Anticancer activity of probiotics – experimental and clinical studies

Anticancer activity of probiotics – experimental and clinical studies

Georgi Alexandrov1, Kaloyan Georgiev2, Marieta Georgieva2, Ivan Iliev3, Maria Peneva4

Abstract

Introduction: Many scientists have been working on the anticancer effect of probiotics for the past decades. In the middle of the last century I. Bogdanov proved experimentally that Lactobacillus subsp. bulgaricus inhibits the growth and proliferation of tumor cells of Sarcoma 180, implanted in rats. Kitadzava (Japan) ascertained good anticancer effect of fermented milk on rats with implanted tumor cells. A. Hosono (Japan) found out that anticancer effect of fermented milk is due to the lactic acid bacteria and polysaccharides contained in it. Our preliminary studies showed that probiotic Laktera Nature® has an anti-proliferative effect against aggressive colon carcinoma cell line HT-29. The probiotic Laktera Nature® contains original strains of Lactobacillus bulgaricus DWT1 and Streptococcus thermophilus DWT4, isolated from spring water in Bulgaria and 21 natural essential and non-essential lactic amino acids. The aim of this study is to determine the anticancer activity of the different ingredients of probiotic Laktera Nature® and to study the effect of the probiotic formula as a nutritional therapy in patients with colorectal cancer.

More information you can find in lecture of prof. Palok Aich \in English\ HERE 

Materials and Methods: Four types of cell lines have been used: 3T3, BJ, HT-29 & MDA-MB-231. Cytotoxicity was determined by MTT-dye reduction assay. The direct cytotoxicity of the tested cell lines, measured at the 24th hour and the antiproliferative activity, measured at the 72nd hour have been studied. Four different samples of Laktera Nature® were used: sample 1 (original probiotic formula containing live cells of Lactobacillus bulgaricus DWT1 and Streptococcus thermophilus DWT4 and 21 lactic amino acids), sample 2 (only the probiotic strains without the amino acids), sample 3 (only the 21 lactic amino acids without the probiotic strains) and sample 4 (the original probiotic formula that has passed γ-sterilization 10 Gy, whereby the probiotic strains are killed, leaving only the cell walls). The clinical study is carried out on 13 patients with surgery for colorectal cancer and undergoing chemo- and radiotherapy. Half of the patients take 15 g daily dose Laktera Nature® (sample 1) as a nutritional therapy.

Results and Discussion: The four samples show a negligible direct cytotoxicity of the tested cell lines measured at the 24th hour. Samples 3 and 4 show a slight concentration- and time-dependent cytotoxic effect. It was interesting to observe that sample 2 (only the probiotic strains) did not have the expected inhibitory effect on carcinoma growth (in that case MDA-MB-231), it even increased its growth. Sample 1 (the original probiotic formula Laktera Nature®, containing live probiotic strains and 21 amino acids) demonstrated a significant growth inhibition of cancerous HT-29 and MDA-MB-231 cell lines with IC50 values – 711,69 ± 82,1 and 934,21 ± 43,5 mcg/ml respectively, measured at the 72nd hour. The parallel conducted clinical trial shows that the nutritional therapy with the original probiotic formula Laktera Nature® allows the full courses of treatment with chemo- and radiotherapy to be carried out and reduces the side effects of the chemo- and radiotherapy.

Conclusion: The original probiotic formula Laktera Nature® inhibits growth of cancerous HT-29 and MDA-MB-231 cell lines. The nutritional therapy with Laktera Nature® improves quality of life and increases the survival rate of patients with colorectal cancer.

Keywords: Probiotic, Laktera Nature®, Lactobacillus bulgaricus, amino acids, antitumor effect, anticancer effect, cytotoxicity, nutritional therapy, colorectal cancer.

1 – Military Medical Academy – Sofia, Bulgaria

2 – Department of Preclinical and Clinical Sciences, Sector Pharmacology and Toxicology, Faculty of Pharmacy, Medical University Varna, Bulgaria

3 – Institute of Experimental Morphology, Pathology and Anthropology with Museum, Bulgarian Academy of Sciences, Sofia, Bulgaria

4 – Medical Centre “Evrozdrave” Sofia, Bulgaria

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Meta-analysis for the effects of probiotic product Laktera with Lactobacillus bulgaricus on gastrointestinal tract

Meta-analysis for the effects of probiotic product Laktera with Lactobacillus bulgaricus on gastrointestinal tract

Summary

The probiotic product Laktera with Lactobacillus bulgaricus regulates and recovers the functions of the gastrointestinal tract and heals the internal human ecosystem.

The aim of Konstantin Ramshev and George Alexandrov from the Military Medical Academy in Sofia and Marieta Georgieva from the Medical University – Varna, is to prove the beneficial effects of a probiotic product Laktera with Lactobacillus bulgaricus on the human gastrointestinal tract.

Researchers have reviewed medical data published in Bulgarian and English languages in twenty two reports, exposés, dissertations and scientific articles for randomized controlled clinical studies on 2550 adult patients and therapeutic naïve individuals. These clinical studies were carried out within 31 years in Military Medical Academy, Medical Universities, Scientific Research Laboratories and Institutes in Member States of the European Union. The clinical studies were preceded by experimental trials on experimental animals. People, subject of these clinical studies were observed for adverse and toxic effects of the probiotic product for a subsequent period of over 10 years and such have not been registered.

The analysis includes clinical trials on humans for evaluation of the probiotic product Laktera with Lactobacillus bulgaricus as a part of tube and/or enteral feeding or part of a complex treatment regimen for sick people with various diseases and severe multiple organ damages and coma,  for healthy people put to extreme physical and mental stress as well as pilots from the Air Force, military sailors and astronauts.

Results

Regular intake of the probiotic product Laktera with Lactobacillus bulgaricus as a part of a complex treatment and enteral feeding leads to a significant reduction of the damage of the gastrointestinal tract, described below with substantial statistical significance. The probiotic products Laktera with Lactobacillus bulgaricus are efficient in cases of the following damage of the functions of the gastrointestinal tract:

  • Disorder of the functions of the gastrointestinal tract in case of space sickness of movement and extreme stress
  • Disbacteriosis and disorders of the intestinal flora of different etiology
  • Gastroenteritis and colitis of toxic, viral and allergic origin
  • Impossibility of active food intake and hypoproteinaemia in comatose, early postoperative period, severe chemical burns and toxo-allergic damages (Lyell syndrome) of the gastrointestinal tract.
  • Impaired digestion
  • Upper dyspeptic syndrome
  • Irritable Bowel Syndrome
  • Increased flatulence
  • Consumptive syndrome and hypoproteinaemia in late postoperative period and due to drug addiction
  • Gastroenteritis and diarrhea as a result of infectious inflammatory damage to the gastrointestinal tract caused by Salmonella, Shigella, coliforms, Proteus and staphylococci
  • Haematemesis of toxic origin (ethyl alcohol)
  • Gastro-enteropathy as a result of lactase deficiency and lactose intolerance
  • Gastro-enteropathy as a result of beta-galactosidase deficiency and galactose intolerance (galactosemia)
  • Disorder of the intestinal motorics
  • Ulcers and gastritis of toxic, toxo-allergic, corrosive or infectious origin (Helicobacter pylori) and stress ulcers in cases of thermal injury

Based on these findings the study authors conclude:

"The probiotic products Laktera with Lactobacillus bulgaricus have healing effect in cases of disorders of the gastrointestinal tract of various etiology and severity. Regular intake of the probiotic Laktera helps the maintenance of the activity and vitality of the human body, improves life quality and maintains the gastrointestinal health."

Researchers: Associate professor Dr. Konstantin Ramshev chief of the Department of Intensive therapy and Reanimation, Military Medical Academy, Sofia, Dr. George Alexandrov, Military Medical Academy, Sofia, Department of Surgery and Associate professor Dr. Marieta Georgieva, Ph. d., Medical University, Varna, Department of Pharmacology.

Reference:

  1. Military Medical Academy, Medical University – Sofia, National Center of Hygiene, Medical Ecology and Nutrition, Medical University of Würzburg - Germany, Elbi-Bulgaricum, JSC; “Complex medical-biological studies of the effects of Biostim LBS on preventive and curative processes in people put to extreme overload in case of sea rescue operations, in Air Force pilots and navigators and patients with acute and chronic damages from drugs”; (136 patients)
  2. Military Medical Institute Academy – Sofia, Department of Toxicology and Allergology at the Higher Military Medical Institute – Sofia, Clinic of Toxicology and Allergology at the Naval Hospital - Varna; Exposé of the report for a clinical testing of a low-lactose lactic acid product Biostim LBS in the treatment of patients with various intoxications; (320 patients)
  3. Report for a clinical testing of the effect of „Biostim LBS“ on the recovery process in cases of different intoxications in the Clinic of Toxicology and Allergology – MMA Sofia and Clinic of Toxicology – Naval Hospital – Varna; (159 patients)
  4. Military Medical Academy, United Aeromedical Research Institute, Department of Toxicology and Allergology; Exposé of a report and scientific articles for a clinical study of the effect of the bio product Biostim LBS-1 on pilots and navigators from Air Force of the Republic of Bulgaria; (92 patients)
  5. High Military Medical Institute – Sofia, Scientific Institute for Emergency “N. Pirogov”; Coordination program „Methods of nutritional therapy – mixed parenteral and enteral nutrition in military field“ /”Calorie”/, adopted by protocol ? 23/1012.1986 by the Scientific Council of the Higher Military Medical Institute - Sofia, Bulgaria; (top secret)
  6. Command of the Air Force and the Medical Department at the Headquarters of the Air Forces, Air Hygiene Laboratory to OAMNII – MMA; Finding for a study of two lyophilized lactic acid products „Bulgaricum with pectin“ and „Biostim with pectin“, developed on the basis of specific strains of Lactobacillus bulgaricus - manufacturer SF „ELBI ENGINEERING“ - SOFIA; Aim of the task: to include these two products in the ration of the flight crews of the Air Force depending on the results of the study; (34 patients)
  7. Report of Gen. Major Ass., N. Alexandrov, Ph.D., Col. Dr. R. Zlatev and Col. Dr. V. Minkov to the Command of the Air Force concerning further development of the scientific topic studying the effect of the lactic acid product „Biostim LBB“ on the lipid metabolism and the immune system in pilots and navigators; (34 patients);
  8. Military Medical Academy – Sofia, Scientific Institute for Emergency “N. Pirogov” - Sofia; Exposé of a final report entitled „Methods of nutritional therapy – mixed parenteral and enteral nutrition“ adopted by protocol ? 23/1012.1986 by the Scientific Council of the Higher Military Medical Institute - Sofia, Bulgaria; (47patients);
  9. Military Medical Academy, Institute for Emergency „N. Pirogov“, Central Experimental laboratory for production, implantation and design; Patent Library AIS „SIREN“, reg. ? 82307019, Final Report on: „Diagnosis and treatment of multiple organ deficiency“, Program task: Development and clinical testing of food and means for enteral nutrition on the basis of lyophilized lactic acid lactose-free products; (103 patients);
  10. Ministry of Health, Institute for Emergency „N. I. Pirogov“; Report for the clinical testing of a lyophilized lactic acid product „Biostim“ produced by „Central Experimental Laboratory for production – implantation – design“ at SO „Dairy Industry“ Sofia; (102 patients);
  11. National Center of Hygiene, Ecology and Nutrition, Medical University of Würzburg - Germany, Military Medical Academy, Medical University – Sofia, SF Elbi; „Complex medical-biological studies in a prolonged vestibular overload in case of a shipwreck“; Aim – study of the effect of the prolonged vestibular overload and the other extreme sea factors on the body and approbation of the lactic acid product of SF Elbi – Biostim LBB as food with which rescue vehicles in water and air transport can be equipped; (56 patients);
  12. Journal „Medicine and Pharmacy“, issue 4 / 2003, „Treatment of poisoning with organic-phosphorus pesticides“;(378 patients);
  13. Journal „Medicine and Pharmacy“, issue 9-10 / 2004, „Alcoholic disease, clinic and treatment“;(151 patients);
  14. Medical University-Sofia, Dissertation of Daniela Petrova Peneva, Social integration - primary objective of a complex program for treatment of a heroin addiction; Sofia, 2006 for conferment of an educational and scientific degree „Doctor“ (152 patients);
  15. Department of Microbiology, Medical University of Sofia; Evaluation of inhibitory activity of Lactobacillus delbrueckii subsp. Bulgaricus on Helicobacter pylori growth in vitro; Lyudmila Boyanova, Maria Stephanova-Kondratenko and others; International symposium on Original Bulgarian Yogurt, 2005, Sofia (4 patients);
  16. Biostim LBS: a special-diet and therapeutic low-lactose powdered food product, Military Medical Academy, Sofia, ELBY Engineering-G State Company, Sofia, B. Gyosheva, N. Alexandrov and others, published in Biotechnology & BioE 6/1992/4 Appendix (100 patients);
  17. Scientific practical conference on disasters and working injuries protection, organized by Council of Ministers; 1992 Sofia, Bulgaria; Biostim LBS in case of acute poisoning as a therapeutic food, B. Gyosheva, N. Alexandrov (376 patients);
  18. University of Food Industry – Plovdiv; Dissertation of Petranka Dimitrova Ivanova, Technologies for obtaining of lactic acid foods for babies (0-1 years old) and children with intolerance to milk sugar; Plovdiv, 1991(25 children);
  19. Report in front of Comecon – Council of Mutual Economic Assistance on a symposium Problems of dietary feeding of children, 1989, Sofia; Characteristic of food products for children with enzyme-deficiency; B. Gyosheva and others (20 children);
  20. Military Medical Academy, Sofia, 1991, Dissertation of Nikola Georgiev Alexandrov, Clinical researches on injured people from chemical catastrophes in Bulgaria in the period 1981 – 1990, for conferment of an educational and scientific degree „Doctor of science“ (197 patients);
  21. Minisrty of National defense, meeting of the member states of the Warsaw Pact, 1988, Varna, Report of N. Alexandrov (top secret);
  22. Report on a International congress of nutrition, Varna, 05.2004, Application of probiotics with Lactobacillus bulgaricus in the early postoperative period, G. Alexandrov, D Damianov (64 patients);

Effect of probiotic product Laktera Vision+ with Lactobacillus bulgaricus and extract from bilberry on dry eye syndrome and tired eye syndrome

Effect of probiotic product Laktera Vision+ with Lactobacillus bulgaricus and extract from bilberry on dry eye syndrome and tired eye syndrome

Technology development and penetration of computers in life lead to refractive anomalies of the eyes in more young people and they start to wear glasses or lenses for vision correction at a young age, before the occurrence of age-related changes (presbyopia). Excessive congestion of eyes at a young age led to the occurrence of new syndromes in ophthalmology – dry eye syndrome and tired eye syndrome.

There are lots of myths as well as lots of studies for the positive effect of bilberry on the eye health.

Bilberry (Vaccinum myrthilus) is a perennial shrub with a height up to 35 cm, which grows in forests and mountain regions of Northern Europe, West Asia and North America. Its fruits are small with a black-violet colour and a pleasant sweet sour taste. (1)

The ancient Roman army physician Pedanius Dioscorides (40 AD), who is considered to be the founder of botany, in his book „De materia medica“ recommends bilberry in treatment of diarrhea and scurvy. (2)

100 g bilberry fruit contain 86,5 g water, 1,1 g proteins, 8,6 g carbohydrates, 2,2 g fibres, 1,2 g free organic acids, 51 mg potassium, 6 mg sodium, 16 mg calcium, 6 mg magnesium, 13 mg phosphorus, 7 mg iron, 0,9 mg copper, 0,01 mg Vit. B1, 0,02 mg Vit. B6, 0,3 mg Vit. PP, 10 mg Vit. C. They are very rich in anthocyanides from the group of flavonoids. (1) Anthocyanides are very important for the eye health. Eye lens and retina are exposed to prolonged intense light radiation and singlet oxygen more than any other tissue in the human body. Thus free radicals are formed and they combine with the lipids in the photoreceptor cell membrane (rods and cones), responsible for vision. To keep the eye tissue healthy it must have an effective antioxidant protection. Anthocyanides in the bilberry are powerful antioxidants and help in the fight against cellular degeneration of the retinal cells caused by free radicals. Anthocyanides help to maintain the normal blood flow in the fine blood vessels (capillaries) that feed the eye tissues. (3)

Bilberry is often associated with the improvement of vision at night. The story that during the World War II pilots have taken bilberry jam to sharpen their vision in night missions is very popular. An American clinical study in 2000 on pilots from the U.S. Navy did’t find such effect. (4)

Experimental research of rats show that the use of bilberry extract can inhibit the development of macular degeneration and senile cataracts.(5)

Object of observation were 41 people aged between 21 and 44 years. 24 of them were men and 21 – women. All of them wear glasses for vision correction, as 17 (41.46%) are shortsighted with dioptre from -0.5 to -3d, and 24 (58,54%) are far-sighted with dioptre + 0.5 to + 2d. 28 (68,29%) of them are with astigmatism. 24 (58,13%) of them wear glasses and lenses for vision correction. All of them work in offices (government officials, IT specialists, accountants, etc.) from 4 to 9 hours a day in front of computers an average 5 hours a day. All wear anti-reflecting glasses while working on a computer.

Most frequent syndromes that occur in these people are the dry eye syndrome and the tired eye syndrome. The dry eye syndrome manifests with reduced production of tears, irritation and itching of eyes. The syndrome of tired eye which is a kind of dryness of eyes occurs after continuous work on a computer with red eyes, marking and expansion of capillaries, blurred vision.

The study was carried out within 8 weeks (56 days). 35 of people have taken 2 capsules Laktera Vision + every day before meal. 6 of the observed people have taken Laktera Vision + irregularly – on average 2.5 times a week. Each capsule (320 mg) contains over 20 billion live cells of Lactobacillus delbrueckii spp. bulgaricus, Streptococcus thermophilus, Lactobacillus helveticus, Lactobacillus lactis (isolated from spring water in Bulgaria), 21 amino acids, milk vitamins, minerals and 10 mg dry extract of bilberry (Vaccinium myrtillus), standardized to 25% anthocyanides.

The results from the study are presented in Table 1.

Days % patients with dry eye syndrome % patients with tired eye syndrome
0 70,73 85,36
7 17,07 21,95
14 14,63 19,51
28 12,20 17,07
42 14,63 17,07
56 12,20 14,63

The table shows that the syndromes of dry and tired eye can be put under control very quickly, in the first week after administration of Laktera Vision +. During the eighth week of the study the dry eye syndrome was observed in 12.2 % (5) of the cases and the tired eye syndrome – in 14.63% (6) of the cases. The medical history for those who have not taken the probiotic product with bilberry extract regularly shows that when they have not taken the product for 3-4 days the syndromes occur again. After administration of the product manifestations of the syndromes are put under control for a period of 12 – 24 hours.

Usually the free market offers products which contain in 1 capsule from 5 to 150 mg bilberry extract. In our observation the effect is achieved with 20 mg bilberry extract daily. The smaller necessary daily dose of dry bilberry extract is probably due to a specific amino acid or group of amino acids in the probiotic product, that acts as a transport carrier of the molecules of the extract and potentiate the effect of the anthocyanides in bilberry. Identification of the amino acid or the group of amino acids, responsible for that process, is a subject of further study.

The probiotic product with bilberry extract controls successfully the syndromes of dry and tired eye, occurring in young people wearing glasses for vision correction, who work on a computer more than 4 hours a day.

  1. Serbin AG, Medical Botany, 2003, – S 150. – 364 p. – ISBN 966-615-125-1
  2. Antonova A, Botany // Encyclopedic dictionary Brockhaus and Efron: in 86 volumes (82 items and 4 suppl.) — 1890—1907.
  3. Gross PM, Scientists zero in on health benefits of berry pigments, Natural Products Information Center, July 2007
  4. Muth ER, Laurent JM, Jasper P (April 2000). Alternative Medicine Review 5(2): 164-73. PMID 10767671. “The effect of bilberry nutritional supplementation on night visual acuity and contrast sensitivity”
  5. Furtsova A, Gesarevich DB, Gonchar AM, Trofimova NA, Kolosova NG, 2005, Progress in gerontology, 16:76-9. PMID 16075680


Experimental and clinical researches of Daflorn’s products

  1. Military Medical Academy – Sofia, Scientific Institute for Emergency “N. Pirogov” – Sofia; Exposé of a final report entitled „Methods of nutritional therapy – mixed parenteral and enteral nutrition“ adopted by protocol ? 23/1012.1986 by the Scientific Council of the Higher Military Medical Institute – Sofia, Bulgaria
  2. Central Experimental Laboratory for production, implantation and design; Contract with Institute for Emergency „Pirogov“ – Sofia for delivery of Biostim – bioproduct for enteral nutrition
  3. Central Experimental Laboratory for production, implantation and design; Patent Library AIS „SIREN“, reg. ? 82307019, Report on: “Development of Food for enteral feeding based on freeze-dried lactic acid lactose-free products“, stage II, 3 “Study of amino acid and salt composition of food, a proposal to use the results in practice”
  4. Military Medical Academy, Institute for Emergency „N. Pirogov“, Central Experimental laboratory for production, implantation and design; Patent Library AIS „SIREN“, reg. ? 82307019, Final Report on: „Diagnosis and treatment of multiple organ deficiency“, Program task: Development and clinical testing of food and means for enteral feeding on the basis of lyophilized lactic acid lactose-free products
  5. Ministry of Health, Institute for Emergency „N. I. Pirogov“; Protocol for the clinical testing of a lyophilized lactic acid product „Biostim“ produced by „Central Experimental Laboratory for production – implantation – design“ at SO „Dairy Industry“ Sofia
  6. Military Medical Academy, Institute for Emergency „N. Pirogov“, Central Experimental laboratory for production, implantation and design; Patent Library AIS „SIREN“, reg. ? 82307019, Final Report on: „Diagnosis and treatment of multiple organ deficiency“, Program task: Development and clinical testing of food and means for enteral feeding on the basis of lyophilized lactic acid lactose-free products
  7. Military Medical Academy, Medical University – Sofia, National Center of Hygiene, Ecology and Nutrition, Medical University of Würzburg – Germany, Elbi Bulgaricum JSC; Research topic: Complex medico-biological studies of the effects of Biostim LBS on prophylactic and healing processes in people put to extreme strain in sea rescue operations, pilots and navigators from the Air Force and in people with acute and chronic drug damages
  8. National Center of Hygiene, Ecology and Nutrition, Medical University of Würzburg – Germany, Military Medical Academy, Medical University – Sofia, SF Elbi; „Complex medical-biological studies in a prolonged vestibular load in case of a shipwreck“; Aim – study of the effect of the prolonged vestibular load and the other experimental sea factors on the body and approbation of the lactic acid product of SF Elbi – Biostim LBB as food with which rescue vehicles in water and air transport can be equipped
  9. Command of the Air Force and the Medical Department at the Headquarters of the Air Forces, Air Hygiene Laboratory to OAMNII – MMA; Finding for a study of two lyophilized lactic acid products „Bulgaricum with pectin“ and „Biostim with pectin“, developed on the basis of specific strains of Lactobacillus bulgaricus – manufacturer SF „ELBI ENGINEERING“ – SOFIA; Aim of the task: depending on the results of the study to include these two products in the ration of the flight crews of the Air Force
  10. Written statement for the clinical testing of the effect of „Biostim LBB“ on the recovery process in cases of different intoxications in the Clinic of Toxicology and Allergology – MMA Sofia and Clinic of Toxicology – Naval Hospital – Varna
  11. Military Medical Academy – Sofia, Department of Toxicology and Allergology at the Higher Military Medical Institute – Sofia, Clinic of Toxicology and Allergology at the Naval Hospital – Varna; Exposé of the protocol for a clinical testing of a low-lactose lactic acid product Biostim LBB in the treatment of patients with various intoxications
  12. Contract between Meiji Milk Products Tokyo, Elbi Bulgaricum JSC and Prof. N. Alexandrov for a study on: Complex medical biological studies of the effects of Biostim LBB on lipid metabolism and immune system in people put to extreme mental and physical stress
  13. Report of Gen. Major Ass., N. Alexandrov, Ph.D., Col. Dr. R. Zlatev and Col. Dr. V. Minkov to the Command of the Air Force concerning further development of the scientific topic studying the effect of the lactic acid product „Biostim LBB“ on the lipid metabolism and the immune system in pilots and navigators
  14. Military Medical Academy, United aero-medical Research Institute, Department of Toxicology and Allergology; Exposé of protocol and scientific articles for a clinical study of the effect of the bioproduct Biostim LBS-1 on pilots and navigators from Air Force of the Republic of Bulgaria
  15. Department of Preclinical and Clinical Pharmacology and Biochemistry, Medical University – Varna; Department of Toxicology and Allergology, MMA – Sofia; University Hospital „St. Anna – Varna“ JSC, Hospital Pharmacy; Clinical Laboratory, Hospital „St. Marina“ – Varna; Department of Nephrology, Haemodialysis and Toxicology, Hospital „St. Marina“ – Varna – Study of the effects of „Biostim LBS“ and silymarin on an experimental model of carbon tetrachloride induced hepato-toxicity in rats
  16. Department of Preclinical and Clinical Pharmacology and Biochemistry, Medical University – Varna; Department of Toxicology and Allergology, MMA – Sofia; University Hospital „St. Anna – Varna“ JSC, Hospital Pharmacy; Clinical Laboratory, Hospital „St. Marina“ – Varna; Study of the effect of the probiotic Biostim LBS on the lipid metabolism
  17. Department of Preclinical and Clinical Pharmacology and Biochemistry, Medical University – Varna; Department of General and Clinical Pathology, Medical University – Varna; Department of Toxicology and Allergology, MMA – Sofia; University Hospital „St. Anna – Varna“ JSC, Hospital Pharmacy; Clinical Laboratory, Hospital „St. Marina“ – Varna; Study of the effect of the probiotic Biostim LBS in experimentally induced hyperlipidemia
  18. Medical Faculty – Sofia, Department of Pharmacology; Written statement for a comparative pharmacological and biochemical study of the effects of Laktera, Laktera/Panax and Laktera /Hypericum on ulcer formation and ethanol-induced oxidative stress in rats
  19. Department of Preclinical and Clinical Pharmacology and Biochemistry, Medical University – Varna; Department of General and Clinical Pathology, Medical University – Varna; Protective effect of the probiotic Biostim LBS in a model of indomethacin-induced ulcer-genesis
  20. Department of Preclinical and Clinical Pharmacology and Biochemistry, Medical University – Varna; Department of Pathophysiology, Medical University – Varna; Department of Anatomy, Histology, Embryology, Medical University – Varna; Effects of the probiotic Biomilk on indomethacin-induced oxidative damage of some tissues
  21. Department of Preclinical and Clinical Pharmacology and Biochemistry, Medical University -Varna; Department of General and Clinical Pathology, Medical University – Varna; Department of Toxicology and Allergology, MMA – Sofia; Effect of the probiotic Biostim LBS on histopathological changes in liver in experimental hypercholesterolemia
  22. Department of Preclinical and Clinical Pharmacology and Biochemistry, Medical University – Varna; Department of General and Clinical Pathology, Medical University – Varna; Department of Toxicology and Allergology, MMA – Sofia; Hepato-protective effect of the probiotic Biostim LBS on experimental model of carbon tetrachloride-induced hepatotoxicity in rats
  23. Department of Preclinical and Clinical Pharmacology and Biochemistry, Medical University – Varna; Department of General and Clinical Pathology, Medical University – Varna; Department of Toxicology and Allergology, MMA – Sofia; Department of Radiology, Medical University – Varna; Ray-protective effect of the probiotic Biomilk containing Lactobacillus bulgaricus
  24. Abstract of Dissertation of Daniela Petrova Peneva for conferment of an educational and scientific degree „Doctor“
  25. Abstract of Dissertation of Marieta Petrova Georgieva for conferment of an educational and scientific degree „Doctor“
  26. Statement of Colonel Dr. Vencislav Minkov Minkov, Head of „Medical Department“ at division 22800 to the Chief of the General Staff of the Air Force for a study of two products – Bulgaricum with pectin and Biomilk with pectin used by pilots from the Air Force
  27. MMA, An extract from the minutes from Vice Head of MMA for educative and scientific affairs, Ass. Prof. Dr. A. Andreev on ongoing clinical tests of low-lactose dry milk products with trademarks „Biomilk“ and „Laktera“
  28. An opinion of the Head of the Department of Toxicology and Allergology at the Military Medical Academy – Sofia, Prof. Dr. Nikola Alexandrov Ph. D., about replacing yogurt with wholefood supplement with probiotic effect Biomilk and Biostim in conditions harmful for health.
  29. Certificate of MMA to give “Biomilk” and “Laktera”, produced by Daflorn Ltd., as wholefood supplements and functional food to the contingent of the Bulgarian Army. And their use in Military Medical Academy for more than 10 years as a food therapy of seriously ill and operated patients.
  30. MMA, Head of the Clinic of Toxicology and Allergology, Associate Dr. N. Alexandrov; Recommendation for the use of probiotics in cases of serious toxicological and allergic diseases (Epidermolis necroticans bulosa Syndrome), as well as in other cases of toxicological and allergic diseases with beneficial effect.
  31. MMA, Head of the Clinic of Toxicology and Allergology, Associate Dr. N. Alexandrov; Protocol for the use of the products from the series „Biomilk“ and „Biostim“, containing Lactobacillus Bulgaricus
  32. MMA, Deputy for the health affairs of MMA, Ass. Prof. Ramshev; Recommendation for the use of different probiotics with trademark Biomilk and Laktera with a beneficial effect in patients requiring stimulation of the immune system of the body because of different viral or bacterial diseases.
  33. Statement of the Ministry of Labour and Social Affairs to the Ministry of Health and the General Headquarters of the branch syndicates – Proposal to include the wholefood supplement Laktera in the nomenclature of the antidotes and protective food
  34. Recommendation from Alexander Chakmakov, Ex. Director of „Titan Zlatna Panega Cement“, JSC for the benefit of the probiotics Biomilk and Laktera as a protective food
  35. MMA, Certificate for clinical tests and Certificates of Analysis of the probiotic wholefood supplements Biomilk, Laktera and Magic Power.
  36. Certificate of MMA to give “Biomilk” and “Laktera”, produced by Daflorn Ltd., as wholefood supplements and functional food to the contingent of the Bulgarian Army located in Iraq. As well as their use in the Military Medical Academy for more than 10 years as a food therapy of seriously ill and operated patients.
  37. Full Doping control of the product series Biomilk in German Sports University of Cologne – Doping Laboratory
  38. Ukrainian Research Institute of Medical Transport, State Department of maritime and river transport of Ukraine, Medical University – Varna – Effects of Biomilk Hepanorm in simulated shipwreck
  39. Journal „Medicine and Health“ – „Ray-protective effect of a lactic acid product containing a Lactobacillus bulgaricus strain“
  40. Annual book of IMAB for Bulgaria, Israel, Germany, Norway, France, Italy and USA – Study of the probiotic Biostim for teratogenic effect
  41. International Conference in Lyon – Lactic acid fermentation and biotechnological method of obtaining a low-lactose lactic acid product for special diets and therapeutic food
  42. Journal „Biotechnology“ – „Effect of the low-lactose lactic acid products Bulgaricum and Biostim on the immune system“
  43. National Cancer Center, National Centre of Haematology and Transfusiology; Study of the ray-protective effect of Gerisan and Biostim LBS on the basic leukocyte populations in cases of radiotherapy of cancer.
  44. Balkan Military Medical Committee; The 4th Congress of B.M.M.C.; June 6-10, 1999, Bucharest, Romania; EFFECT OF THE BIOSTIM LBB YOGHOURT-TYPE PRODUCT ON LIPID METABOLISM IN AIR-FORCE AVIATORS; Author(s): Alexandrov, N., Petrov, A., Zlatev, R., Zhekova, M., Mikhaylova, Z., Kotseva, M. and Alexiev, L. Institution: Military Medical Academy, Sofia Institution For Aviation Medicine;
  45. FEBS; 20th MEETING OF THE FEDERATION OF EUROPEAN BIOCHEMICAL SOCIETIES, organized by the Hungarian Biochemical Society; Budapest, Hungary, August 19-24, 1990; Freeze dried dietetic lactic acid product “Biostim LBS”; Gyosheva B., Alexandrov N., Ivanova P., Manafova N., Georgiev G.; Central Exper. Lab. Dairy Ind. 1618 Sofia; Emergency Inst. “N. Pirogov” 1612 Sofia, Bulgaria.
  46. An International Journal for the Rapid Publication of Short Reports on Biochemical Mechanisms of Mammalian Toxicity; Clinical Research on the Victims of Chemical Accidents in Bulgaria; N. Alexandrov, D. Jordanova and M. Georgieva, Military Medical Academy, Sofia, Bulgaria; VI INTERNATIONAL CONGRESS OF TOXICOLOGY, Rome, 28 June – 3 July 1992
  47. Therapeutic Drug Monitoring; A Journal Devoted to Therapeutic Drug Monitoring and Clinical Drug Toxicology, volume 15, number 2, 1993, Philadelphia, USA; Influence of Nootropic Agents and Hyperbaric Oxygenation in the Treatment of Acute Poisonings with Halogenated Hydrocarbons; N. Alexandrov, M. Georgieva, Military Medical Academy, Sofia, Bulgaria.
  48. Addictive drugs and addictive states, The state of the art; Addiction Research Foundation of Italy (ARFI), Padova in collaboration with University of Florence – Department of Preclinical and Clinical Pharmacology “Mario Aiazzi Mancini”; International congress of Toxicology in Florence, 07.1992; Clinical research on narcotic and non-narcotic drug addiction. Comparative analysis; N. Alexandrov, M. Georgieva, D. Jordanova.
  49. Biotechnology Journal; “Biostim LBS – a special diet and therapeutic low-lactose powdered product”, issue 4, series V, 92 – english version. Biostim LBS – a special diet and therapeutic low-lactose powdered product; B. Gyosheva, N. Alexandrov, N. Manafova, P. Ivanova, G. Georgiev; ELBY Engineering –G, Sofia and Military Medical Academy, Sofia, Bulgaria.
  50. Biotechnology Journal, 2/1994; Effect of dairy products, containing Lactobacillus strain LBL4 on some biochemical parameters in humans; M. Zhekova¹, K. Avramova², B. Gyosheva³; Aviomedical Research Institute, Higher Medical Academy, Sofia; National Center of Hygiene, Medical Ecology and Nutrition, Sofia; ELBY Engineering-G, Sofia.
  51. Biotechnology Journal, 4/1992; Biostim LBS: a special diet and therapeutic low-lactose powdered food product; B. Gyosheva, N. Alexandrov, N. Manafova, P. Ivanova, G. Georgiev; ELBY Engineering – G State Company, Sofia; Military Medical Academy, Sofia.
  52. Cancer Letters Journal 114 (1997) 93-95; Preventive role of lyophilic dairy products bulgaricum and biostim; T. Ivanova, M. Jekova; Transport Medical Institute, Sofia, Bulgaria; Medical Military Aviation Institute, Sofia, Bulgaria.
  53. Contract between Joint MLB Promotion Planning Committee between MEIJI MILK, Tokyo and LB Bulgaricum PLC, Sofia and Prof. Nikola Georgiev Alexandrov D.Sc. – Head of Toxicologic and Allergologic Department at Military Medical Academy; Clinical-Immune Researches over some effects of the yoghurt product “Biostim LBB” on pilots from the Army-Air Forces; N. Alexandrov¹, K. Plotchev², K. Penkova², R. Vladimirova², K. Mladenov², P. Zlatev³, Dzh. Nedyalkova; ¹ Military Medical Academy, Sofia – CLINIC OF TOXICOLOGY; ² Military Medical Academy, Sofia – CENTRAL LABORATORY ON CLINICAL IMMUNOLOGY; ³ OAMNII, Military Medical Academy, Sofia.
  54. Fundamental & Clinical Pharmacology; The 3rd Meeting of the Federation of the European Pharmacological Societies (EPHAR); 6-9 July 2001, Lyon, France; Effects of the chronic dietary supplementation of the probiotic Biostim LBS on carbon tetrachloride-induced hepatotoxicity in rats; K. Marazova¹, N. Alexandrov², ¹Department Pharmacology Toxicology, Medical Faculty, Sofia, Bulgaria, ²Department Toxicology Allergology, Military Medical Academy, Sofia, Bulgaria.
  55. Journal „Medicine and Pharmacy“, issue 2 / 2002, „Clinical features and treatment of acute intoxications with heroin”
  56. Journal „Medicine and Pharmacy“, issue 4 / 2003, „Treatment of organophosphorus pesticides poisoning“
  57. „Medicine and Pharmacy“, issue 2 / 2003, „Study of the probiotic Biostim LBS in experimentally induced hyperlipidemia“
  58. VIIth National Congress of Nutrition with International participation; Nutrition science for human health protection; Varna – St. Konstantin and Elena, Bulgaria, May 15-17, 2003; Probiotics application before and in the early post-operative period; Alexandov G., D. Damianov, Clinic of Surgery, UNBAL “Queen Jovana” – Sofia
  59. NATO Symposium; May 2005, Munich, Germany; Complex treatment of organophosphorus pesticide poisoning as a model of chemical warfare agents; Alexandrov N., Ks. Stoyancheva, Military Medical Academy, Toxicology Department.
  60. International Symposium on original Bulgarian yogurt under the patronage of President of the Republic of Bulgaria, Sofia, 2005 ; The application of probiotics for restoration of vaginal environment; Stefan Kovachev¹, Aneta Nacheva¹, Daniela Petrova², Milena Kovacheva²; ¹MC “Gynart” MMA – Sofia; ²MC “Evrozdrave” MMA – Sofia
  61. International Symposium on original Bulgarian yogurt under the patronage of President of the Republic of Bulgaria, Sofia, 2005; Farmacological study of the probiotic Biomilk, containing Lactobacillus bulgaricus, for antioxidant activity, Georgieva M.¹, Bekyarova G.², Alexandrov N.³, Manolov N.?; ¹Department of preclinical and clinical pharmacology and biochemistry, Medical University “Prof. Dr. Paraskev Stoyanov” – Varna, ²Department of Pathophysiology, Medical University – Varna, ³Department of toxicology and allergy, MMA – Sofia, ?Clinic of Vascular Surgery, University Hospital “St. Anna” – Varna
  62. International Symposium on original Bulgarian yogurt under the patronage of President of the Republic of Bulgaria, Sofia, 2005; Good bacteria of Bulgarian youghurt – a base of symbiotic and functional food, Alexandrov N.¹, Gyosheva B.², Ivanova P.³; ¹Military Medical Academy, ²Bulgarian Association of Dairy Processors, ³LB Bulgaricum
  63. International Symposium on original Bulgarian yogurt under the patronage of President of the Republic of Bulgaria, Sofia, 2005; Probiotics – base of functional nutrition and nutritive treatment of XXI century; Nikola Alexandrov¹, Phd, Daniela Petrova², Milena Kovacheva¹; ¹Emergency Clinic of Toxicology and Allergy, MMA – Sofia, Bulgaria, ²MC “Evrozdrave” MMA Sofia, Bulgaria
  64. International Symposium on original Bulgarian yogurt under the patronage of President of the Republic of Bulgaria, Sofia, 2005; Probiotics – lifestyle, M. Georgieva¹, E. Softova², P. Borisova, N. Alexandrov³, N. Manolov?; ¹Department of preclinical and clinical pharmacology and biochemistry, Medical University “Prof. Dr. Paraskev Stoyanov” – Varna, ²Department of General and Clinical Pathology, Medical University “Prof. Dr. Paraskev Stoyanov” – Varna, ³Department of Toxicology and Allergy, MMA – Sofia, ?Clinic of Vascular Surgery, University Hospital “St. Anna” – Varna
  65. International Symposium on original Bulgarian yogurt under the patronage of President of the Republic of Bulgaria, Sofia, 2005; Synbiotics – their place in modern treatment of abusers, Daniela Petrova¹; Milena Kovacheva²; ¹MC “Evrozdrave” – MMA, Sofia, ²Clinic of Emergency Toxicology and Allergy, MMA – Sofia
  66. Journal „Military Medicine“, issue 4 / 2007, „Study of the antioxidant activity of the probiotic Biomilk under experimental conditions“
  67. Journal „Military Medicine“, issue 2 / 2007, „Ray-protective effect of the probiotic Biomilk“
  68. Journal „Medicine and Pharmacy“, issue 9-10, „Alcoholic disease, clinic and treatment“
  69. Journal „Medicine and Pharmacy“, issue July/ 2008, „Probiotics and vaginal flora”
  70. Journal „Medicine and Pharmacy“, issue 1 / 2009, „Prevention and treatment of atherosclerosis with probiotic lactic acid food and bacteria“
  71. EXPERIMENTAL CLINICAL STUDY OF THE EFFECT OF THE PROBIOTIC BIOMILK IN CASES OF LIVER INJURIES; VI National Congress of Pharmacology 2009; Georgieva M.¹, Popov G.², Plochev K.², Alexandrov N.²; ¹ Department of Preclinical and Clinical Pharmacology and Toxicology, Medical University of Varna; ² Military Medical Academy
  72. Effect of a probiotic product with Lactobacillus bulgaricus DWT1 in avoiding adverse effects during adjuvant treatment of patients with tumors of the stomach, colon and rectum. Medical University, Zagreb, Republic of Croatia, Pimezon Polyclinic; Prof. Dr. Mirko Shamija; December 2012
  73. Forensic Medicine 9/2013, A meta-analysis of the effects of a probiotic product with Lactobacillus bulgaricus Laktera in the gastrointestinal tract; Ramshev C. 1 D. Alexandrov 1; 1 MMA Sofia
  74. MU Varna power. Magazine., 2013, Effect of probiotic product Laktera Vision with Lactobacillus bulgaricus and bilberry extract on syndromes dry and tired eyes Georgieva M.1 Alexandrov D.2; 1 MU Varna, 2 Sofia MMA
  75. World Congress of probiotics Athens 2014, newly origin of probiotics in the Balkans; Petrov N.1 G. Alexandrov 1 Peneva M. 2; 1 MMA Sofia 2Plovdiv University of Plovdiv
  76. Bulgarian Red Cross 2014 Recommendation on the use of probiotic foods Laktera of victims of disasters and accidents.
  77. World Journal of Pharmacy and Pharmaceutical Sciences, volume 4, issue 06, 2015q 130-136, Antiproliferative Effect of Bulgarian Spring Water Probiotics (Laktera Nature Probiotic®) Against Human Colon Carcinoma Cell Line, Kaloyan Georgiev, Marieta Georgieva, Ivan Iliev, Maria Peneva, Georgi Alexandrov; Department of Preclinical and Clinical Sciences, Sector Pharmacology and Toxicology, Faculty of Pharmacy, Medical University Varna; Institute of Experimental Morphology, Pathology and Anthropology with Museum, Bulgarian Academy of Sciences, Sofia; Medical Centre Evrozdrave Sofia; Military Medical Academy Sofia.
  78. 6th Southeast European Conference of Chemotherapy, Infectious and Cancer Diseases, November 2015, Thessaloniki, Greece; Anti-tumor Effect of probiotics – experimental and clinical research, Georgi Alexandrov, Kaloyan Georgiev, Marieta Georgieva, Ivan Iliev, Maria Peneva; Military Medical Academy Sofia; Department of Preclinical and Clinical Sciences, Sector Pharmacology and Toxicology, Faculty of Pharmacy, Medical University Varna; Institute of Experimental Morphology, Pathology and Anthropology with Museum, Bulgarian Academy of Sciences, Sofia; Medical Centre Evrozdrave Sofia.
  79. 3rd Biennial Conference of Probiotic Association of India (PAi), International Symposium on Stress, Microbiome & Probiotics, March 11-13, 2016, NISER, Bhubaneswar, India; Application of Lactobacillus bulgaricus in Clinical Practice; Georgi Alexandrov, Maria Peneva; Military Medical Academy Sofia; Medical Centre Evrozdrave Sofia.
  80. Excerpt from Abstract of dissertation, Sofia 2016, p. 27-29; Epidemiological study of patients with food allergies; Maria Peneva, Medical Centre Evrozdrave Sofia.
  81. Scripta Scientifica Pharmaceutica, ISSN 2367-6000, volume 4, 2017, supplement 2, page 51, Medical University Prof. Dr. Paraskev Stoyanov, Varna, Bulgaria, “Hepatoprotective effect of probiotic food, containing lactobacillus bulgaricus DWT1 in acute ethanol intoxication in experimental animals”, Gabriela Kehayova1 , Marieta Georgieva, Kaloyan Georgiev, G. Aleksandrov, M. Peneva.
  82. World Journal of Pharmaceutical Research, volume 7, Issue 12, 2018, Research Article, page 35-42, “Hepatoprotective effect of probiotic, containing Lactobacillus bulgaricus DWT1, in acute paracetamol-induced liver damage in rats”, Gabriela Kehayova, Marieta Georgieva and Kaloyan Georgiev, Medical University, Varna
  83. Monography, Varna 2018, publishing house Steno, pages 167, “Probiotics – Medicine and Food”, Marieta Georgieva
  84. Journal of Functional Foods, Volume 56, May 2019, Pages 232-245, “A probiotic formulation containing Lactobacillus bulgaricus DWT1 inhibits tumor growth by activating pro-inflammatory responses in macrophages”, Dipanjan Guhaa, Arka Banerjeea, Raktim Mukherjeea, Biswaranjan Pradhana, Maria Peneva, Georgi Aleksandrov, Sujit Suklabaidya, Shantibhushan Senapati, PalokAich,
  85. Probiotics in the Prevention and Management of Human Diseases,Academic Press, 2022, Pages 431-448, Chapter 29 – Probiotics: past, present, and future challenges, Marieta Georgieva, Kaloyan Georgiev, Nadezhda Hvarchanova,
  86. Educational event III international science conference “Human microbiota” FSBSI “Institute of experimental medicine”, October 7-8, 2021, St. Petersburg, “Newly found origin of probiotics in the Balkans” , Nikolai Petrov, Georgi Aleksandrov, Maria Peneva
  87. National Conference on Clinical Toxicology with International Participation on “Toxicology Today“ June 2-4, 2022, Pleven Medical University, “Probiotics – contemporary challenges”, Marieta Georgieva, Georgi Alexandrov, Maria Peneva, Daniela Petrova
  88. National Conference on Clinical Toxicology with International Participation on “Toxicology Today“ June 2-4, 2022, Pleven Medical University, “MORBUS LYELL – clinical case”, Katerina Stefanova, Evgenia Burzashka, Olimpiada Atmazhova, Ani Aleksandrova, Nadya Vasileva,
  89. National Conference on Clinical Toxicology with International Participation on “Toxicology Today“ June 2-4, 2022, Pleven Medical University, “Toxic-necrotic changes after SARS-COV 19 a clinical case”, Olimpiada Atmazhova, Evgenia Burzashka, Katerina Stefanova, Ani Aleksandrova, Nadya Vasileva, Vladislav Tenev
  90. 11th National Congress of nutrition with international participation “Nutrition science with evaluation of the present and look to the future”, 2022, Bulgarian Society of nutrition and dietetics, “Antioxidant and hepatoprotective activity of probiotic food against paracetamol-induced liver damage in rats”, M. Georgieva, K. Georgiev, N. Hvarchanova, S. Stoeva, M. Radeva-Ilieva, V. Tzankova, M. Kondeva-Burdina, D. Aluani, M. Tzaneva
  91. Patent No. 4294 dated 06.07.2022, registered in the Patent Office of the Republic of Bulgaria for a biologically active product used as nutritional therapy in the treatment of patients with COVID-19, including pregnant women.
  92. Scientific project No. 21-00038 to study the immune- stimulating and protective effect of probiotic food Laktera in extreme stress situations during the upcoming trip, stay and return of our expedition to Antarctica on Livingston. The project is carried out by the VVMU “N. Y. Vaptsarov” together with the University of Varna and the University of St. Kliment Ohridski” for the period 2022-2023.
  93. 16th International Scientific Conference on Probiotics, Prebiotics, Gut Microbiota and Health – IPC2023, 20-22 June 2023, Bratislava, Slovakia, Application of a probiotic formula containing Lactobacillus bulgaricus DWT1 in patients with COVID-19, Maria Peneva, Ioannis Misopoulos, Evgenia Burzashka, Tsvetan Lukanov, Tsetsa Doichinova, Anna Petkova, Marieta Georgieva, Georgi Alexandrov, Pencho Tonchev, Medical Center “Eurohealth-Bulgaria”,  Sofia, Bulgaria, Mika Nutri-Pharma OOD, Petrich, Bulgaria, UMBAL Pleven “Dr. Georgi Stranski”, Pleven, Bulgaria, University Multispecialty Hospital for Active Treatment St. Anna Sofia, Sofia, Bulgaria, Varna Medical University “Prof. Dr. Paraskev Stoyanov”, Varna, Bulgaria
  94. V International Scientific Conference ”Microbiota of Humans and Animals”, Institute of Experimental Medicine, October 11-13, 2023, St. Petersburg, Effects of Lactobacillus bulgaricus DWT1 on cell immunity in patients with COVID-19, Georgiy Alexandrov, Maria Peneva, Ioannis Misopoulos, Evgenia Barzashka, Tsvetan Lukanov, Tsetse Doichinova, Anna Petkova, Marieta Georgieva, Pencho Tonchev
  95. National Clinical Toxicology Conference “Toxicology in the lens of the present and the future”, November 2-4, 2023, Bulgaria, Probiotics and modulation of cellular immunity in COVID-19, Olympiada Atamadjova, Katerina Stefanova, Ani Alexandrova, Georgi Alexandrov, Maria Peneva, Anna Petkova, Marieta Georgieva, Pencho Tonchev


Nutritional therapy for oncological diseases

Effect of a probiotic product containing a strain of Lactobacillus bulgaricus DWT1 on preventing adverse effects during adjuvant therapy for patients with tumours of the stomach, colon and rectum

Summary

Previous experience in oncology. The studies of some strains of Lactobacillus bulgaricus have shown that they have antimutagenic effect. Some experiments on animals show a certain beneficial effect in cases of colorectal cancer in rodents. A population study noted lower levels of occurrence of colorectal cancer among a considerable group of fermented dairy products consumers.

Tumours in the digestive system are of exclusive importance because of their high frequency rate and because of the great importance of the digestive system for the whole body. Nutritional therapy that improves digestion is very important for the treatment of the underlying disease as well as for the symptomatic treatment. On the other hand, adjuvant therapy, i.e. the specific oncological treatment /chemo- or radiotherapy/ often leads to a number of healthy tissue damages and to serious complications and side effects. These complications inhibit the beneficial effects of treatment and even make it impossible. Cytostatics cause nausea and vomiting during and after application of chemotherapy and overtaking vomiting due to inadequate antiemetic prevention in previous chemotherapy. Mucositis is an extremely serious side effect after both chemotherapy and irradiation in the area of head and neck. It concerns inflammatory changes of the mucous membrane of the digestive system. It often occurs in parallel with myelosuppression and leukopenia. The most dangerous consequence of leukopenia /decrease in the number of leukocytes/ is febrile neutropenia that requires hospitalization, blood transfusion and often leads to discontinuation of cytostatics and the quality of life is getting worse. Anorexia and cachexia /weight loss/ are frequent in patients with tumors of the digestive system and adversely affect the successful treatment, tolerance of treatment and indirectly affect survival. Radiotherapy causes adverse effects depending on the total dose, fraction size, field of irradiation, interval between fractions, type of radiotherapy and specificity of the irradiated tissues. The toxic effects of radiotherapy are dyspepsia, gastritis, ulceration /ulcers/ and radiation enteritis.

Results. The study included 80 patients with tumours of the stomach, colon and rectum. All of them have undergone a patho-histological examination and the disease stage was precisely defined. In this randomized study patients were divided into two groups of 40 patients. The first group of 40 patients was undergoing chemotherapy or chemoradiotherapy. The other group of 40 patients was taking probiotic food, containing strain of Lactobacillus bulgaricus DWT1 – 15 mg once a day before breakfast for three months, in addition to the chemotherapy or chemoradiotherapy.

The obtained results show that probiotics, containing a strain of Lactobacillus bulgaricus DWT1, have a potential beneficial effect as taken in addition to the treatment of cancer patients with adjuvant chemotherapy or chemoradiotherapy. Administration of that probiotic product influences on the reduction of nausea and vomiting and maintains appetite of patients during the adjuvant therapy. There is a clear bond between reduction of the frequency and severity of disorders and the intake of Lactobacillus bulgaricus DWT1.

Nutritional therapy with probiotic product, containing a strain of Lactobacillus bulgaricus DWT1, helps the prevention of the adverse effects during adjuvant therapy.

Researchers: Prof. Dr. Mirko Shamia, PhD and associates, Zagreb, December 2012


Newly found origin of probiotics in the Balkans

Newly found origin of probiotics in the Balkans

Download the presentation of newly found origin of probiotics in the balkans prestented at the congress of International Probiotics Association (Athens, May 2014).

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Newly found origin of probiotics in the Balkans


Cytotoxicity of Bulgarian spring water probiotics (probiotic Laktera Nature®) on colon carcinoma cell line

Cytotoxicity of Bulgarian spring water probiotics (probiotic Laktera Nature®) on colon carcinoma cell line

Abstract

Colorectal carcinoma is a malignant disease, from which more than 1 million people worldwide suffer. Over the past two decades, its social significance attracts more attention. Colorectal cancer is the second leading cause of death from malignancy and the third most frequently diagnosed malignancy. It has been estimated that nutrition is the main reason for the high incidence of cancer. Probiotics are live microorganismswith proven health benefits in a variety of gastrointestinal diseases, and disorders outside the gastrointestinal tract. In this study, we wanted to explore cytotoxicity and anti-proliferative activity of the new probiotic Laktera Nature® on non-cancerous cell lines – 3T3 and BJ and colon adenocarcinoma cell line – HT-29. Probiotic Laktera Nature® has weak direct cytotoxicity against 3T3 and BJ cells, but inhibits their proliferation in a dose-dependent manner. On carcinoma cell line – HT-29, in low concentration range (2-125 µg/ml) probiotic Laktera Nature® stimulates proliferation; at high concentrations (250-1000 µg/ml) we detected concentration-dependent inhibition of proliferation. Probiotic Laktera Nature® has shown promising primary screening results, which should be clarified as mechanisms in further works.

Kaloyan Georgiev – Department of Preclinical and Clinical Sciences, Sector Pharmacology and Toxicology, Faculty of Pharmacy, Medical University Varna, Marin Drinov str. 55, 9002 Varna, Bulgaria. Correspondence to Dr. KaloyanGeorgiev, Department of Preclinical and Clinical Sciences, Sector Pharmacology and Toxicology, Medical University, Marin Drinov 55, 9002 Varna, Bulgaria. Phone: +359898343274, E-mail: kalgeorgiev@hotmail.com

Marieta Georgieva – Institute of Experimental Morphology, Pathology and Anthropology with Museum, Bulgarian Academy of Sciences, Sofia, Bulgaria

Maria Peneva – Medical Centre “Evrozdrave” Sofia, Sofia, Bulgaria

Georgi Alexandrov – Military Medical Institute Academy – Sofia, Sofia, Bulgaria


Application of Lactobacillus Bulgaricus in Clinical Practice

Application of Lactobacillus Bulgaricus in Clinical Practice

Georgi Alexandrov1, Maria Peneva2

Abstract

Introduction: Lactic acid fermentation has been known to mankind for thousands of years. Ceramic pots dating back to 6500 years BC, containing residues of fermented dairy products, were found during archeological excavations in the territory around the Black sea. The first written evidence of lactic acid fermentation is by Hrodotus (V Century BC). He wrote that the Thracians (the population inhabited the present Bulgarian land) prepared special fermented dairy foods which were „a gift from Gods”. In 1905 Lactobacillus Bulgaricus was isolated from Bulgarian yoghurt. In 2010 N. Alexandrov and D. Petrova isolated Lactobacillus Bulgaricus from pure spring water from the mountain Stara planina in Bulgaria for the first time in the world. They think that there is a relation between probiotics, the human individual bacterial system, diseases and longevity of life. Beneficial effects of different strains of Lactobacillus Bulgaricus are scientifically proven on over 2500 patients. Probiotics with Lactobacillus Bulgaricus were used in the Space program Intercosmos.

Materials and Methods: During our clinical practice in 625 patients with different diseases we have applied probiotic formulas, containing different strains of Lactobacillus Bulgaricus. All strains of Lactobacillus Bulgaricus were repeatedly multiplied on the territory of Bulgaria.

Results and Discussion: In cases of alcohol intoxication, liver damage is leading, so it is used as a model of chronic liver injury. A double blind randomized study was carried out in 32 patients with alcoholic disease. 17 of them have taken the probiotic formula, containing strains L. Bulgaricus DMR1 and DMR2 at a daily dose of 30 g (over 1 billion CFU/g) and also a combination of that probiotic formula and an extract of silymarin (600 mg/24h) for a period of 6 months as a part of a complex treatment regimen. Adding that nutritional therapy to the main treatment leads to a significantly more efficient normalization of the values of ASAT, ALAT, GGTP and of the ultrasound image of liver and to a restoration of the functions of the liver cell in patients with alcoholic disease. Similar results were obtained when the same daily dose of probiotic foods, containing L. Bulgaricus DWT1, was given to other 78 patients with alcoholic disease and L. Bulgaricus Lb4 – given to 152 patients with heroin addiction and 208 patients with viral hepatitis.

A 28 year old patient with decompensated liver cirrhosis (diagnosed in Germany) and referred for liver transplantation was administered probiotic formula, containing strains L. Bulgaricus DMR1 and DMR2 at a daily dose of 60 g (over 1 billion CFU/g) for a period of 6 months, as a nutritional therapy added to the basic treatment.

We applied a probiotic formula, containing strains L. Bulgaricus DMR1 and DMR2 at a daily dose of 60 g (over 1 billion CFU/g) for a period of 6 months, as a nutritional therapy added to the basic treatment of a patient of 28 years with decompensated liver cirrhosis (diagnosed in Germany) and referred for liver transplantation. The patient avoided the liver transplantation and has recovered almost completely both clinically and in regard of her blood and instrumental tests and restored her working ability.

We carried out a comparative observation of the effects of 3 strains of Lactobacillus Bulgaricus DWT1, DMR1 and DMR2 all in combination with Bulgarian rose oil in 77 patients with proven by ELISA method food allergies. 23 of them have taken a daily dose of a probiotic formula, containing L. Bulgaricus DMR1 and DMR2 (over 1 billion CFU/g) and 40 mg Bulgarian rose oil. The other 54 patients have taken a daily dose of a probiotic formula, containing L. Bulgaricus DWT1 (over 1 billion CFU/g) and 40 mg Bulgarian rose oil. The duration of probiotic intake was 2 months. All patients have stopped the consumption of the proven food allergens and have not taken any antihistamine medications. For a period of 2 to 6 weeks depending on the severity of the manifestations the classic symptoms of allergy and the newly established by us symptoms of food allergy such as hair loss, dandruff, periodontal disease and changes in the prolactin levels in women, were put under control. We have not observed a statistically significant difference regarding the anti-allergic effects of the probiotic formulas, described above.

Local vaginal application of a probiotic formula, containing L. Bulgaricus DMR1 and DMR2 at a daily dose of 500 mg (over 1 billion CFU/g) in 32 women with vaginal discharge as a part of the anti-infective treatment shows a significant increase in the normal lactobacillaceae flora when the probiotic is applied during and 5 days after the anti-infective treatment of vaginal discharge. Similar results were obtained in case of application of the same daily dose of probiotic formulas, containing L. Bulgaricus DWT1 in 12 women and L. Bulgaricus Lb4 in 33 women with vaginal discharge.

Conclusion: Therapy with probiotic formulas, containing strains Lactobacillus Bulgaricus DMR1, DMR2 and DWT1 alone or in combination with plant extracts and oils, as a part of a complex treatment regimen leads to significantly more effective recovery of the liver functions, vaginal flora and putting the food allergy symptoms under control.

The beneficial effects of the probiotic microorganisms are mainly determined by their subspecies as the various strains show certain prominent features.

A proportional relationship is observed between the beneficial effect of the probiotic and the dose and period of its administration.

Keywords: probiotic, Lactobacillus Bulgaricus, allergy, liver, vaginal application

1 – Military Medical Academy – Sofia, Bulgaria

2 – Medical Centre “Evrozdrave” Sofia, Bulgaria


Hepatoprotective effect of probiotic food, containing Lactobacillus Bulgaricus DWT1 in acute ethanol intoxication in experimental animals

Gabriela Kehayova1, Marieta Georgieva1, Kaloyan Georgiev2, G. Aleksandrov3, M. Peneva4

1 Department of Pharmacology, Toxicology and Pharmacotherapy, Faculty of Pharmacy, Medical University of Varna

2 Department of Pharmaceutical Technologies, Faculty of Pharmacy, Medical University of Varna

3 Department of Abdominal Surgery, Military Medical Academy, Sofia, Bulgaria

4 MC Evrozdrave, Sofia, Bulgaria

Probiotics are live microorganisms, which have a beneficial effect on human health and contribute to the intestinal balance. In 1905 the Bulgarian Dr. Stamen Grigorov discovered the microorganism Lactobacillus bulgaricus in yogurt. Later Russian physiologist Ilya Mechnikov concluded that the secret of longevity of Bulgarians lies in eating yogurt. Lactobacillus bulgaricus is the only microorganism, named after a particular geographic territory. It reproduces only in the territory of Bulgaria. Nowadays probiotics are subject of increasing interest because of their proven hepatoprotective, immunostimulating, antioxidant and anti-tumor effect. For the first time in the world, Professor Nikola Aleksandrov managed to isolate natural probiotic bacteria from water source in Stara Planina. The probiotic association contains wild strains of lactic bacteria.

In the present experiment, we first investigated a hepatoprotective effect of a new strain of Lactobacillus bulgaricus DWT1. We used 30 male Wistar albino rats. The rats were treated within 14 days with Laktera Nature probiotic, and on the 12th day an acute liver damage was induced with a high dose of ethanol. After laboratory and histological analysis of rat blood and liver, we found that animals receiving only ethanol had extensive necrosis zones and elevated liver enzymes. In the Laktera Nature pretreated and then subjected to ethanol groups, ALT and alkaline phosphatase levels were significantly lower than those in the ethanol group. Histologically, a preserved liver structure was established. The present experiment demonstrates the hepatoprotective effect of Laktera Nature. It can improve significantly the treatment of the patients suffering from liver disease.

Keywords: probiotics, ethanol, liver damage, hepatoprotective effect

Medical University Prof. Dr. Paraskev Stoyanov, Varna, Bulgaria, Scripta Scientifica Pharmaceutica, ISSN 2367-6000, volume 4, 2017, supplement 2, page 51


Daflorn Patents

United States Patent of Daflorn Ltd Patent No: US 9,131,708 B2 Date of Patent: Sep. 15, 2015 Probiotics for dietary diary product

European Patent of Daflorn Ltd Patent No: EP 3 172 973 Probiotic formula, process of its preparation and use

United States Patent of Daflorn Ltd

The present invention relates to compositions containing probiotic microorganisms and methods of making and using probiotic associations containing these microorganisms. More particularly, the invention relates to an association of probiotic lactic acid microorganisms including newly identified strains of microorganisms and their use in dietary products. These microorganisms are cultured to produce a protective shell that enhances self-stability of compositions and products of the invention. The isolated microorganisms include new strains of Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus helveticus, Lactobacillus delbrueckii subsp. lactis, and Streptococcus thermophilus.

All products according to this US Patent contain original probiotic strains, isolated from pure spring water in Bulgaria. All products have approved health claim according to Commission Regulation (EU) No 432/2012 : Live probiotic cultures L. bulgaricus and St. thermophilus improve lactose digestion in individuals who have difficulty digesting lactose.

European Patent of Daflorn Ltd

Patent No: EP 3 172 973The invention relates to a probiotic formula involving cooperation of strains of probiotic microorganisms, isolated as it is, from a traditional diary product in Rhodope mountains, to a process of preparation of the probiotic formula and its use as a dietary food for people with impaired liver function and structure, for controlling the symptoms of food allergy and for recovering the normal vaginal lactobacillacae flora of women. The probiotic cooperation of strains microorganisms includes newly identified strains of Lactobacillus bulgaricus and Streptococcus thermophilus. This probiotic cooperation is used for fermentation of a standardized and pasteurized goat`s milk for preparation of the probiotic formula, which after lyophilization and storage for 24 months at a room temperature up to 24°C, contains alive cells of the probiotic cooperation, in concentration from 1.1×109 cfu/g to 1.6×109 cfu/g.

All products according to this EU Patent contain unique goat`s milk probiotic with original strains. All products have approved health claim according to Commission Regulation (EU) No 432/2012: Live probiotic cultures L. bulgaricus and St. thermophilus improve lactose digestion in individuals who have difficulty digesting lactose.