Research Summary for Lactobacillus bulgaricus G-LB-44

Bacteria are the most common form of life on planet earth. The bacteria normally present in or on the human body outnumber the cells that make up our bodies by ten-fold and play an important role in keeping us healthy. These “good” bacteria provide nutrients for us, prevent pathogenic or “bad” bacteria from growing in or on our bodies, and help our immune system develop normally. Many of these “good” bacteria belong to a group called lactobacilli. The food that we eat, water we drink and even the air that we breathe all contain bacteria and are an important way to replenish the “good” bacteria living in and on our bodies. Most people are only aware of the bacteria that cause infections, while the bacteria that help us stay healthy are largely unknown to the general public.

It has been recognized for some time that certain “good” bacteria are active in suppressing the growth of other, more harmful bacteria. A great deal of scientific research has been directed at these helpful, or probiotic, bacteria. Probiotic bacteria have been included in a variety of foods that we eat, such as yogurt, to replenish the bacteria in our bodies and promote better health. In fact, the FDA requires that specific strains of “good” bacteria, such as Lactobacillus bulgaricus, must be present in order for fermented dairy products to be called yogurt. Recent scientific research has shown that probiotic activity tends to be specific to particular strains of bacteria that are often part of the group (genus) called Lactobacillus. We have evaluated the inhibitory effects of one probiotic strain, Lactobacillus delbrueckii subsp. bulgaricus G-LB-44 in our laboratory using organic juice products. These products normally contain a variety of bacteria acquired during the growth and harvest of the ingredients, some of which can be harmful for human health. We tested whether the addition of this strain of probiotic bacteria could inhibit or kill potentially pathogenic organisms. In these studies we added the probiotic strain, Lactobacillus delbrueckii subsp. bulgaricus G-LB-44, to juice products that contained specific “bad” bacteria including those most commonly associated with food borne diseases such as Listeria, E.coli, Salmonella and Shigella that may be present in improperly prepared or cleaned food. The results showed that Lactobacillus delbrueckii subsp. bulgaricus G-LB-44 could reduce the growth of potentially harmful bacteria that cause disease in humans. In most cases, the reduction in the numbers of “bad” bacteria was greater than 99%. The broad based activity of Lactobacillus delbrueckii subsp. bulgaricus G-LB-44 is unusual among Lactobacillus species and suggests that there are unique cellular components that may account for this activity. The ability of one organism to inhibit the growth of another organism is due to protein substances called bacteriocins. Such compounds are common in nature but tend to be directed at very specific strains of other bacteria that compete for nutrients with the probiotic bacteria in a natural setting such as the gastrointestinal tract. It is therefore unusual to find a single strain of Lactobacillus that produces an inhibitory effect for a broad array of harmful bacteria, since these bacteria would rarely be found at the same time within the same natural setting. We are continuing to evaluate the inhibitory properties of this strain, as well as testing the usefulness of adding Lactobacillus delbrueckii subsp. bulgaricus G-LB-44 to certain food products. Since this sub-species of Lactobacillus bulgaricus has been safely used in foods for over 100 years with no indications of overdose or side effects, we believe that if it is added to unpasteurized juice it will serve both as natural preservative capable of inhibiting “bad” bacteria and as a way to replenish the healthy bacteria found in our bodies.

Sincerely,

Andrew B. Onderdonk

Pathogen InhibitionTest

Purpose: To determine if Lactobacillus delbrueckii subsp. bulgaricus G-LB-44 inhibits the growth of Listeria monocytogenes 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 (Bulgarian brand received 06/05/14 from K Petkov)
3.  Tryptic Soy Agar Plates with 5% sheep blood (TSA)
4.  Listeria monocytogenes – ATCC BAA-751
5.  Listeria monocytogenes – BWH 432

Bacterial Inoculum
L. monocytogenes ATCC BAA-751
Previously prepared frozen stock culture of L. monocytogenes BAA-751 was used for these experiments. The bacterial concentration was 9.9×108/ml.

For the L. monocytogenes BAA-751 control and test samples:
The frozen tube containing L. monocytogenes BAA-751 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 9.9×104/ml. 5ul of this dilution was added to 5 ml each of the control and test samples to achieve a concentration of approximately 1×102/ml.

L. monocytogenes BWH 432
Previously prepared frozen stock culture of L. monocytogenes BWH 432 was used for these experiments. The bacterial concentration was 1.68×109/ml.

For the L. monocytogenes BWH 432 control and test samples:
The frozen tube containing L. monocytogenes BWH 432 was removed from the freezer and allowed to thaw at room temperature. The bacteria were diluted (1:100000) in sterile phosphate buffered saline to achieve a concentration of approximately 1.68×104/ml. 30ul of this dilution was added to 5 ml each of the control and test samples to achieve a concentration of approximately 1×102/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:

Incubation for 48 hours at 37^o C

Incubation for 48 hours at 37^o C

Sincerely,

Andrew B. Onderdonk

August 1, 2014

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

Sincerely,

Andrew B. Onderdonk

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 (4^oC) 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 37^o C for 24 hours before enumeration; all counts were recorded as CFU/ml. All treatment and control samples were then place at 37^o C for 48 hours. Following incubation, the bacterial concentration for each sample was determined as previously described.

Results:

Incubation for 48 hours at 37^o C

Incubation for 48 hours at 37^o C

Incubation for 48 hours at 37^o C

Sincerely,

Andrew B. Onderdonk

September 19, 2014

Letter to Editor

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

Borislav Vladimirov¹, Jana Valerieva ¹, Ivan Terziev², Kiril Petkov³

¹Department of Gastroenterology, University Hospital „Queen Yoanna- ISUL“, Sofia

²Department of Pathology, University Hospital „Queen Yoanna- ISUL”, Sofia

³ProViotic AD, Bratia Buckston 58, Sofia, Bulgaria.

Introduction

The purpose of this study is an assessment of the effect of Lactobacillus delbrueckii subsp. bulgaricus G-LB-44 in Helicobacter pylori (+) patients.

Methods

The 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.

Results

In 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 +HOLFR EDFWHUS\ORUL (+) infection without the use of antibiotics.

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.

Figure 2. Visualization of the API ZYM miniaturized test (BioMerieux, France) of Lactobacillus bulgaricus GLB44

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.

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).

Figure 5. Inhibitory activity against yeasts. (Control – Fungostatin, 100 000 U/ml).

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.