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Clinical Laboratories
75 Francis Street
Boston , Massachusetts 02115
Tel: 617 732-7372, Lab: 617 525-2506
Fax: 617 277-1762
E-mail: aonderdonk@partners.org
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Andrew B. Onderdonk, Ph.D.
Director
Clinical Microbiology Laboratory
Professor of Pathology
Harvard Medical School

Pathogen Inhibition Test

Purpose: To determine if Lactobacillus delbrueckii subsp. bulgaricus G-LB-44 inhibits the growth of Escherichia coli in vegetable juice.
Test Materials:
  1. Lyophilized Lactobacillus delbrueckii subsp. bulgaricus G-LB-44 (powder CFU – 1×109/g)
  2. Vegetable juice (3 bottles of Juice Press Organic Complete Source, 17 fluid ounces each)
  3. Tryptic Soy Agar Plates (TSA)
  4. Esherichia coli – ATCC 25922
Bacterial Inoculum
E.coli Previously prepared frozen stock culture of E. coli was used for these experiments. The bacterial concentration was 1.32×109/mL.
For the E. coli control and test samples: The frozen tube containing E. coli was removed from the freezer and allowed to thaw at room temperature. The bacteria were diluted (1:10000) in sterile phosphate buffered saline to achieve a concentration of approximately 1×105/mL. 50ul of this dilution was added to each of the control and test samples to achieve a concentration of approximately 1×102/mL.
Lactobacillus delbrueckii subsp. bulgaricus G-LB-44 2 grams of Lactobacillus delbrueckii subsp. bulgaricus G-LB-44 was added to 18 mL of sterile water at room temperature. The solution was mixed using a vortex for 5 minutes to ensure the homogeneity of the mixture. 0.5 ml of the prepared solution was added to each of the test samples.
Test Procedure: The following test samples were prepared for the E. coli. Control Samples: 5 bottles containing 50 mL each of vegetable juice Test Samples: 5 bottles containing 50 mL each of boiled vegetable juice plus Lactobacillus delbrueckii subsp. bulgaricus
E. coli was added to the control and test samples as described above. All of the samples were stored in a refrigerator (4oC) for 24 hours. Following refrigeration, the bacterial concentration for each sample was determined. Serial 10 fold dilutions were made in phosphate buffered saline. A 0.1mL aliquot of each dilution

Results:

Bacterial Incubation for 48h

Vegetable JuiceControl - E. coli (CFU)with L. bulgaricus - E. coli (CFU)
187,0960
264,5650
358,8840
430,9030
560,2560
Sample Average60,3410
% Inhibition of 48h Pathogenic Levels by L. bulgaricus100.00%

Sincerely,

1

Andrew B. Onderdonk

logo2
Clinical Laboratories
75 Francis Street
Boston , Massachusetts 02115
Tel: 617 732-7372, Lab: 617 525-2506
Fax: 617 277-1762
E-mail: aonderdonk@partners.org
logo1
Andrew B. Onderdonk, Ph.D.
Director
Clinical Microbiology Laboratory
Professor of Pathology
Harvard Medical School

Pathogen InhibitionTest

Purpose: To determine if Lactobacillus delbrueckii subsp. bulgaricus G-LB-44 inhibits the growth of varying concentrations (1×105/ml, 1×104/ml, 1×103/ml) of Salmonella typhimurium in carrot juice.
Test Materials:
  1. Lyophilized Lactobacillus delbrueckii subsp. bulgaricus G-LB-44 (powder CFU – 1×109/g, received 06/05/14 from K Petkov)
  2. Carrot juice
  3. Tryptic Soy Agar Plates (TSA)
  4. Salmonella typhimuruim – clinical isolate
Bacterial Inoculum
S. typhimurium Previously prepared frozen stock culture of S. typhimurium was used for these experiments. The bacterial concentration was 5.5×108/ml.
For the S. typhimurium 1×105/ml control and test samples: The frozen tube containing S. typhimurium was removed from the freezer and allowed to thaw at room temperature. The bacteria were diluted (1:10) in sterile phosphate buffered saline to achieve a concentration of approximately 5.5×107/ml. 9ul of this dilution was added to 5 ml each of the control and test samples to achieve a concentration of approximately 1×105/ml..
For the S. typhimurium 1×104/ml control and test samples: The frozen tube containing S. typhimurium was removed from the freezer and allowed to thaw at room temperature. The bacteria were diluted (1:100) in sterile phosphate buffered saline to achieve a concentration of approximately 5.5×106/ml. 9ul of this dilution was added to 5 ml each of the control and test samples to achieve a concentration of approximately 1×104/ml.
For the S. typhimurium 1×103/ml control and test samples: The frozen tube containing S. typhimurium was removed from the freezer and allowed to thaw at room temperature. The bacteria were diluted (1:1000) in sterile phosphate buffered saline to achieve a concentration of approximately 5.5×105/ml. 9ul of this dilution was added to 5 ml each of the control and test samples to achieve a concentration of approximately 1×103/ml. Lactobacillus delbrueckii subsp. bulgaricus G-LB-44 1 gram of Lactobacillus delbrueckii subsp. bulgaricus G-LB-44 was added to 9 ml of sterile water at room temperature. The solution was mixed using a vortex for 5 minutes to ensure the homogeneity of the mixture. Either 0.05 ml or 0.5 ml of the prepared solution was added to 5 ml of test sample.
Test Procedure: The following samples were prepared for the S. typhimurium at each concentration

Control Samples:

3 tubes containing 5 ml each of carrot juice

Test Samples:

3 tubes containing 5 ml each of carrot juice plus 0.05 ml Lactobacillus delbrueckii subsp. bulgaricus

3 tubes containing 5 ml each of carrot juice plus 0.5 ml Lactobacillus delbrueckii subsp. bulgaricus

S. typhimurium was added to the control and test samples as described above. All of the samples were stored in a refrigerator (4oC) for 24 hours. Following refrigeration, the bacterial concentration for each sample was determined. Serial 10 fold dilutions were made in phosphate buffered saline. A 0.1mL aliquot of each dilution was plated onto TSA. The agar plates were incubated at 37o C for 24 hours before enumeration; all counts were recorded as CFU/ml. All treatment and control samples were then place at 37o C for 48 hours. Following incubation, the bacterial concentration for each sample was determined as previously described.

Results:

Incubation for 48 hours at 37o C

Carrot JuiceControl - S. typhi (1x105/ml)0.5 ml L. bulgaricus + S. typhi (1x105/ml)0.05 ml L. bulgaricus + S. typhi (1x105/ml)
1420,00000
212,10000
34,40000
Sample Average145,50000
% Inhibition of 48h Pathogenic Levels by L. bulgaricus100%100%

Incubation for 48 hours at 37o C

Carrot JuiceControl - S. typhi (1x104/ml)0.5 ml L. bulgaricus + S. typhi (1x104/ml)
178,0000
2580,0000
397,0000
Sample Average251,6670
% Inhibition of 48h Pathogenic Levels by L. bulgaricus100%

Incubation for 48 hours at 37o C

Carrot JuiceControl - S. typhi (1x103/ml)0.5 ml L. bulgaricus + S. typhi (1x103/ml)
1290,0000
2115,0000
380,0000
Sample Average161,6670
% Inhibition of 48h Pathogenic Levels by L. bulgaricus100%

Sincerely,

1

Andrew B. Onderdonk

September 19, 2014

logo2
Clinical Laboratories
75 Francis Street
Boston , Massachusetts 02115
Tel: 617 732-7372, Lab: 617 525-2506
Fax: 617 277-1762
E-mail: aonderdonk@partners.org
logo1
Andrew B. Onderdonk, Ph.D.
Director
Clinical Microbiology Laboratory
Professor of Pathology
Harvard Medical School
Purpose: To determine if Lactobacillus delbrueckii subsp. bulgaricus G-LB-44 inhibits the growth of Helicobacter pylori Test Materials: 1) Lyophilized Lactobacillus delbrueckii subsp. bulgaricus G-LB-44 (powder CFU – 1x109/g) 2) Brucella broth with 5% Fetal bovine serum (B-FBS) 3) Brucella agar plates with hemin, vitamin K and 5% sheep blood (BMB) 4) Helicobacter pylori #3, #5 and #8 (clinical strains)Bacterial Inoculum H. pylori Previously prepared frozen stock cultures of H. pylori were used for these experiments. The bacterial concentrations were 1.58 x107  CFU/mL for H. pylori #3, 1.11 x107 CFU/mL for H. pylori #5 and 1.44 x107 CFU/mL for H. pylori #8. Control and test samples containing 5 ml of B-FBS were inoculated with each strain to achieve a bacterial concentration of approximately 1x105CFU/mL.Lactobacillus delbrueckii subsp. bulgaricus G-LB-44 1 gram of Lactobacillus delbrueckii subsp. bulgaricus G-LB-44 was added to 9 mL of sterile water at room temperature. The solution was mixed using a vortex for 5 minutes to ensure the homogeneity of the mixture. 50ul (106) or 500ul (107) was added to each of the test samples. For 108 G-LB-44, 500 mg was added to 4.5 ml of B-FBS and vortexed for 5 minutes before inoculating with the H. pylori.Test Procedure: The following test samples were prepared for the H. pylori #3 and H.pylori #5. Control Samples: 3 tubes containing 5 mL each B-FBS Test Samples: 3 tubes containing 5 mL each B-FBS with 106 Lactobacillus delbrueckii subsp. bulgaricusH. pylori was added to the control and test samples as described above. The bacterial concentration for each sample was determined at time 0. Serial 10 fold dilutions were made in phosphate buffered saline and plated onto BMB. The agar plates were placed in Gas-Pak jars containing microaerophilic gas generating sachets and incubated at 37o C for a minimum of 120 hours. Colonies were enumerated and all counts were recorded as CFU/mL. All control and test samples were then place in Gas-Pak jars containing microaerophilic gas generating sachets and incubated at 37o C for a 48 hours. Following incubation, the bacterial concentration for each sample was determined as previously described.The following test samples were prepared for the H. pylori #8. Control Samples: 3 tubes containing 5 mL B-FBS Test Samples: 3 tubes containing 5 mL B-FBS with 106, 107 and 108 Lactobacillus delbrueckii subsp. bulgaricus H. pylori was added to the control and test samples as described above. The bacterial concentration for each sample at time 0 and at 48h was determined as described above.
Sincerely,
1
Andrew B. Onderdonk

ACTA MICROBIOLOGICA BULGARICA

uk
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Letter to Editor

Clinical Experience with Lactobacillus bulgaricus GLB44 in Helicobacter pylori (+) Patients

Borislav Vladimirov1, Jana Valerieva 1, Ivan Terziev2, Kiril Petkov31Department of Gastroenterology, University Hospital „Queen Yoanna- ISUL“, Sofia2Department of Pathology, University Hospital „Queen Yoanna- ISUL”, Sofia3ProViotic AD, Bratia Buckston 58, Sofia, Bulgaria.
IntroductionThe purpose of this study is an assessment of the effect of Lactobacillus delbrueckii subsp. bulgaricus G-LB-44 in Helicobacter pylori (+) patients.
MethodsThe monitoring included twenty-four patients at the average age of 45,46±13,3 years, of which 50% were women. All patients were Helicobacter pylori positive (+). The infection was evident by rapid urease test (RUT), fecal antigen test, a breath test, and histological examination, or by a combination of these methods. Unsuccessful eradication therapy was conducted in six of the patients in the past and the rest of them have not been treated previously. Esophagogastroduodenoscopy was performed in all patients with the following findings: 26.1% had gastroesophageal reflux disease, 65.2% – hiatal hernia, 87% – gastric changes, 4.3% – duodenal erosions, and 21.7% – active duodenal ulcer. Enrolled course conducted by administration of Lactobacillus delbrueckii subsp. bulgaricus G-LB-44 (capsules and tablets) at a daily dose of 15×09 in combination with Rabeprazole 2 × 20 mg or Pantoprazole 2 × 20 mg for seven days followed by Lactobacillus delbrueckii subsp. bulgaricus G-LB-44 individually for three days at the same dosage (15×109). In all patients was carried out the control fecal antigen test for Helicobacter pylori after at least 43 days post treatment.
ResultsIn 22 patients (91.7%) the control fecal antigen test was negative for Helicobacter pylori. In two patients (8.3%) the control study showed persistent Helicobacter pylori infection. Both patients belonged to the group of previously treated patients, who have previously failed eradication with different antimicrobial drugs. The remaining four patients of the group of the previously treated patients (one of them was with autoimmune gastritis) had negative control Helicobacter pylori test. Patients did not manifest adverse reactions or side effects when taking Lactobacillus delbrueckii subsp. bulgaricus G-LB-44 (ProViotic®).This preliminary human trial demonstrated a novel effective method of treating patients with Helicobacter pylori (+) infection without the use of antibiotics.
Conclusion
This preliminary human trial demonstrated a novel effective method of treating patients with + HOLFRDLE EDFWHUS / ORUL (+) infection without the use of antibiotics.
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ANTIFUNGAL ACTIVITY AND ENZYME PROFILE OF LACTOBACILLUS BULGARICUS GLB44

Rositsa Tropcheva1, Yana Evstatieva1, Dilyana Nikolova1, Svetlana Saeva-Kondratenko2, Petko Petkov3, Kiril Petkov3 1Department of Biotechnology, Faculty of Biology, Sofia University St. Kliment Ohridski, 8, Dragan Tsankov Blvd., 1164, Sofia, Bulgaria. 2Genesis Laboratories LTD, Sofia, Bulgaria. 3Proviotic LTD, Sofia, Bulgaria. e-mail: tropcheva12@mail.bg
INTORDUCTION Molds and yeasts are the main spoilage microorganisms, responsible for significant economic losses and several healthy risks in human food chain. The antimicrobial activity is an important criterion for the selection of bio-protective lactic acid bacteria (LAB). A limited data exists on the antifungal activity of Bulgarian LAB and their enzyme profile. With this aim, the activity of the commercially available probiotic Proviotic®, containing the strain Lactobacillus bulgaricus GLB44, against yeasts and deteriorative and toxigenic molds, and the presence of key enzymes, were studied.
MATERIALS AND METHODS Lactobacillus bulgaricus GLB44 (property of Genesis Laboratories LTD) was screened for antifungal activity against five mold species – Aspergillus flavus, Aspergillus niger, Fusarium graminearum, Trichoderma viride and Penicillium claviforme and three yeast species – Saccharomyces cerevisiae, Kluyveromyces marxianus and Rhodotorulla sp., using agar diffusion method. The enzyme profile of the L. bulgaricus GLB44 was determined using API ZYM miniaturized test (BioMerieux, France), following the manufacturer’s instructions. The API strip was inoculated with 24-h-old GLB44 culture, grown in MRS broth and than incubated at 37°C for 4 h. The evaluation of the activity was carried out on 5-grade scale, according to the intensity of coloration.
RESULTS Enzyme profile of Lactobacillus bulgaricus GLB44 Figure 1. API ZYM enzyme profile of Lactobacillus bulgaricus GLB44. The evaluation of the activity was carried out on 5-grade scale, according to the intensity of coloration.
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Figure 2. Visualization of the API ZYM miniaturized test (BioMerieux, France) of Lactobacillus bulgaricus GLB44
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Lactobacillus bulgaricus GLB44 possess a high amino-peptidase, acid-phosphatase and β-galactosidase enzymatic activity and a complete lack of the associated with the colon carcinogenesis β-glucuronidase activity.
Antifungal activity of Lactobacillus bulgaricus GLB44 Figure 3. Antifungal activity of L. bulgaricus GLB44, determined by mold’s hyphal radial growth inhibition after 5-11 days of incubation at 29°C. Results are presented as a percentage of inhibition.
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The in vitro tests revealed a broad spectrum of antifungal activity. L. bulgaricus GLB44 completely (100%) suppress the growth of Aspergillus flavus, Fusarium graminearum, Trichoderma viride, Penicillium claviforme. With regard to Aspergillus niger, a lower inhibitory activity (66.6%), was observed.
Figure 4. In vitro antifungal activity of of Lactobacillus bulgaricus GLB44 (mono-layer agar plate assay).
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Figure 5. Inhibitory activity against yeasts. (Control – Fungostatin, 100 000 U/ml).
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Proviotic® demonstrated a stronger inhibition against Saccharomyces cerevisiae, compared to the used as a control commercial antibiotic Fungostatin.  L. bulgaricus GLB44 does not inhibit the dairy yeast Kluyveromyces marxianus var. bulgaricus, which is a perspective for a future application of the GLB44 in the dairy industry.
CONCLUSTION The demonstrated strong proteolytic activity of Proviotic® makes the Lactobacillus bulgaricus GLB44 interesting for use in the production of antihypertensive and immuno-modulatory products and also in the manufacture of different dairy products. The antifungal activity of Proviotic® is a promising advantage, suggesting its potential applications in different food technologies as a bio-preservative agent and a health promoting products against fungi.