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.

Partners of the Cariplo Project:

• ICRM CNR
• Dept. Biotech, Univ. Milano Bicocca
• ISPA CNR

MIPOL 2017

Milan; 15-16 Feb 2017

Structure of galactomannans (GM)

Guar: Galactose/Mannose ratio 1: 1.5-1.8 Mannose Galactose

Gal/Man ratio in leguminous galactomannans: Cassia 1:4.5 Locust bean 1:3.5 Tara gum 1:2.5 Guar 1:1.5-1.8 Sesbania 1:1.3 Fenugreek 1:1

galactomannans main properties: Ø Soluble in cold or hot water. Ø Develop high viscosities at low polymer concentrations. Ø Versatile, flexible, reactive, can be chemically and biochemically modified.

Guar: the source

Cyamopsis Tetragonolobus 15% 40% 45% Guar Gum Powder (GG)

chemical /biochemical modifications

4.0 4.5 5.0 ppm

c) b) a) d)

̴ ̴ ̴ ̴

G1 M1

1H-NMR of NATIVE GM

MAN : GAL RATIO

TARA 1 : 2.50 GUAR 1 : 1.50 SESBANIA 1 : 1.35 FENUGREEK 1 : 1.0

Enzymatic modifications of galactomannans

Oxidation Debranching Depolymerization Galactose

• xidase/Laccase

α -Galactosidase β -Mannanase

Trinuclear cluster for the reduction of O2 to H2O Site of mono- electronic

• xidation of

substrate

Laccase structure and catalytic mechanism

Ø Laccase are “blue multi copper”

• xidases with wide specificity.

Ø They catalyze mono-electronic

• xidation of various substrates.

Ø They are active on phenolic and non- phenolic substrates (with a mediator)

(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 galactomannan solution Guar galactomannan solution before

• xidation

Rheological profile of the oxidation reaction

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

RHEOLOGICAL PROFILE OF SESBANIA

1.0E-02 1.0E-01 1.0E+00 1.0E+01 1.0E+02 1.0E-01 1.0E+00 1.0E+01 1.0E+02 Frequency ω [rad/s] G', G'' [Pa] G' G'' Sesbania 1.0E-01 1.0E+00 1.0E+01 1.0E+02 1.0E-01 1.0E+00 1.0E+01 1.0E+02 Frequency ω [rad/s] G', G'' [Pa] G' G'' Sesbania Ox

G’ = ELASTIC MODULUS G’’= VISCOUS MODULUS

CROSSOVER: TYPICAL POLYMER PROFILE NO CROSSOVER: ELASTIC GEL PROFILE NATIVE ENZ OX

Tan (δ) Viscous modulus (G”)/elastic modulus (G’) trend of GM:

2 4 6 8 10 1.0E-02 1.0E-01 1.0E+00 1.0E+01 1.0E+02 1.0E+03 Frequency ω [rad/s] tg(δ) Sesbania Tara Guar Fenugreek Sesbania Ox Tara Ox Guar Ox Fenugreek Ox

Tan (δ) < 1

1

LCC + TEMPO, 35°C, 3 h

The proposed mechanism for enzymatic hydrogel formation

Elastic gel formation General galactomannans structure Hemiacetalic bond Carbonyls Hydrated aldehydes

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. Merlini et al., 2015, J. Biotechnol. 198, 31-43.

BIOCHEMICAL & PHYSICAL MODIFICATIONS of GM

NATIVE FENUGREEK SOLUTION ENZYMATICALLY OXIDIZED & LYOPHILIZED FENUGREEK LYOPHILIZED NATIVE FENUGREEK ENZIMATICALLY OXIDIZED FENUGREEK Fenugreek gum

Trigonella foenum-graecum, Fabaceae (Annual)

Protocol for the generation of EOLFG

(Enzymatically Oxididized Lyophilized Fenugreek Gum) and other aerolegs

TEMPO

Carving the plugs from a EOLFG wafer with a cork borer

Plugs of EOLFG Absorption of active substance (1 h, rt) Rinsing 3 times in sterile water Solution of active substance

Loaded aerogel with the active substance

Unloaded aerogel Loaded hydrogel Loaded aerogel Lyophilization

Preparation of active loaded aerogels

Polymyxins (polymyxin B and colistin)

• secondary metabolite non ribosomal peptides produced by Bacillus

polymyxa

• cationic cyclic lipodecapeptides

Polymyxin Antibiotics

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

1st rinse water 2nd rinse water 3rd rinse water

EOLFG EOLGG EOLSG

EOLFG EOLGG EOLSG

Control Loaded with polymyxin Loaded with polymyxin Loaded with polymyxin Control Control Free polymixin

Serratia marcescens Salmonella Typhimurium Escherichia coli Enterobacter cloacae Hafnia alvei

EOLFG with Polymyxin B EOLGG with Polymyxin B EOLSG with Polymyxin B Free polymyxin Free polymyxin Free polymyxin Free polymyxin Free polymyxin Free polymyxin

Release of polymyxin B from aerogels on Gram-negative bacteria

Peschel & Sahl, 2006. Nature Reviews Microbiology, 4(7), 529-536.

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

• f action

NISIN

Release of nisin from aerogels on Gram-positive bacteria

Clostridium tyrobutirycum Enterobacter faecalis

Free nisin Free nisin

1 1 2 2

EOLGG with nisin EOLFG with nisin EOLSG with nisin

1 2 3 3 3

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

2CH3CHOHCOOH > CH3CH2CH2COOH + 2CO2 + 2H2

LYSOZYME

Release of lysozyme from EOLFG and inhibition of Clostridium tyrobutyricum

Free lysozyme Lysozyme released from EOLFG Control EOLFG

105 CFU/ml per plate

20 40 60 80 100 1 2 3 4 5 6 Activity (% theoretical max) Time (h) EOLFG EOLSG EOLGG

Serine protease uptake & release

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
• f 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

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). Bianca Rossi Fiorenza Viani Luca Merlini Yves M. Galante Paola Campia Tiziana Silvetti Milena Brasca Antonella Caterina Boccia Raniero Mendichi

Funding from:

Lucio Melone Nadia Pastori Carlo Punta Erika Ponzini Stefania Brocca Rita Grandori Alessandra Polissi Alessandra Martorana Stefano Farris, Unimi Food Packaging Lab DeFENS, University of Milan