New type of biofertilizers: microspheres/microcapsules loaded with chemical and biological agents

Transcription

1 New type of biofertilizers: microspheres/microcapsules loaded with chemical and biological agents 9th CASEE Conference Associate Professor Marko Vinceković, PhD University of Zagreb Faculty of Agriculture Department of Chemistry Coordinator of Instalation Scientific Project (Croatian Science Foundation)

2 INTRODUCTION (UNIVERSITY OF ZAGREB FACULTY OF AGRICULTURE) the oldest and leading higher education agricultural institution in the Republic of Croatia founded in 1919 as the Faculty of Agriculture and Forestry in Zagreb Faculty of Agriculture (FAZ) becomes independent in 1959

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4 Organizational Units Faculty of Agriculture owns 6 experimental stations used for scientific, technical activities, research and practical work for teachers and students

5 Department of Chemistry Department of Plant Pathology Department of Vegetable Crops New biopolymer based microcapsules for plant protection and nutrition

6 Introduction Definition of encapsulation Objective of investigation Encapsulation processes Encapsulation relates to technologies which enable loading of one active compound (or more) inside individualized particles (spheres/capsules) with a specific geometry and properties Encapsulation of agrochemicals in biopolymer matrix a tool for ecological and sustainable plant production. Biocontrol agents - biological control of plant pathogens (encapsulation enhancement of shelf-life and application efficiency). Design of biopolymer based delivery systems optimization of many parameters (the most important type and concentrations of both: biopolymer and gelling cation as well bioactive agent compatibility). Main goal Preparation, characterization and application of novel biopolymer microspheres and microcapsules simultaneously loaded with chemical and biological agents.

7 Objective of investigation Preparation and characterization of novel alginate microparticles (microspheres and microcapsules) simultaneously loaded with copper or calcium ions and Trichoderma viride spores The specific goals are: investigation of intermolecular interactions in systems with oppositely charged biopolymers, investigation of interactions between bioactive agents and the delivery system, investigation of conditions for simultaneous encapsulation of biological and chemical agents, in vitro investigations of microcapsule formulations, in vivo testing of optimal microcapsule formulations on conventionally and hydroponically grown lettuce and tomato. Ca 2+ or Cu 2+

8 Strategy of investigation Phytopatology (viability of Trichoderma viride) Physicochemical properties of microparticles Microspheres/microcapsule size and morphology (light microscopy, CLSM, SEM) Interactions between chemical and biological agents in microspheres/microcapules (FTIR) Kinetics and mechanisms of bioactive agents release from microsphere/microcapsules Application of optimal microspheres/microcapules formulation on different plant cultures

9 Microspheres were prepared by the ionic gelation technique at ambient temperature. Microcapsules were prepared in two stages. The first stage comprises the formation of microspheres loaded with copper or calcium cations (ALG/Cu), (ALG/Ca) loaded with Trichoderma viride (ALG/(Cu+Tv)), (ALG/(Ca+Tv)). The second stage includes the coating of microspheres by polyelectrolyte complexation with chitosan: CS/(ALG/(Cu+Tv)) and CS/(ALG/(Ca+Tv)). Microspheres/microcapsules preparation

10 Preparation with BUCHI B-390 encapsulator

11 Phytopathology (viability of Trichoderma viride) Trichoderma viride (Tv) biocontrol agent (an opportunistic avirulent plant symbiont, a mycoparasite of plant pathogenic fungi). Agricultural importance good antagonistic abilities: a) against soilborne plant pathogenic fungi thanks to different mechanisms of antagonism, b) the production of antifungal metabolites (antibiosis), competition for space and nutrients, c) induction of defense responses in plant, and mycoparasitism. Microphotograph of mycelium formed around microcapsules loaded with copper ions and Trichoderma viride after 10 days aging at room temperature CLSM microphotograph of microsphere part with distributed T. viride spores inside alginate matrix and protruded germ tube (marked with black arrows) Microspheres/microcapsules provide an environment supportive to T. viride sporulation

12 d / m Morphology and size of microspheres/microcapsules CLSM microphotographs of wet CS/(ALG/(Cu+Tv)) microcapsules (a) SEM microphotographs of (a) ALG/Ca microsphere surface and (b) ALG/(Ca+Tv) microsphere surface with spores prepared at calcium chloride concentration, c(cacl 2 ) = 1 mol dm -3 (b) CLSM microphotographs of (a) CS/(ALG/Cu+Tv)) and (b) CS/(ALG/Ca+Tv)) microcapsules in fluorescent mode CS/(ALG/Ca+Tv)) wet dry 0,4 0,6 0,8 1,0 1,2 1,4 1,6 1,8 2,0 2,2 c i / mol dm -3 Variation of microcapsule size with calcium chloride concentration The concentration of gelling cation affects the structure of the microcapsule (homogeneity, water content)

13 Transmittance Transmittance Interactions between chemical and biological agents in mixtures 1,1 1,1 1,0 0,9 0,8 0,7 0,6 0,5 0,4 0,3 0,2 Trichoderma viride CuSO 4 x 5H 2 O Trichoderma viride + CuSO 4 x 5H 2 O Wavenumber (cm -1 ) 1,0 0,9 0,8 0,7 0,6 0,5 0,4 0,3 Tv + CaCl 2 Tv CaCl 2 0, FTIR spectra of Trichoderma viride spores (black line), copper sulfate or calcium chloride (cyan line), and their mixture (red line) Wavenumber (cm -1 ) main functional groups detected in the FTIR spectrum relate well with the the chemical structure of T. viride cell wall. disappearance of bands and shifting of peaks towards the lower or towards higher frequencies indicated at least amine, hydroxyl, carbonyl and amide bonds interact with divalent cations electrostatic attractions. broadening and more intense stretching vibrations around 3330 cm -1 (amino (-NH) group superimposed on the side hydroxyl (-OH) groups) wih copper addition enhanced intermolecular hydrogen bonds. Calcium addition narrowing of the peak around 3330 cm -1 and an increase in the peak intensity at 1625 cm -1 (stretching mode of carbonyl group (C=O) conjugated to -NH deformation mode indicative of amide bond formation).

14 Effect of copper or calcium ions concentration on T. viride spores charge 0-2 / mv (a) 8 (b) 4 / mv -4-6 Cu + Tv ζ, Tv + CaCl 2 Variation of the average zeta potential, ζ, with increasing divalent cation concentrations (a) Cu 2+ and (b) Ca ,01 0,1 1 c Cu / mol dm ,0 0,5 1,0 1,5 2,0 c i (CaCl 2 ) / mol dm -3 T. viride spores dispersed in water had negative charge (average zeta potential - 9 mv) prevailing spore surface hydrophilicity. The negative charge on the cell surface arises from various functional groups (carboxyl, hydroxyl, amine, phosphate). The increase of copper or calcium ions concentration less negatively charged spore surface (electrostatic interactions) and consequently aggregation. A reversal of charge around 0.7 mol dm -3 of calcium ions besides electrostatic interactions some other mechanisms are responsible for calcium binding (probably ion exchange and/or some other physicochemical interactions between cations and functional groups on the fungal cell wall).

15 Transmittance Transmitance Interactions between chemical and biological agents in microcapsules 1,00 0,95 0,90 0,85 0,80 0,75 0,70 0,65 0,60 CS/(ALG/Cu) CS/(ALG/(Cu+Tv)) CS Wavenumber (cm -1 ) (a) 1,1 1,0 0,9 0,8 0,7 0,6 0,5 0,4 0,3 CS/(ALG/(Ca+Tv) c i (CaCl 2 ) / mol dm Tv wavenumber (cm -1 ) (b) FTIR spectra of (a) CS/(ALG/Cu) (blue line) and CS/(ALG/Cu+Tv) (red line) microcapsules, and chitosan (black line), and (b) CS/(ALG/Ca+Tv) microcapsules prepared with increasing calcium ions concentration and T. viride (violet line) FTIR spectra of microcapsules loaded with copper or calcium as well as with T. viride, revealed interactions between various functional groups all components interact with each other. The most significant changes were observed in the alginate functional groups region (hydroxyl and carboxylate) prevailing electrostatic interactions and hydrogen bonding.

16 f(cu) In Vitro copper cations release 1,0 0,8 0,6 0,4 0,2 0,0 CS/(ALG/(Cu) (0.45 mm) CS/(ALG/Cu ) (2 mm) CS/(ALG/(Cu + TV)) (0.45 mm) CS/(ALG/(Cu + TV)) (2 mm) The copper cation release an initial burst followed by a slower release The amount of copper cations released depends on microcapsule size and loaded active agents All release profiles can be described by Korsmeyer Peppas equation: t / days Fraction of released copper cations, f(cu), from CS/(ALG/Cu) (open symbols) and CS/((ALG/(Cu+Tv)) (solid symbols) microcapsules at initial copper cation concentration c i = 18 mmol dm 3 with time (t) f Cu = R t = kt n R tot f(cu) represents the fraction of released copper cations, R t is the amount of copper cations released at time t, R total is the total amount of Cu loaded in capsules, k is a constant characteristic of the active agents/polymer system that considers structural and geometrical aspects of the system, n is the release exponent representing the release mechanism

17 Values of the release constant (k) and exponent (n) of copper cations from CS/(ALG/Cu) and CS/(ALG/(Cu+Tv)) microcapsules The values of the release constant (k) and exponent (n) of copper cations encapsulated in CS/(ALG/Cu) and CS/(ALG/(Cu+Tv)) microcapsules microcapsule size (mm) k (day -1 ) n CS/(ALG/Cu) CS/(ALG/Cu) CS/(ALG/(Cu + Tv)) CS/(ALG/(Cu + Tv)) Large microcapsules - n<0.43 the release mechanism of copper cation involved is controlled by a classical Fickian diffusion. Smaller microcapsules n> copper cation release followed non- Fickian kinetics (a combination of the diffusion mechanisms and polymer stress relaxation (transition of glassy structure to a rubbery state), due to rapid swelling and partial dissolution of microcapsules.

18 In Vitro release of Trichoderma viride spores 0,25 f Tv 1,8 1,6 1,4 1,2 1,0 0,8 0,6 0,4 0,2 0,0 ALG/(Cu + Tv) d / mm = 0.45 c i / mmol dm t / h (a) Release profiles for microspheres f Tv (a) f Tv 0,20 0,15 0,10 0,05 0,00 = kt n CS/((ALG/(Cu + Tv)) d / mm = 0.45 c i / mmol dm t / h (a) Fraction of released T. viride spores, f(tv), with copper cation concentration (c i ) from (a) microspheres (ALG/(Cu+Tv)) and (b) chitosan coated microcapsules CS/(ALG/(Cu+Tv)) (b) Release profiles for microcapsules f Tv = k(t l) n, where l denotes the lag time (equivalent to the time required for the microcapsule to hydrate and reach equilibrium before erosion, and the advance of solvent through the microcapsule occur) 1. The higher amount of T. viride spores detected in water than concentration loaded in microspheres germination of T. viride and formation of germ tube biomass in the surrounding medium 2. Chitosan layer significantly reduced the release of both active agents, T. viride spores and copper cations.

19 The values of the release constant (k) and exponent (n) of T. viride spores encapsulated in microspheres (ALG/(Cu + Tv)) and microcapsules (CS/(ALG/(Cu+Tv)) c i /mmol dm -3 k/h -n n R 2 k/h -n n R 2 ALG/(Cu+ Tv) 0.45 mm 2 mm CS/(ALG/(Cu+Tv) The concentration of copper cations and the presence of chitosan layer influenced the kinetics and mechanism of T. viride spores release from microspheres/microcapsules. The increase in the copper cation concentration promoted, but the chitosan layer on microcapsule surface slowed T. viride spores release. Fickian diffusion was found to be the ratecontrolling mechanism for T. viride spores release from microspheres and small microcapsules coated with chitosan layer. Modification of Korsmeyer Peppas empirical model showed that the release from large microcapsules is controlled by a combination of diffusion and the polymer swelling and relaxation.

20 In Vitro calcium cations release from microspheres f Ca 0,0024 c i (CaCl 2 ) / mol dm -3 0,0021 0,0018 0,0015 0,0012 0,0009 0, ALG/Ca (a) f Ca 0,0012 0,0008 0,0004 ALG/(Ca+Tv) c i (CaCl 2 ) / mol dm (b) Fraction of released calcium cations (f Ca ) with time (t) from (a) ALG/Ca and (b) ALG/(Ca+Tv) microspheres. Initial calcium cation concentrations, c i (CaCl 2 ), are denoted. The error bars indicate the standard deviation of the means t / h t / h Fitting to simple Korsmeyer Peppas empirical model the underlying release mechanism is Fickian diffusion. Microspheres without T. viride a decrease in release rate with increasing calcium cation concentration (the effect on the microsphere structure the increase in crosslink density and homogeneity). The presence of T. viride spores affects kinetics and amount of calcium released (change of microspheres structure due to electrostatic repulsions between alginate chains and spores as well as by mechanical interactions).

21 In Vitro release of Trichoderma viride spores from microspheres f Tv 2,5 c i (CaCl 2 ) / mol dm -3 2,0 1,5 1,0 0, ALG/(Ca+Tv) The release profiles for T. viride spores are characterized by rapid initial burst effect showing good fit into power law equation (f Tv = kt n ). After one week of observation, a fraction of released T. viride spores increased above 1 the germination and germ tube formation. 0, log (t / h) The fraction of released T. viride spores, f(tv) with time, t, at various initial calcium chloride concentrations, c i (CaCl 2 ), from ALG/(Ca+Tv) microspheres. The error bars indicate the standard deviation of the means The release constant, k, (incorporated the overall solute diffusion coefficient and geometric characteristics of a microsphere) is almost constant and somewhat increased at the highest calcium concentration. Value n > 0.43 the controlling release mechanisms is a combination of diffusion and polymer swelling and relaxation.

22 Application of on different plant cultures Microspheres/microcapsules were applied on: a) grapevine (Vitis vinifera), b) tomato (Solanum lycopersicum), c) lettuce (Lactuca sativa) - on test filds at Faculty of Agriculture Lactuca sativa Vitis vinifera Solanum lycopersicum

23 Enhancement of Vitis vinifera bioactive potential Calcium, magnesium alginate microspheres loaded with Trichoderma viride were applied on Vitis vinifera plants at two growth stages before flowering and berries pea-size. Physicochemical characteristics of leaves after the two growth stages and grapes bioactive components content and antioxidant activity. After the treatments vine leaves reached a significant increase in almost all measured parameters (total polyphenols, antioxidant capacity via DPPH and ABTS, β- carotene and total chlorophyll) compared to the control. The highest total chlorophyll content after the treatment with microspheres containing Mg 2+ and Ca 2+ cations, and T. viride. No significant influence was found on the grapes but all of the treated samples had somewhat elevated values of total phenols and antioxidant activity compared to the control after applying microcapsules. Vitis vinifera leaves with enhanced bioactive potential a functional food.

24 Preliminary results of the analysis Tomato (Salanum lycopersicum) Calcium or copper alginate microspheres without and with T. viride were applied on tomato at greenhouse Physicochemical characteristics of tomato fruit bioactive components content and antioxidant activity. After the treatments a significant increase in almost all measured parameters (total polyphenols, antioxidant capacity via ABTS, β-carotene, lykopen, amount of calcium or copper) compared to the control.

25 the first harvest, the second harvest, the third harvest o

26 Lettuce (Lactuca sativa) Calcium or copper alginate microspheres without and with T. viride were applied on lettuce at open field Physicochemical characteristics of lettuce bioactive components content and antioxidant activity. After the treatments an increase in almost all measured parameters (antioxidant capacity via DPPH and ABTS, chlorophyll, β-carotene, carotenoids, amount of calcium or copper) compared to the control.

27 Lettuce (Lactuca sativa) copper

28 Conclusions The results revealed alginate microspheres and chitosan/alginate microcapsules can simultaneously incorporate T. viride spores and copper or calcium cations without inhibiting their activities and even promoted T. viride germination. Investigation of intermolecular interactions between oppositely charged biopolymers and bioactive agents using FTIR spectroscopy revealed complex interaction between functional groups of all components in microspheres and microcapsules pointing electrostatic interactions and hydrogen bonding. Physicochemical properties of microspheres/microcapsules could be controlled by adjusting their structure with a concentration of crosslinking cation and microsphere/microcapsule size

29 Kinetics and mechanism (Fickian diffusion or a combination of the diffusion and polymer relaxation) of active agents release depend on gelling cation (copper or calcium) concentration, size of microspheres and microcapsules and the presence of chitosan layer due to changes in their structures. The investigation pointed out that proper selection of formulation variables helps in designing microcapsules with controlled release of bioactive agents. Promising results of microspheres application on the selected plants opened up perspectives for the future use of alginate microspheres/microcapsules simultaneously loaded with biological and chemical agents in the plant nutrition and protection.

30 Some directions of research Encapsulation of propolis

31 Preparation of perfume formulation for plants (roses)

32 Encapsulation of Lactobacilus sakei at different concentration of CaCl 2

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