Safety aspects of application of Lactic Acid Bacteria SVETOSLAV TODOROV

1. Safety aspects of application of Lactic Acid Bacteria SVETOSLAV TODOROV Federal University of Viçosa Viçosa, MG, Brazil

2. Lactic acid bacteria Early definition of LAB as a group, based on the ability to ferment and coagulate milk, included coliform bacteria with the lactic. The description of Lactobacillus organism by Beijerinck in 1901 as Gram-positive bacteria separated the coliforms from LAB. According to Orla Jensen in 1919 “the true LAB form a natural group of Gram-positive non-motile, non-spore forming, rod- and coccus-shaped organisms that ferment carbohydrates and higher alcohols to form chiefly lactic acid.”

3. Lactic acid bacteria Rods Cocci Lactobacillus Carnobacterium Lactococcus, Enterococcus, Streptococcus, Leuconostoc, Weisella, Pediococcus Streptococcus gallolyticus subsp. macedonicus Desemzia, Isobacilum, Paralactobacillus, Tetragenococcus, Trichococcus, Oenococcus, Melissococcus

4. Lacticacidbacteria

5. medicine chemistry foodandfeed industries USES Lacticacidbacteria

6. Lacticacidbacteria FUNCTIONAL INGREDIENTS

7. medicine chemistry foodandfeed industries USES Lacticacidbacteria FUNCTIONAL INGREDIENTS probiotics enzymes vitamins Antimicrobialagents starter cultures sweeteners exopolysaccharides medical sector dairy biopreservatives non-dairy

8. medicine chemistry foodandfeed industries USES Lacticacidbacteria FUNCTIONAL INGREDIENTS probiotics enzymes vitamins Antimicrobialagents starter cultures sweeteners exopolysaccharides medical sector dairy biopreservatives non-dairy

9. Antimicrobial agents produced by LAB organic acids carbone dioxide hydrogen peroxide diacetyl bacteriocins low molecular weightsubstances All act as biopreservatives in food, with records dating back to approximately 6000 B.C. (Pederson, 1971; De Vuyst and Vandamme, 1994).

10. Bacteriocins & Probiotics www.scopus.com

11. Bacteriocins & Probiotics www.scopus.com

12. Bacteriocins & Probiotics www.scopus.com

13. Bacteriocins & Probiotics www.scopus.com

14. What are bacteriocins? Classical definition Bacteriocins produced by LAB are polypeptides synthesized in ribosomes, that exhibit bactericidal or bacteriostatic effect against genetically closely related bacteria Klaenhammer, 1988, De Vuyst & Vandamme, 1994

15. Bacteriocins: developing innate immunity for food Paul D. Cotter, Colin Hill & R. Paul Ross Nature Reviews Microbiology 3, 777-788 (October 2005)

16. Bacteriocins of LAB Mechanisms of action Cell wall Cell membrane Bacteriocins: developing innate immunity for food Paul D. Cotter, Colin Hill & R. Paul Ross Nature Reviews Microbiology 3, 777-788 (October 2005)

17. Spectrum of activity Bacteriocin Closely related LAB Other LAB, included LAB from same ecological niche Important Food or Human Pathogens A Unusual activity: Gram-negative, viruses, TB, Yeast and Fungi B

18. M 1 2 3 4 5 300 bp 100 bp Lactobacillus plantarum ST8Sh Shpek Agarose gel showing DNA fragments obtained after PCR with species-specific primers. Lines 1 and 2: strain ST8SH, lines 3 and 4: Lactobacillus plantarum LMG 13556, line 5: No DNA and line M: O’GeneRulertm 100 bp DNA Ladder (Fermentas). Lactobacillus plantarum

19. Plantaricin S Nisin Plantaricin NC8 Pediocin PA-1 Plantaricin W Lactobacillus plantarum ST8Sh

20. Plantaricin S Nisin Plantaricin NC8 Pediocin PA-1 Plantaricin W Lactobacillus plantarum ST8Sh

21. Spectrum of activity recorded for bacteriocin ST8Sh

22. Growth of Lactobacillus plantarum ST8SH in MRS broth (-▲-), changes in pH (-●-) and production of bacteriocin expressed in AU/ml (histogram bars) at 370C (A), 300C (B) and 250C (C).

23. Effect of different concentrations of bacteriocin ST8SH on • Listeria monocytogenes ScottA, • Lactobacillus sakei ATCC 15521 and • Enterococcus faecalis ATCC 19433 • recorded at 3h, 9h and 18h presented as % of lysis of test microorganisms.

24. Effect of bacteriocin ST8SH on exponentially growing (a) Listeria monocytogenes ScottA, (b) Lactobacillus sakei ATCC 15521 and (c) Enterococcus faecalis ATCC 19433. (-♦-) without added bacteriocin; (-▲-) with added 10% (v/v) bacteriocin ST8Sh

25. Growth of a mixed culture of Lactobacillus plantarum ST8SH and Listeria monocytoegnes ScottA (--) and Listeria monocytogenes ScottA (--). Changes in pH is shown by --. Production of bacteriocin ST8SH is indicated by the histogram

26. The dose makes the poison, a principle of toxicology, was first expressed by Paracelsus. It means that a substance can produce the harmful effect associated with its toxic properties only if it reaches a susceptible biological system within the body in a high enough concentration (dose). Paracelsus (Philippus Aureolus Theophrastus Bombastus von Hohenheim, 11 November or 17 December 1493 – 24 September 1541) was a German-Swiss Renaissance physician, botanist, alchemist, astrologer, and general occultist

27. Toxicity of bacteriocin ST8Sh Cell-free supernatant of Lactobacillus plantarum ST8Sh Partial purification of the bacteriocin ST8Sh Semi-purified fractions (60% iso-propanol and 80% iso-propanol) of bacteriocin ST8SH were tested on Huh7.5 cells for their cytotoxicity profile

28. Toxicity of bacteriocin ST8Sh Cell-free supernatant of Lactobacillus plantarum ST8Sh Partial purification of the bacteriocin ST8Sh Semi-purified fractions (60% iso-propanol and 80% iso-propanol) of bacteriocin ST8SH were tested on Huh7.5 cells for their cytotoxicity profile At concentration of 25 µg/ml 60% fraction demonstrated to be highly cytotoxic, reducing the cell viability by approximately 80%, but not with lower tested concentrations of the bacteriocin ST8Sh Regarding the 80% iso-propanol Fraction of bacteriocin ST8Sh, cell viability was not reduced in both tested concentrations (25 µg/ml and 5 µg/ml)

29. Toxicity of bacteriocin ST8Sh • Toxicological studies showed that nisin intake does not cause toxic effects to the human body with an estimated lethal dose (LD50) of 6950 mg/kg, which is similar to that of salt, when administered orally (Jozala, Andrade, Arauz, Pessoa Junior & Vessoni-Penna, 2007). • In general, some authors have associated LD50 of bacteriocins with digestive enzymes capable of rapidly inactivating these substances such as trypsin and chymotrypsin produced in the pancreas (Deegan, Cotter, Hill, & Ross, 2006; Motta, Cannavan, Tsai & Brandelli, 2007; Vaucher et al., 2011).

30. Beneficial properties of Lactobacillus plantarum ST8Sh Survival in GIT conditions

31. Beneficial properties of Lactobacillus plantarum ST8Sh Survival in GIT conditions Growth/survival in presence of low pH and oxbile

32. Beneficial properties of Lactobacillus plantarum ST8Sh Survival in GIT conditions Growth/survival in presence of low pH and oxbile Aggregation propertis

33. Beneficial properties of Lactobacillus plantarum ST8Sh Survival in GIT conditions Growth/survival in presence of low pH and oxbile Aggregation propertis Interaction with antibiotics and commercial drugs

34. Beneficial properties of Lactobacillus plantarum ST8Sh Survival in GIT conditions Growth/survival in presence of low pH and oxbile Aggregation propertis Interaction with antibiotics and commercial drugs Hydrophobicity

35. Beneficial properties of Lactobacillus plantarum ST8Sh Survival in GIT conditions Growth/survival in presence of low pH and oxbile Aggregation propertis Interaction with antibiotics and commercial drugs Hydrophobicity β-galactosidase

36. Beneficial properties of Lactobacillus plantarum ST8Sh Survival in GIT conditions Growth/survival in presence of low pH and oxbile Aggregation propertis Interaction with antibiotics and commercial drugs Hydrophobicity β-galactosidase Adherence to cell lines Caco2 & HT29

37. Beneficial properties of Lactobacillus plantarum ST8Sh Survival in GIT conditions Growth/survival in presence of low pH and oxbile Aggregation propertis Interaction with antibiotics and commercial drugs Hydrophobicity β-galactosidase Adherence to cell lines Caco2 & HT29 Cholesterol reduction

38. Mub Mab EF2662-cbp EF1249-fbp EF2380-maz EF-tu prgB (surface protein) Beneficial properties of Lactobacillus plantarum ST8Sh Some genes

39. Mub Mab EF2662-cbp EF1249-fbp EF2380-maz EF-tu prgB (surface protein) Beneficial properties of Lactobacillus plantarum ST8Sh Some genes

40. Safety of Lactobacillus plantarum ST8Sh Lactobacillus plantarum ST8Sh

41. Safety of Lactobacillus plantarum ST8Sh Antibiotic resistance Production of biogenic amines Virulence factors Horizontal gene transfer

42. ddl E. faecalis aac(6′)-Ii E. faecium mur-2ed E. durans mur-2 E. hirae int-Tn vanC1 vanC2/C3 vanC1 (D-ala-D-ser ligase) vanC2 (D-ala-D-ser ligase) vanA ermA ermB1 ermC ermB2 vanB tetK tetL tetM tetO tetS aac(6′)-Ie-aph (2″)-Ia aph(3′)-IIIa ant(4′)-Ia (adenylyltransferase) aph(2″)-Id (phosphotransferase) aph(2″)-Ic (phosphotransferase) ant(6)-Ia catA (PIP501) vatE ccf cob bcrB bcrD bcrR cpd sprE ace esp efaA cylA hdc2 odc aph(2″)-Ib (phosphotransferase) hdc1 tdc fsrC fsrA gelE asa1 fsrB int hyl Safety of Lactobacillus plantarum ST8Sh Antibiotic resistance Production of biogenic amines Virulence factors Horizontal gene transfer

43. ddl E. faecalis aac(6′)-Ii E. faecium mur-2ed E. durans mur-2 E. hirae int-Tn vanC1 vanC2/C3 vanC1 (D-ala-D-ser ligase) vanC2 (D-ala-D-ser ligase) vanA ermA ermB1 ermC ermB2 vanB tetK tetL tetM tetO tetS aac(6′)-Ie-aph (2″)-Ia aph(3′)-IIIa ant(4′)-Ia (adenylyltransferase) aph(2″)-Id (phosphotransferase) aph(2″)-Ic (phosphotransferase) ant(6)-Ia catA (PIP501) vatE ccf cob bcrB bcrD bcrR cpd sprE ace esp efaA cylA hdc2 odc aph(2″)-Ib (phosphotransferase) hdc1 tdc fsrC fsrA gelE asa1 fsrB int hyl Safety of Lactobacillus plantarum ST8Sh Antibiotic resistance Production of biogenic amines Virulence factors Horizontal gene transfer

44. Are we going to open the Pandora’s box???

45. Lucius Annaeus Seneca (often known simply as Seneca; ca. 4 BC – AD 65) was a Roman Stoic philosopher, statesman, dramatist, and in one work humorist, of the Silver Age of Latin literature. He was tutor and later advisor to emperor Nero. I know that I know nothing, but you even don’t know this.

46. slavi310570@abv.bg Svetoslav D. Todorov