WORK PACKAGE WP1

Microwave assisted extraction (MAE) is, according to Azmir et al (2013), an innovative extraction technology that has significant advantages in extracting sensitive bioactive ingredients from plant tissues. It is an extraction technology that is selective for extracting organic and organometallic substances without destroying them and is also recognized as a “green” technology as it does not use organic solvents. (Alupului, 2012).

According to Cravottoa et al. (2008) this microwave extraction is characterized by smaller extraction times, reduced equipment size, greater efficiency and less exposure of bioactive components to thermal destruction. Aghdase Sadeghi (2017) presents in a review article the advantages of MAE (Microwave Assisted Extraction) stating that Manabe et al. (1998) used MAE to extract pectin from orange peel and found that they could extract 5% more pectin in 15 min than would be extracted in 60 min by conventional methods.

In addition according to Sanchez-Aldana et al, (2013) using MAEs as an alternative technology of conventional extraction technologies we gain in extraction time, we have a higher extraction rate, better product and lower operation cost, reduced up to 85 times energy consumption and much lower amount of waste (Hao, L., Han, W., Huang, S., Xue, B, Deng, X. 2002; Yan, M.M., W. Liu, Y.J. Fu, Y.G. Zu and C.Y. Chen et al. 2010).

This  work package, named WP1, is to be carried out by the research group of the University of Thessaly (sub-contractor) under the supervision of the project beneficiary POLYHEALTH S.A. The research team of the University of Thessaly is to use for a fee a set of industrial extractors operating under the principle of state-of-the-art Vacuum Microwave-Assisted extraction , which are established in factory in Northern Greece owned by a company that cooperates with the UTH group.

Laboratory scale MAE experiments at atmospheric conditions as well as experiments of dynamic industrial vacuum microwave extraction of pomegranate and orange pomace were conducted at the premises of PELLAS NATURE Co. POSITION SEREMS, GR 58200 in the suburb of Edessa (Figure 1) while the corresponding measurements of polyphenols, flavonois and antioxidant capacity for the collected pomegranate and orange pomace extract samples were carried out in the Laboratory of Food and Biosystems Engineering of the University of Thessaly.

As a raw materials solid waste from pomegranate juice production process involving Wonderful variety as well as solid waste from orange juice production process involving NAVAL variety were used. The two solid wastes were obtained during the juice production process of Alberta S.A. in Argos area and they were transported to the Laboratory of Food and Biosystems Engineering of UTH. The solid waste sampels after grinding them to a particle size of 3 mm they were placed in plastic vacuum bags of polypropylene (PP) with 2 Kg capacity each and kept frozen at -25 ^oC until they were used for the production of extracts by MAE.

In a subsequent step the pomegranate and orange pomace samples were extracted by microwave assisted extraction using both a Laboratory scale extractor as well as an industrial-size vacuum microwave assisted extractor and natural extracts of both solid pomegranate and orange residue were obtained which were then analysed to determine their antioxidant properties, and namely (a) Total polyphenols by the Folin Ciocalteau method (b) Total antioxidant strength IC50 by DPPH or ABTS and (c) total flavonoids by the AlCl3 method.  The results of the antioxidant parameters were then mathematicaly analysed using statistical optimization methods using the Response Surface Methodology (RSM) to determine their optimal values both on economic grounds as well as  concerning achievement of maximum  yield of total polyphenols and flavonoids,

Finally, by using the obtained optimal values of the three extraction parameters (a) Microwave power (b) Water-to-solid waste ratio and (c) Extraction time to achieve the maximum extraction rate, bulk extractions were performed and the collected optimized pomegranate and orange pomace were extracts were concentrated by reverse osmosis technology in spiral wounded modules. The obtained concentrate after optimization of the RO process were used by POLYHEALTH S ,A, for the production of liquid mixed extracts to be tested for plant protection applications and also for the production of the lyophilized encapsulated synergistic powders intended for applications in food or cosmetics.

The main research results are summarized below:

• Although, additional measurements were carried out using a small laboratory extractor to assess its use for easier process optimization in the future, it was found that optimization conditions for industrial use cannot be determined in this way and reliable tests are required directly on an industrial scale VMA extractor.
• The optimal extraction conditions of total polyphenols from pomegranate juice industry solid residue by using an industrial dynamic rotating vacuum microwave extractor were found to be:

MICROWAVE POWER: 4961.07W
WATER TO SOLID POMACE/ RATIO: 29.90
EXTRACTION TIME: 119.53 min

The optimal extraction conditions of total flavonoids from pomegranate juice industry solid residue using an industrial dynamic rotating vacuum microwave extractor were found to be:

MICROWAVE POWER: 4147.76  Watt
WATER TO SOLID POMACE/ RATIO: 19.32
EXTRACTION TIME:  63.32  min

The optimal extraction conditions of total polyphenols from orange juice industry solid residue using an industrial dynamic rotating vacuum microwave extractor were found to be:

MICROWAVE POWER:  5999.997  Watt
WATER TO SOLID POMACE/ RATIO: 26,09
EXTRACTION TIME: 120,00 min

The optimal extraction conditions of total flavonoids from orange juice industry solid residue using an industrial dynamic rotating vacuum microwave extractor were found to be:

MICROWAVE POWER:: 2000 Watt
WATER TO SOLID POMACE/ RATIO: 24.12
EXTRACTION TIME: 53.45 min

• The extraction of total polyphenols is favored by high microwave power while the extraction of total flavonoids from low.
• The extraction of total polyphenols is favored by high extraction times while the extraction of total flavonoids by significantly lower extraction times (approximately 30% reduced in comparison with polyphenols).

In addition to the above optimal conditions which ensure high recovery of total polyphenols and total flavonoids and are useful to be applied in case the post extraction solid waste is intended to be further utilized as fermentation substrate to avoid undesirable retard of the starter culture by natural antioxidant residuals, additional optimization was carried out on economic grounds (to maximize the productivity of bioactives i.e. the rate of production of natural antioxidants), This second set of optimal extraction conditions , listed in Table 1., below , represents the most useful goal of the research for POLYHEALTH S.A., as the operation with them provide the desired maximum financial benefit to the company. THE CONDITIONS, LISTED  IN TAMPLE 1., ARE THOSE  USED BY POLYHEALTH S.A. FOR THE PRODUCTION OF THE PRIMARY POMEGRANATE AND ORANGE POMACE EXTRACTS WHICH AFTER RO CONCENTRATION USED IN MIX AS RAW MATERIAL FOR THE PRODUCTION OF EXPERIMENTAL SAMPLES OF NOVEL LIQUID PHYTOPROTECTANTS AS WELL AS FOR THE PRODUCTION OF THE ENCAPSULATED POWDERS INTENDED TO BE TESTED AS NATURAL ANTIMICROBIALS IN FOOD AND COSMETIC APPLICATIONS.

TABLE 1. Extraction conditions for the most economical operation

• Total polyphenols and the total IC50/DPPH antioxidant capacity of pomegranate and orange solid pomace extracts are directly correlated. Therefore, the minimum value IC50/DPPH (associated to maximum of TAC), which was considered by POLYHEALTH S.A., equivalent to high antimicrobial activity on the basis of extensive measurements carried out previously, can be effectively substituted as antimicrobial activity index by the maximum value of total polyphenols which in fact is determined by a much simpler measurement.
• The maximum economically feasible concentration of both orange and pomegranate extracts is 5:1 and the optimal operating RO pressure is 20 bar, This is because at this pressure we have the possibility of operating for 90 min total as the flux does not fall below 15 Kg m^-2 hr^-1 . On the contrary. in the case of pressures 30 and 40 bar we have an approximation of this minimum economically accepted value of 15 Kg m^-2 hr^-1 much earlier concluding that the overall amount of concentrates of each RO concentration cycle at a pressure of 20 bar is greater compared to pressures 30 and 40 bars. This way by operation at 20 bars the the premature destruction of the membranes as well as the excessive consumption of washing detergents are nor compromised. In addition, this pressure ensures lower energy consumption and more safety during operation.