Aerogels of enzymatically oxidized galactomannans from leguminous - PowerPoint PPT Presentation

“Aerogels of enzymatically oxidized galactomannans from leguminous plants: versatile delivery systems of antimicrobial compounds and enzymes” Yves M. Galante Associate Istituto di Chimica del Riconoscimento Molecolare, C.N.R., Via Mario Bianco 9, 20131 Milano, Italy. MIPOL 2017 Milan; 15-16 Feb 2017 Partners of the Cariplo Project: • ICRM CNR • Dept. Biotech, Univ. Milano Bicocca • ISPA CNR

Structure of galactomannans (GM) Guar: Galactose/Mannose ratio Galactose 1: 1.5-1.8 Mannose galactomannans main properties: Gal/Man ratio in leguminous galactomannans: Ø Soluble in cold or hot water. Ø Develop high viscosities at low Cassia 1:4.5 Locust bean 1:3.5 polymer concentrations. Tara gum 1:2.5 Ø Versatile, flexible, reactive, can Guar 1:1.5-1.8 Sesbania 1:1.3 be chemically and Fenugreek 1:1 biochemically modified.

Guar: the source Cyamopsis 15% Tetragonolobus 40% 45% Guar Gum Powder (GG) chemical /biochemical modifications

̴ ̴ ̴ ̴ 1 H-NMR of NATIVE GM MAN : GAL RATIO TARA 1 : 2.50 a) b) GUAR 1 : 1.50 SESBANIA 1 : 1.35 c) FENUGREEK 1 : 1.0 d) G1 M1 5.0 4.5 4.0 ppm

Enzymatic modifications of galactomannans Galactose oxidase/Laccase β -Mannanase α -Galactosidase Oxidation Depolymerization Debranching

Laccase structure and catalytic mechanism Site of mono- Ø Laccase are “ blue multi copper ” electronic oxidases with wide specificity. oxidation of substrate Ø They catalyze mono-electronic oxidation of various substrates. Ø They are active on phenolic and non- phenolic substrates (with a mediator) Trinuclear cluster for the reduction of O 2 to H 2 O (2,2,6,6-­‑Tetramethylpiperidin-­‑1-­‑yl)oxyl ¡ ¡ or ¡TEMPO.

Kinetics of guar gum oxidation in water monitored by Brookfield viscosity Complete reaction mixture: guar gum + laccase + TEMPO ( full circles) Control: guar gum + laccase (empty symbols) Single point pH values are indicated.

Effect of oxidation Oxidized guar Guar galactomannan galactomannan solution solution before oxidation

Rheological profile of the oxidation reaction Kinetics of the oxidation reaction monitored by Time Oscillation Test. tang δ G ’ (elastic modulus) of oxidized galactomannan ( ♦ ) G’’ (viscous modulus) of oxidized galactomannan ( ◊ );

RHEOLOGICAL PROFILE OF SESBANIA 1.0E+02 NATIVE Sesbania G' G'' G’ = ELASTIC MODULUS 1.0E+01 G’’= VISCOUS MODULUS G', G'' [Pa] CROSSOVER: 1.0E+00 TYPICAL POLYMER PROFILE 1.0E-01 1.0E+02 Sesbania Ox ENZ OX G' G'' 1.0E-02 1.0E-01 1.0E+00 1.0E+01 1.0E+02 1.0E+01 Frequency ω [rad/s] G', G'' [Pa] NO CROSSOVER: 1.0E+00 ELASTIC GEL PROFILE 1.0E-01 1.0E-01 1.0E+00 1.0E+01 1.0E+02 Frequency ω [rad/s]

Tan ( δ ) Viscous modulus (G”)/elastic modulus (G’) trend of GM: 10 Sesbania Tara Guar Fenugreek 8 Sesbania Ox Tara Ox Guar Ox Fenugreek Ox 6 tg( δ ) 4 Tan ( δ ) < 1 2 1 0 1.0E-02 1.0E-01 1.0E+00 1.0E+01 1.0E+02 1.0E+03 Frequency ω [rad/s]

The proposed mechanism for enzymatic hydrogel formation Hydrated aldehydes LCC + TEMPO, 35°C, 3 h Carbonyls General galactomannans structure Elastic gel formation Parikka and Tenkanen, 2009, Carb. Pol. 344, 14-20. Lavazza et al., 2011, J. Biotechnol. 156, 108-116. Mikkonen et al, 2014, RSC Adv. 4, 11884-11892. Hemiacetalic bond Merlini et al., 2015, J. Biotechnol. 198, 31-43.

BIOCHEMICAL & PHYSICAL MODIFICATIONS of GM Fenugreek gum NATIVE ENZIMATICALLY FENUGREEK SOLUTION OXIDIZED FENUGREEK LYOPHILIZED ENZYMATICALLY OXIDIZED & NATIVE FENUGREEK LYOPHILIZED FENUGREEK Trigonella foenum-graecum, Fabaceae (Annual)

Protocol for the generation of EOLFG (Enzymatically Oxididized Lyophilized Fenugreek Gum) and other aerolegs TEMPO

Preparation of active loaded aerogels Absorption of active Carving the plugs from a Rinsing 3 times Plugs of EOLFG substance (1 h, rt) in sterile water EOLFG wafer with a cork borer Loaded aerogel with the active substance Unloaded Loaded Loaded Solution of active aerogel hydrogel aerogel substance Lyophilization

Polymyxin Antibiotics Polymyxins (polymyxin B and colistin) - secondary metabolite non ribosomal peptides produced by Bacillus polymyxa - cationic cyclic lipodecapeptides Green: linear tri-peptide segment Red: polar residues of the hepta-peptide ring Blue hydrophobic motif within the hepta-peptide ring Velkov et al. (2010) J.Med.Chem 53:1898

Release of polymyxin B from the aerogels on Pseudomonas aeruginosa 2 nd rinse water 1 st rinse 3 rd rinse water water Control EOLFG Loaded with polymyxin EOLGG EOLFG Loaded with polymyxin Control EOLSG Free polymixin Control Loaded with polymyxin EOLGG EOLSG

Release of polymyxin B from aerogels on Gram-negative bacteria Serratia marcescens Salmonella Typhimurium Escherichia coli Free polymyxin Free polymyxin Free polymyxin Free polymyxin Enterobacter cloacae Hafnia alvei EOLFG with Polymyxin B EOLSG with Polymyxin B Free polymyxin Free polymyxin EOLGG with Polymyxin B

NISIN Natural antimicrobial polypeptide produced by Lactococcus lactis EU No. 1129/2011: Food additive (E234) Active against Gram-positive bacteria ( Lactococcus spp. , Streptococcus spp. , Staphylococcus spp. , Pediococcus spp. , Lactobacillus spp ., Listeria spp .) , spores (Bacillus spp., Clostridium spp) and Gram-negative bacteria, if combined with chelating agent ( E. coli , P. aeruginosa ) Mechanisms of action Peschel & Sahl, 2006. Nature Reviews Microbiology , 4 (7), 529-536.

Release of nisin from aerogels on Gram-positive bacteria Enterobacter faecalis Clostridium tyrobutirycum 1 1 3 3 Free nisin Free nisin 2 2 1 EOLFG with 2 EOLSG with 3 EOLGG with nisin nisin nisin

LYSOZYME Natural antimicrobial enzyme from hen egg albumen EU No. 1129/2011: Food additive «E1105» Active against Gram-positive bacteria particularly Clostridium spp. and LAB (Lactic Acid Bacteria) Mechanism of action: lysozyme is a glycoside hydrolase, that damages bacterial cell walls by catalyzing hydrolysis of 1,4-beta-linkages. Used to prevent butirric acid fermentation which causes the “ late blowing ” of cheese wheels 2CH 3 CHOHCOOH > CH 3 CH 2 CH 2 COOH + 2CO 2 + 2H 2

Release of lysozyme from EOLFG and inhibition of Clostridium tyrobutyricum Free lysozyme Lysozyme released Control from EOLFG EOLFG 10 5 CFU/ml per plate

Serine protease uptake & release EOLFG EOLSG EOLGG 100 Activity (% theoretical max) 80 60 40 20 0 0 1 2 3 4 5 6 Time (h)

OUR RECENT PUBLICATIONS ON ENZYMATIC OXIDATION OF GM BY LACCASE AND GENERATION OF AEROGELS AS DELIVERY SYSTEMS

SUMMARY OF RESULTS • Laccase/TEMPO oxidation of 5 species of GM solutions (locust beam, tara, guar, sesbania, fenugreek) causes an increase in viscosity and formation of elastic, stable gels. • This phenomenon is more pronounced with fenugreek, sesbania and guar than with tara and locust bean, suggesting that the primary hydroxyl groups that undergo oxidation are exclusively or preferentially those of the galactose side chains. • Carbonyl groups are generated by the enzymatic reaction, which eventually form hemiacetalic bonds with adjacent free OH ’ s, causing internal cross-linking of the GM and their “ structuring ” to yield elastic gels. • Instead, chemical oxidation causes a sharp, but only transient, increase in viscosity of GM solutions, immediately followed by “ melting ” of the intermediate gels to low viscosity liquids. NMR of the reaction products reveals the presence of carboxyl groups and absence of intermediate carbonyls. • Rheological profiles offer a further confirmation that enzymatic oxidation by laccase/ TEMPO causes a transition of GM from viscous solutions to structured, elastic gels. • If enzymatic oxidation is followed by lyophilization, water-insoluble aerogels are obtained, capable of absorbing water over 20 times their own weight. • Active compounds, such as anti microbial peptides and enzymes, can be loaded and entrapped in the aerogels and released in active form. • We propose that these original materials, composed of modified polysaccharides from renewable sources, might represent versatile “ delivery systems ” of various active principles (e.g., chemical biocides, enzymes, peptides, anti-inflammatories, antibiotics, etc.).

ACKNOWLEDGEMENTS & FUNDING Bianca Rossi Fiorenza Viani Luca Merlini Yves M. Galante Paola Campia Tiziana Silvetti Milena Brasca Funding from: Antonella Caterina Boccia Raniero Mendichi Stefano Farris, Unimi Food Packaging Lab DeFENS , University of Milan Erika Ponzini Stefania Brocca Rita Grandori Lucio Melone Alessandra Polissi Nadia Pastori Alessandra Martorana Carlo Punta This project was supported by the programs: “Suschem Lombardia: prodotti e processi chimici sostenibili per l’industria lombarda”. Accordo Quadro Regione Lombardia-CNR, 16/07/2012 (protocol no. 18096/RCC); by Cariplo Foundation (grant 2014-0478) and by MIUR (PRIN 2010-2011, PROxi project 2010PFLRJR_005).