WORK PACKAGE WP2

WP2.1. Production of powders by mixing olive fruit polyphenol extracts with pomegranate and orange pomace extracts using ultrasonic homogenization and encapsulation by cryogenic lyophilization.

The powders are to be prepared in house by POLYHEALTH S.A. by encapsulating liquid olive fruit polyphenol extract in combination with:
(a) pomegranate pomace extract in different ratios (b) orange pomace extract in different ratios (c) mixed pomegranate and orange extracts, mixed in different ratios.

The commercially available preparation of olive fruit polyphenol marketed by the company under the name MEDOLIVA is to be used as the olive fruit polyphenol extract. Encapsulation is to be performed by using industrial-type freeze drying after initial homogenization of the liquid mixture of extracts together with the encapsulating agent. The encapsulation agent will be selected to be edible and clean label (e.g. maltodextrin or other edible and clean label biopolymers).

The optimization parameters  in stage WP2.1. are going to be the following:

• The homogenization time (in min) in the ultrasonic homogenizer
• The weight ratio of active material and encapsulation material (loading of active material)
• Type of the used water-soluble and clean label encapsulating material
• Total solid content in the solution to be lyophilized.
• The initial freezing temperature and the time profile of pressure and temperature dependence during the lyophilization cycle.

The produced powders will be subsequently tested for antimicrobial action against pathogenic and allogeneic microorganisms of food and cosmetics initially with in vitro and eventually with in vivo tests.

WP 2.2. Production of homogenised liquid mixtures of olive polyphenol extract and pomegranate and orange juice solid waste extracts for use in plant protection.

The different combinations of the liquid olive fruit polyphenol extracts, pomegranate  pomace and orange pomace extracts will be tested in the WP3  by in-vitro and in vivo tests as potential phytoprotectants in plant protection applications. Their production, as mentioned above, will be carried out by using the advanced ultrasonic homogenizer available in the company and afterwards will be given to the research group of the subcontractor (University of Thessaly) in order to be used for the in vitro and in vivo tests against commercially important soil and folliar  phyto-pathogens of plants and trees.

This work task will be executed by the research group of POLYHEALTH S.A. To complete this block of research work the optimum conditions of VMAE extraction of pomegranate and orange pomace extracts obtained in WP1link at industrial scale were used in order to be produced the required concentrated extracts of pomegranate and orange pomace.  The state-of-the-art vacuum microwave-assisted extractors which are established in Northern Greece in the factory of a subcontractor that cooperates with the research group of UTH will be used for this purpose. Subsequently, the produced extracts in bulk will be used by POLYHEALTH S.A. as the raw materials for the production of the novel mixed liquid plant protection products.

2. ACTIONS IN THE CONTEXT OF THE WORK PAKET & RESULTSΑ

For the production of homogenized mixtures of liquid extracts for use in plant protection, the ultrasonic homogenizer of POLYHEALTH S.A. was used.  (Picture 1) which is equipped with a cooling system for the homogenised fluid. The settings of the homogenizer during the homogenization of liquid mixtures was at its maximum power and with the cooling facility activated. In addition, the company’s industrial scale lyophilizers were used for the production of the novel antimicrobial powders from the homogenized mixed extracts. ( Picture 2.)

Picture 1. The continuous mode Ultrasonic homogenizer of POLYHEALTH S.A.
Picture 2 The morphology of the industrial lyophilizers of  POLYHEALTH S.A.

The time required for satisfactory homogenization of the liquid mixtures was determined for each of the 71 samples (68 mixtures + 3 controls) using UV-Vis spectroscopy measurements every 1 min for a total time period of 5-10 min for each sample.

The optimum homogenization time was determined as the point of reduction and stabilisation of the absorption of the sample at 200 nm. It should also be stressed that as for all 71 samples the optimized homogenization times of liquid mixtures ranged from 3-4 min, the required time of effective homogenization of the liquid mixtures of the three natural olive-rose and orange extracts is also in the range of 3-4 min, using the maximum power of the ultrasonic homogenizer and with the cooling system of the homogenized liquid activated.

Therefore the basic conclusion is that the use of ultrasonic homogenization time =4 min at the maximum power setting of the continuous ultrasonic homogenizer and with the cooling system activated can effectively homogenize each of the liquid mixed extracts respectively as well as the plain extracts enabling them to be used as efficient natural liquid plant protection products.

As regards the results of optimization of the production of antimicrobial natural powders, the relevant experimentation has revealed the following:

(a) The optimal ultrasonic homogenization time for the mixture of maltodextrin and mixed extracts intended to be used for the production of lyophilized antimicrobial powder is 4 min
(b) The optimal addition of maltodexrin DE18 was found to be 20% w/v in the liquid mixture in order to produce stable free-flowing powders.
(c) The best performing encapsulating agent at a productive and economic level was found to be maltodexrin DE18.
(d) The optimal total solids content of the solution to be lyophilized was found to be 30% w/v and eventually
(e) The optimum initial freezing temperature was found to be -35 ^oC

In addition, the optimal temperature and vacuum time profile for achieving production of free flowimg stable powder in the minimum time is:

STAGE A: from -35oC to -10oC with a vacuum of 0.5 mbar
STAGE B: from -10oC to 5oC with a vacuum of 0.5 mbar
STAGE C: from 5oC to 43oC with a gap of 0.15 mbar

while the optimal total lyophilization time achieved by adopting the above-mentioned optimal lyophilization conditions was found to be 21 hours.

In addition, an important conclusion drawn from the measurement of the total polyphenols of the produced 71 mixed extracts in powder form, is that the relative ratios of solids of the three extracts corresponding to the higher concentrations of total polyphenols is  the following:

In best performance order:

Olive solids: 90% w/w., Pomegranate solids: 7% w/w. Orange solids: 3% w/w.

followed by: Olive solids: 90% w/w., Pomegranate solids: 10% w/w.Orange solids: 0 % w/w.
followed by: Olive solids: 80% w/w., Pomegranate solids: 20% w/w., Orange solids: 0% w/w.
followed by: Olive solids: 80% w/w.. Pomegranate solids: 14% w/w., Orange solids: 6% w/w.

This means that the extracts most likely to act as natural antimicrobial powders are those containing more olive polyphenol (80-90% of solids) and that in addition, the relative composition of the solids of the pomegranate and orange extracts in the powder plays an important role in the final active concentration of total polyphenols determined by the Folin-Ciocalteu method, despite their smaller percentage.

This observation is particularly important for optimization and justifies the selected multiple point optimization in this research project, which, in fact, could be applied more generally by POLYHEALTH S.A. to develop additional applications of this kind in the future.

Finally, in the context of the Loterature search, a major review of the applications of natural pomegranate and orange extracts to food, cosmetics and plant protection as antimicrobials and antioxidants was carried out. Morover, in the context of this research, a significant amount of bibliographical information on the antimicrobial action of various natural substances has been collected for future reference by the Research and Development Department of POLYHEALTH S.A towards developing of synergistic natural antimicrobial and antioxidant formulations.