiotic foods because of their proven health benefits to the body [6]. But not all strains of lactobacilli and bifidobacteria can be used as components of probiotics and probiotic foods, but only those that exhibit certain properties. Probiotic microorganisms should be of human origin, resistant to gastric acid, bile and to the antibiotics, administered in medical practice, non-pathogenic; they should also have the potential to adhere to the gut epithelial tissue and produce antimicrobial Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XI, Issue 4 – 2012 23 substances; they should allow the conduction of technological processes, in which high concentrations of viable cells are obtained as well as to allow industrial cultivation, encapsulation and freeze- drying and they should remain active during storage [5]. This leads to the mandatory selection of strains of the genera Lactobacillus and Bifidobacterium with probiotic properties. The purpose of this paper is to identify the strain Lactobacillus F3, isolated from naturally fermented sourdough, and to examine some of its technological properties – survival in the model conditions of the gastrointestinal tract and ability for industrial cultivation. 2. Experimental 2.1. Microorganisms The studied Lactobacillus strain, Lactobacillus F3, is isolated from naturally fermented sourdough. Reference microorganisms: Lactobacillus acidophilus DSM 20079, Lactobacillus delbrueckii ssp.bulgaricus DSM 20081, Lactobacillus casei ssp.casei DSM 20011, Lactobacillus casei ssp.paracasei DSM 20312, Lactobacillus casei ssp.rhamnosus LMG 6400, Lactobacillus fermentum DSM 20052, Lactobacillus helveticus DSM 20075, Lactobacillus plantarum DSM 20174. 2.2. Media Saline solution. Composition (g/dm3): NaCl - 5. Sterilization - 20 minutes at 121ºC. LAPTg10-broth medium. Composition (g/dm3): peptone - 15, yeast extract - 10; tryptone - 10, glucose - 10. pH is adjusted to 6.6 - 6.8 and Tween 80 - 1cm3/dm3 is added. Sterilization - 20 minutes at 121ºC. LAPTg10-agar. Composition (g/dm3): LAPTg10-broth medium and 2% agar. Sterilization - 20 minutes at 121ºC. MRS – broth medium (Scharlau) 2.3.Identification Isolation of total DNA The isolation of DNA is performed by the method of Delley et al. [2]. PCR reactions and visualization All PCR reactions are performed using the PCR kit - Ready To GoTM PCR beads (Amersham Biosciences), in a volume of 25 µl in a Progene cycler (Techne, UK). The resulting products are visualized on a 2% agarose gel stained with ethidium bromide solution (0.5 µg/ml), using an UVP Documentation System (UK). 16S rDNA amplification and 16S rDNA ARDRA (Amplified Ribosomal DNA Restriction Analysis) The method ARDRA involves enzymatic multiplication of the gene encoding the 16S rRNA, using primers complementary to the conservative regions at both ends of the 16S rRNA gene and the product of the multiplication is then restricted with restriction enzymes. The resulting profile is highly specific for the particular studied species. DNA of the studied strain is amplified using universal primers for the 16S rDNA gene - fD1 and rD1 [9]. The amplification program includes: denaturation - 95°C for 3 minutes, 40 cycles - 93°C for 30 s, 48°C for 60 s, 72°C for 60 s, final elongation - 72°C for 5 min. The resulting PCR product from the 16S rDNA amplification of the tested strain is treated with the endonucleases Eco RI, Hae III and Alu I (Boehringer Mannhem GmbH, Germany). Reactions are carried out according to the following quantities: PCR products - 10µl, enzyme solution - 10 µl (1 µl of the respective enzyme, 2 µl buffer, 7 µl dH2O). Incubation for 1 night at 37°C is performed. The resulting restriction products are visualized on a 2% agarose gel. 2.4. Purification of the product of the PCR-reaction – 16S rDNA – from TAE- agarose gel Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XI, Issue 4 – 2012 24 The purification of 16S rDNA is conducted using DNA-purification kit (GFX MicrospinTM) according to the manufacturer’s instructions: 1) Sample capture. After visualizing the product of the 16S PCR-amplification reaction on a 2% agarose gel with UV light with wavelength 302 nm, the gel is visualized with UV light with wavelength 365 nm. The 16S PCR product is cut from the gel and placed in a DNA-free microcentrofuge tube. Through weighing the microcentrofuge tube before and after the gel fragments are put in them, the weight of the fragments is calculated and 10µl Capture buffer is added to every 10mg of the gel. The microcentrofuge tube are mixed gently and incubated at 60°C for about 20 minutes until the full dissolution of the gel fragments. 2) Sample binding A GFX MicrospinTM column is labelled and placed in a collection tube and the centrofuged (shortspin) samples in the eppendorf tubes from 1) are poured in the GFX MicrospinTM columns (no more than 600µl). The GFX MicrospinTM columns are allowed to wet for about 60 seconds and centrofuged until the whole volume passes through the column. The liquid from the column is disposed and the GFX MicrospinTM column is placed in the same collection tube. If a sample is more than 600µl, all the steps from the sample binding are repeated until the whole sample is eluated. 3) Wash and dry 500 µl of wash buffer type 1 are poured in each GFX MicrospinTM column, the columns are centrofuged (shortspin), the collection tubes are disposed and each GFX MicrospinTM column is placed in a new 1,5 ml DNAase free microcentrofuge tube. 4) Elution 10-50µl Elution buffer type 4 or type 6 are poured in each GFX MicrospinTM column. The column is allowed to wet at room temperature for 60 seconds and the microcentrofuge tubes with the GFX MicrospinTM columns are centrofuged for about 60 seconds. The eluate (containing purified 16S rDNA) is collected and freezed at -20°C. 2.5. DNA-sequencing Sequencing of the gene encoding the 16S rRNA is performed by „Macrogen Europe Laboratory”, the Netherlands using the Sanger method for DNA-sequencing. 2.6. Determination of the resistance to low pH in the presence of pepsin and to weakly alkaline pH in the presence of pancreatin [1] Fresh 24 - hour culture of the studied strain is centrifuged for 15 min at 5,000 x g. The resulting sludge biomass is washed twice with PBS - buffer and resuspended to the initial volume in PBS - buffer. 0.2 cm3 of the cell suspension are incubated with 5 cm3 buffer solution with pH = 2 containing 0,5% NaCl and pepsin (at a concentration of 3.2 g/dm3) (Sigma, 2,500 - 3,500 U / mg protein), buffer with pH = 4,5 + pancreatin and buffer with pH = 7 + pancreatin at a suitable temperature for the studied strain (37°C) for 24h. At the 0, the 2nd, the 4th and the 24th hour aliquots for the determination of the number of viable cells are taken (cfu/cm3). 2.8. Determining the tolerance to bile salts [3] Fresh 24 - hour culture of the studied strain is centrifuged for 15 min at 5,000 x g. The resulting sludge biomass is washed twice with PBS - buffer and resuspended to the initial volume in PBS - buffer. 0.2 cm3 of the cell suspension are incubated with 5 cm3 of the MRS-broth medium with different concentrations of bile salts - 0%, 0.15%, 0.3%, 0.6% and 1% - for 24h at the optimum temperature for the strain (37°C), and aliquots for the determination of the number of viable cells (cfu/cm3) at the 0, the 2nd, the 4th, the 6th, the 8th and the 24th hour are taken. Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XI, Issue 4 – 2012 25 2.9. Batch cultivation in a bioreactor with continuous stirring and in a thermostat at static conditions The laboratory cultural vessel (Fig.1) is a cylinder with geometric volume of 2 dm3 and displacement – 1,5 dm3. Figure 1. Scheme of the laboratory bioreactor 1 - vessel with geometric volume of 2 dm3; 2- four repulse devises; 3–thermo-strength Pt100; 4–heater; 5-heat exchanger for cold water ; 6– turbine stirrer; 7–pH electrode; 8–exit for CO2; 9–filter; 10–peristaltic pump for pH correction; 11– reagent for pH correction – 20% KOH; 12– motor; 13-control links; 14–control device "Applikon". The periodic cultivation processes are conducted in MRS-broth without pH adjustment. The medium is sterilized at 118ºC for 15 min. After cooling to 39-40ºC the prepared medium in the bioreactor (MRS-broth) is inoculated with 5% (v/v) inoculum. The process of cultuvation is conducted at 37ºC, stirring speed of 100 rpm, without air supply. During the cultivation pH, Eh, number of colony- forming units and tirable acidity are examined. Along with the carried out periodical cultivation with constant stirring (in a bioreactor), static cultivation (in an incubator) under the same conditions is carried out as well. The number of viable cells of Lactobacillus plantarum F3 is determined through appropriate tenfold dillusions of the samples and plating on coloured LAPTg10 – agar medium. The Petri dishes are cultivated for 72 hours at 37°C until single colonies can be counted. The titratable acidity is determined using 0,1N NaOH. 5 cm3 of each sample are mixed with 10 cm3dH2O and titrated with 0,1N NaOH, using phenolphtalein as an indicator, until the appearance of pale pink colour, which retains for 1 minute. The value for the titratable acidity is obtained by multiplying the millilitres 0,1N NaOH by the factor of the 0,1N NaOH and the number 20. 3. Results and Discussion The strain Lactobacillus F3 is isolated from naturally fermented sourdough. Identification of Lactobacillus F3 The identification of Lactobacillus F3 is performed using ARDRA analysis, followed by sequencing of the gene encoding the 16S rRNA. ARDRA analysis. As a result of the ARDRA analysis with the enzymes Eco RI (Fig.1), Hae III (Fig. 2) and Alu I (Fig. 3) the studied strain is determined to be a representative of the species Lactobacillus plantarum. DNA-sequencing of Lactobacillus F3 is conducted by Macrogen Europe Laboratory, the Netherlands by the method of chain termination (method of Sanger). After careful comparison of the obtained sequence with the public online nucleotide BLAST database, the strain Lactobacillus F3 is confirmed to be a Lactobacillus plantarum strain (Fig. 4). Cooling water Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XI, Issue 4 – 2012 26 Fig. 1. Restriction profile of the 16S rDNA with EcoRI 1. Lactobacillus F3 2. Lactobacillus acidophilus DSM 20079 3. Lactobacillus delbrueckii ssp.bulgaricus DSM 20081 4. Lactobacillus casei ssp.casei DSM 20011 5. Lactobacillus casei ssp.paracasei 6. Lactobacillus casei ssp.rhamnosus 7. Lactobacillus fermentum DSM 20052 8. Lactobacillus helveticus DSM 20075 9. Lactobacillus plantarum DSM 20174 10. M Fig. 2. Restriction profile of the 16S rDNA with HaeIII 1. Lactobacillus F3 2. Lactobacillus acidophilus DSM 20079 3. Lactobacillus delbrueckii ssp.bulgaricus DSM 20081 4. Lactobacillus casei ssp.casei DSM 20011 5. Lactobacillus casei ssp.paracasei 6. Lactobacillus casei ssp.rhamnosus 7. Lactobacillus fermentum DSM 20052 8. Lactobacillus helveticus DSM 20075 9. Lactobacillus plantarum DSM 20174 10. M Fig. 3. Restriction profile of the 16S rDNA with Alu I 1. Lactobacillus F3 2. Lactobacillus acidophilus DSM 20079 3. Lactobacillus delbrueckii ssp.bulgaricus DSM 20081 4. Lactobacillus casei ssp.casei DSM 20011 5. Lactobacillus casei ssp.paracasei 6. Lactobacillus casei ssp.rhamnosus 7. Lactobacillus fermentum DSM 20052 8. Lactobacillus helveticus DSM 20075 9. Lactobacillus plantarum DSM 20174 10. M 1 2 3 4 5 6 7 8 9 10 1500 kb 1000 kb 900 kb 800 kb 700 kb 600 kb 500 kb 1 2 3 4 5 6 7 8 9 10 1500 kb 1000 kb 900 kb 800 kb 700 kb 600 kb 500 kb 1 2 3 4 5 6 7 8 9 10 1500 kb 1000 kb 900 kb 800 kb 700 kb 600 kb 500 kb Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XI, Issue 4 – 2012 27 > ref|NR_042394.1| Lactobacillus plantarum strain NRRL B-14768 16S ribosomal RNA, partial sequence Length=1474 Score = 1977 bits (1070), Expect = 0.0 Identities = 1072/1073 (99%), Gaps = 0/1073 (0%) Strand=Plus/Minus Query 11 GTCCACCTTAGGCGGCTGGTTCCTAAAAGGTTACCCCACCGACTTTGGGTGTTACAAACT 70 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 1467 GTCCACCTTAGGCGGCTGGTTCCTAAAAGGTTACCCCACCGACTTTGGGTGTTACAAACT 1408 Query 71 CTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGA 130 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 1407 CTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGA 1348 Query 131 TCCGCGATTACTAGCGATTCCGACTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTG 190 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 1347 TCCGCGATTACTAGCGATTCCGACTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTG 1288 Query 191 AGAATGGCTTTAAGAGATTAGCTTACTCTCGCGAGTTCGCAACTCGTTGTACCATCCATT 250 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 1287 AGAATGGCTTTAAGAGATTAGCTTACTCTCGCGAGTTCGCAACTCGTTGTACCATCCATT 1228 Query 251 GTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTC 310 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 1227 GTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTC 1168 Query 311 CTCCGGTTTGTCACCGGCAGTCTCACCAGAGTGCCCAACTTAATGCTGGCAACTGATAAT 370 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 1167 CTCCGGTTTGTCACCGGCAGTCTCACCAGAGTGCCCAACTTAATGCTGGCAACTGATAAT 1108 Query 371 AAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACC 430 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 1107 AAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACC 1048 Query 431 ATGCACCACCTGTATCCATGTCCCCGAAGGGAACGTCTAATCTCTTAGATTTGCATAGTA 490 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 1047 ATGCACCACCTGTATCCATGTCCCCGAAGGGAACGTCTAATCTCTTAGATTTGCATAGTA 988 Query 491 TGTCAAGACCTGGTAAGGTTCTTCGCGTAGCTTCGAATTAAACCACATGCTCCACCGCTT 550 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 987 TGTCAAGACCTGGTAAGGTTCTTCGCGTAGCTTCGAATTAAACCACATGCTCCACCGCTT 928 Query 551 GTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGCCTTGCGGCCGTACTCCCCAGGCGGAA 610 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 927 GTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGCCTTGCGGCCGTACTCCCCAGGCGGAA 868 Query 611 TGCTTAATGCGTTAGCTGCAGCACTGAAGGGCGGAAACCCTCCAACACTTAGCATTCATC 670 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 867 TGCTTAATGCGTTAGCTGCAGCACTGAAGGGCGGAAACCCTCCAACACTTAGCATTCATC 808 Query 671 GTTTACGGTATGGACTACCAGGGTATCTAATCCTGTTTGCTACCCATACTTTCGAGCCTC 730 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 807 GTTTACGGTATGGACTACCAGGGTATCTAATCCTGTTTGCTACCCATACTTTCGAGCCTC 748 Query 731 AGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGC 790 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 747 AGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGC 688 Query 791 ATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTTTCCCAGTTTCC 850 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 687 ATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTTTCCCAGTTTCC 628 Query 851 GATGCACTTCTTCGGTTGAGCCGAAGGCTTTCACATCAGACTTAAAAAACCGCCTGCGCT 910 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 627 GATGCACTTCTTCGGTTGAGCCGAAGGCTTTCACATCAGACTTAAAAAACCGCCTGCGCT 568 Query 911 CGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGG 970 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 567 CGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGG 508 Query 971 CACGTAGTTAGCCGTGGCTTTCTGGTTAAATACCGTCAATACCTGAACAGTTACTCTCAG 1030 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 507 CACGTAGTTAGCCGTGGCTTTCTGGTTAAATACCGTCAATACCTGAACAGTTACTCTCAG 448 Query 1031 ATATGTTCTTCTTTAACAACAGAGTTTTACGAACCGAAACCCTTCTTCACTCA 1083 |||||||||||||||||||||||||||||||| |||||||||||||||||||| Sbjct 447 ATATGTTCTTCTTTAACAACAGAGTTTTACGAGCCGAAACCCTTCTTCACTCA 395 Figure 4. Comparison of the nucleotide sequences of the 16S rDNA of Lactobacillus F3 and the partial sequence of the 16S rDNA of Lactobacillus plantarum NRRL B-14768. Probiotic properties of Lactobacillus plantarum F3 Survival in the model conditions of the gastrointestinal tract The resistance of the cells of Lactobacillus plantarum F3 in the model conditions of the gastro - intestinal tract is examined: survival at pH = 2 + pepsin, at pH = 4,5 + pancreatin and at pH = 7 + pancreatin. The results of the experimental studies are presented on Fig. 5. Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XI, Issue 4 – 2012 28 Figure 5. Survival of the cells of the strain Lactobacillus plantarum F3 at pH = 2 + pepsin, at pH = 4,5 + pancreatin and at pH = 7 + pancreatin. It is observed that the sensitivity of Lactobacillus plantarum F3 to pH = 2 + pepsin, pH = 4,5 + pancreatin and pH = 7 + pancreatin is comparable – the reduction of the number of viable cells is about 4logN (Fig. 5) by the 24th hour of cultivation. But the concentration of active cells even by the 24th hour remains high – 1,6x106cfu/cm3 at pH = 2 + pepsin, 3x105cfu/cm3 at pH = 4,5 + pancreatin, 1,1x105cfu/cm3 at pH = 7 + pancreatin, which makes the strain appropriate for incorporation in probiotics. Another factor of great importance that influences the survival of probiotic strains in the gastrointestinal tract are bile salts. About three hours after ingestion of food the concentration of bile salts in the small intestine reaches about 0.3%. This requires study of the influence of different concentrations of bile salts on the survival of Lactobacillus plantarum F3 in MRS- broth medium with different concentrations of bile salts, 0%, 0.15%, 0.3%, 0.6% and 1% for 24 hours of incubation. The number of viable cells of Lactobacillus plantarum F3 starts decreasing since the beginning of the cultivation of the strain in MRS-broth medium with different concentrations of bile salts (Fig. 6). The degree of reduction is different at the different concentrations of bile salts – it is greater at concentrations 0,6% and 1% and smaller at 0,15% and 0,3% bile salts. At 0,15% bile salts the reduction is 1,3logN and at 0,3% it is 1logN. At 0,6% bile salts the degree of reduction is considerably higher – 3logN and at 1% bile salts it is about 3,5logN. Figure 6. Survival of the cells of Lactobacillus plantarum F3 at different concentrations of bile salts. But by the end of the experiment the concentration of viable cells remains between 1,9x105 cfu/cm3 (at 1% bile salts) and 1x107cfu/cm3 (at 0,15% bile salts), which allows the inclusion of Lactobacillus plantarum F3 in the composition of probiotic preparations. Batch cultivation in a bioreactor with continuous stirring and at static conditions of Lactobacillus plantarum F3 The strain Lactobacillus plantarum F3 is cultivated in MRS-broth at 37ºC in a laboratory bioreactor with continuous stirring and in a thermostat. It is observed that the time to reach high concentration of viable cells during cultivation in the bioreactor with continuous stirring is reduced in comparison to cultivation at static conditions (Fig. 7, Fig. 8). At the 6th hour the number of cells reaches 8,3x1010cfu/cm3 (Fig. 7), while under static conditions, the same concentration of cells is reached at the 12th hour from the beginning of the process (Fig. 8). The number of active cells of Lactobacillus plantarum F3 obtained in cultivation in a Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XI, Issue 4 – 2012 29 bioreactor with continuous stirring and at static conditions by the 24th hour is comparable – 3x1012cfu/cm3 in the bioreactor and 7,8x1012cfu/cm3 at static conditions. The titratable acidity of the medium in the bioreactor increases from 46,2°T to 241,7°T, while at static conditions it reaches 240,1°T by the 24th hour and 248,3°T by the 48th hour. 0 6 12 18 24 8 9 10 11 12 13 0 6 12 18 24 40 80 120 160 200 240 280 0 6 12 18 24 520 560 600 640 680 720 0 6 12 18 24 3,2 3,6 4,0 4,4 4,8 5,2 5,6 log N lo g N time, h TK, oT T K , O T Eh, mV E h, m V pH p H Figure 7. Batch cultivation of Lactobacillus plantarum F3 in MRS-broth in a bioreactor with constant stirring. 0 6 12 18 24 30 36 42 48 8 9 10 11 12 13 0 6 12 18 24 30 36 42 48 40 60 80 100 120 140 160 180 200 220 240 260 log N lo g N time, h TK, oT T K , 0 T Figure 8. Static cultivation of Lactobacillus plantarum F3 in MRS-broth The redox potential of the system starts increasing since the beginning of the batch process. It starts from +526mV and reaches +716 mV (Fig. 7). The strain Lactobacillus plantarum F3 allows industrial cultivation with accumulation of high concentrations of viable cells. 4. Conclusion The strain Lactobacillus F3 is identified as belonging to the species Lactobacillus plantarum. Lactobacillus plantarum F3 has the ability to survive in the model conditions of the gastro - intestinal tract and allows industrial cultivation with accumulation of high concentrations of viable cells. Thus, it can be defined as a potential probiotic culture, which after further research can be incorporated in the composition of probiotic preparations for treatment and prevention. 5. References [1]. CHARTERIS W., KELLY P., MORELLI L., COLLINS J., Development and application of an in vitro methodology to determine the transit tolerance of potentially probiotic Lactobacillus and Bifidobacterium species in the upper human gastrointestinal tract. J.of Appl.Micr. 84 (5), pp. 759–768, 1998. [2]. DELLEY M., MOLLET B., HOTTINGER H., DNA probe for Lactobacillus delbrueckii. Appl. Environ. Microbiol. 56:1967–1970, 1990 [3]. DENKOVA Z., Production and application of probiotics, D.Sc. Thesis, 2005 [4]. KALLIOMAKI M., SALMINEN S., ARVILOMMI H., KERO P., KOSKINEN P., ISOLAURI E., Probiotics in primary prevention of atopic disease: a randomised placebocontrolled trial, Lancet 357. 1076–1079, 2001 [5]. KIRTZALIDOU E., PRAMATEFTAKI P., KOTSOU M., KYRIACOU A., Screening for lactobacilli with probiotic properties in the infant gut microflora, Anaerobe 17. 440 – 443, 2011 [6]. MARTEAU P., DE VRESE M., CELLIER C., SCHREZENMEIR J. Protection from gastrointestinal diseases with the use of probiotics. Am. J.of Clin. Nutr.73 (Suppl.2). 430S–436S, 2001 [7]. RYBKA S., KAILASAPATHY K., The survival of culture bacteria in fresh and freeze-dried AB yoghurts, The Aust. J. of Dairy Technology 50(2). 51–57, 1995 [8]. SHAH N., Functional cultures and health benefits, Int. Dairy J. 17. 1262–1277, 2007 [9]. WEISBURG W., BARNS S., PELLETIER D., LANE D., 16S ribosomal DNA amplification for phylogenetic study. J. Bacteriol.173. 697-703, 1991