PRODUCTS LIST MONOBUTYRIN HYDRO C4-30 WPSA SILO MEETING MONOBUTYRIN GLYCEROL + BUTYRIC ACID = BUTYRIC ACID MONOGLYCERIDE

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1 WPSA SILO MEETING October 8,21 Ozzano dell Emilia Molecular structure and mode of action of SILO monoglycerides Manuela Parini SILO SpA 1 2 Up-dated May, 21 PRODUCTS LIST AVAILABLE PRODUCTS, BASED ON: - MONOPROPIONIN Butterfly 21 - Hydro C4 3 - MONOCAPRYLIN Energy COCO-8 - MONOCAPRIN Energy COCO-1 - MONOLAURIN Energy COCO-12 PRODUCTS already manufactured on SILO pilot plant ESTERIFICATION OF DIFFERENT MONOGLYCERIDES BUTYRIC ACID C8-C1 MONO-C8-C1 - MONOFORMIN Force 1 - MONOACETIN Aceto Sweet PRODUCTS in fieri - MONOFUMARIN - MONOLACTIN - MONOBENZOIN Patent pending - Strong antibactirial action (non ph dependent) in: * water * feed * gizzard stomach intestine - Gastric By-pass (= oils) - Non corrosive - No bad smell LAURIC ACID MONOLAURIN 4 HYDRO C4-3 GLYCEROL + BUTYRIC ACID = BUTYRIC ACID MONOGLYCERIDE lipophilic hydrophilic Undissociated Butyric acid Molecular weight 88 Molecular weight 92 Mix of + Free LIQUID & POWDER The Hydrogen atom H of glycerol is bound with the OH of the butyric acid 1

2 Molecular weight 162 A new molecule!! Covalent bond The chemical bond but. Acid / glycerol is not a ionic bond (ph dependent), but a covalent bond (ph independent) Covalent bond = force of attraction between 2 atoms that share a couple of electrons Characteristics of the chemical bond butyric acid+glycerol: Covalent bond (the attraction force between 2 atoms that share a couple of electrons) Very stable (up-to 23 C) Stability non-ph dependent (practically no problem of dissociated or undissociated form) Odorless Absorbance Absorbance,, % ph = 4. STABILITY AT VARIOUS ph VALUES 5 1 Time, minutes Absorbance Absorbance,, % ph = Time, minutes SODIUM BUTYRATE ph 7= Dissociated butyric acid Ionic bond = attraction force between ions charged with opposite signes (+;-) ph dependent Sodium NON PROTECTED SODIUM BUTYRATE Non protected sodium butyrate is absorbed very quickly at the level of crop and gizzard 11 Inclusion in the feed% Crop (anathomic part) Small intestine sections DETERMINATION OF NON PROTECTED SALTS (ACIDS) IN THE GIT Acetic(*) Propionic(*) Butyric(*) 2,4 2,4 2,6 99% 4% 6% Section 1 2% (of starting amount=,5%) Section 2 1,7% (of starting amount =,4%),5% (of starting amount=,1%) Section 3 Section 4 (*)Administered in form of Ca salts Adapted from The digestibility of acetic, propionic and butyric acids by the fowl Bolton and Dewar 1965 (*) Sodium or calcium butyrate 2

3 Na Butyrate Unprotected Region where product is ac LIMITS OF PROTECTED SODIUM BUTYRATE If the Sodium Butyrate is protected (micro-encapsulated) it is buffered in the gut by calcium carbonate. At ph 6 7 (ph of the gut) Sodium Butyrate is present in dissociated form losing its antibacterial efficacy ONLY UNDISSOCIATED ACIDS KILL BACTERIA Cell membrane ❶ RCOOH RCOO - H + Nucleus ph lowering ADP + P > ATP Ribosomes ❷ R-COOH = undissociated acid ph EFFECT ON DISSOCIATION RATE OF ORGANIC ACIDS Acid type Ratio dissociated: non dissociated pk a ph 4 ph 6 ph 7 Formic acid :1 178:1 1778:1 Lactic acid :1 148:1 1479:1 Benzoic acid :1 65:1 646:1 Butyric acid :1 15:1 138:1 Propionic acid :1 13:1 132:1 Conclusion: organic acids are most active as antimicrobials at low ph Minimum inhibitory concentrations Organism E. Coli Staf. Aureus B. Cereus E. Coli Staf.Aureus Listeria Acid Propionic Propionic Propionic Sorbic Sorbic Lactic Undissociated ,6 5 um Dissociated F. Boyen F.Van Immerseel - Veterinary Microbiology 27 Overview of the MIC values of formic acid, acetic acid, butyric acid, tested at ph4, 5, 6 using 54 porcine SALMONELLA TYPHIMURIUM strains. Number of strains with MIC of (mm),625 1,25 2, Formic Acid ph ph5 54 ph6 54 Acetic Acid ph4 54 ph ph Propionic Acid ph ph ph Butyric Acid ph ph ph Adapted from Presser et al. (1997)

4 Comment to the previous slide: At ph 4, 5 mm butyric acid, correspondending to.4% in water, are sufficient to obtain the MIC on 49 strains of Salmonella Typhymurium At ph 6, 16 mm butyric acid, correspondending to 1.4% in water, are needed to obtain the MIC on 53 strains of Salmonella Typhymurium MONOGLYCERIDES NEW IDEAS TO APPROACH THE PROBLEM?? MONOGLYCERIDES proved to have a significantly stronger microbicidial action than relevant acid In conclusion, at ph 6 an amount of butyric acid 32 times higher than that at ph 4 is necessary 19 & MONOPROPIONIN Istituto Zooprofilattico Lombardia - Emilia Romagna Bruno Ubertini Brescia dr. L. Alborali FARM TRIAL WITH PIGS Istituto Zooprofilattico Lombardia - Emilia Romagna Bruno Ubertini Brescia dr. L. Alborali F. Foresti Observation period: 6/23 8/1/21; age of pigs: from 41 up to 91 d. 678 pigs in the control group and 653 in the group MIC DETERMINATION Dilution expressed as % in water ph S. typhimurium S.choleraesuis E. coli O %.12%.12% Hydro C4 Monopropionin Hydro - C3 7.3%.12%.6% N u m b e r o f i n j e c ti o n s Number of injections to control diarrhea: 53 in the control group and 5 in the group Gruppo di Controllo,2% di Monobutirrina in acqua Compo unds Lauric acid 1- Monolaur in 1-3 Dilaurin MIC OF ACIDS OR GLYCERIDES Undissociated form Pneum o-cocci Streptoc occus Group A Streptoc occusbet a- Hemoliti c non- A Coryneba cteria Nocar dia astero ides Microc occi Candi da µmoles/ml S. Aure us S. epider mis fold! Nl Nl Nl Nl Nl Nl Nl Nl Nl Trilaurin Nl Nl Nl Nl Nl Nl Nl Nl Nl J.J. Kabara S.Leeson (Guelph University, personal communication) CLOSTRIDIUM p. Positive Control ppm % Butyric acid Hydro C % Bacteria Growth + = 24 h ++ = 36 h +++ = 96 h Inoculum = % % %

5 S.Leeson (Guelph University, personal communication) CLOSTRIDIUM p. Positive Control ppm % Butyric acid Hydro C % Bacteria Growth + = 24 h ++ = 36 h +++ = 96 h 3.3% 4 Inoculum = 1 5 EXERTS A DIRECT EFFECT: One of the MECHANICAL modes of action RUPTURE of OF THE is BACTERIAL the rupture OUTER of cell MEMBRANE membranes AND WALL Compatibility with hydrophilic and lipophilic membranes Cell membrane Cytoplasm Cell membrane / wall 26 ANOTHER MODE OF ACTION OF Inactivation of pathogenicity island in the bacteria ACTIVATION OF SALMONELLA PATHOGENICITY = Salmonella Pathogenicity island (genes enconding for Salmonella pathogenicity) Effector protein SALMONELLA External stimuli: food, temperature, ph. etc Activation of virulence genes present in Needle complex component (TTSS type three secretion system) TTSS complex component (needle) injecting effector proteins into host animal cells) Delivery of inside the bacterial cell through aquaporins Many bacteria can metabolize glycerol as an energy source Bacteria possess aquaporins, that is channels designed to absorb glycerol from their surroundings ( uptake facilitators ) (Antonio H. Romano 199) Tactic of thetrojan Horse: employ glycerol as a carrier to transport butyrate into the bacterial cell. Infected animal cell Bacterial cell

6 INACTIVATION OF SALMONELLA PATHOGENICITY A non-specific bacterial lipase breacks the gliyerol-butyric acid bond (R. Gupta et al. 24) Lipase = Salmonella Pathogenicity island SALMONELLA Butyric Inactivation of genes 31 Gantois,, R. Ducatelle, Butyrate Specifically Down-Regulates Salmonella Pathogenicity Island 1 Gene Expression. Applied and Environmental Microbiology The TTSS complex is present in gram negative bacteria: Yersinia, Shigella, Salmonella, Pseudomonas e E. coli TTSS complex injecting effector proteins into host cell ANTIBACTERIAL ACTION - is active at any ph values, from ph 4 to ph 7; it is active in crop, gizzard and intestine conditions In the presence of monobutyrin.. Infected animal cell the formation of the TTSS complex is inhibited - If administered in the drinking water, activity starts already in the water Monobutirrina Area dove la Monobutirrina è attiva 35 6