Method for extracting anthocyanin derivatives from a plant source

Abstract

The present document describes a method for extracting anthocyanin derivatives from a plant source. The method comprises the step of separating a homogenized extract of plant source in a polar acidified solvent for obtaining a residual solid, and a filtrate mixed with the acidified polar solvent. The separation includes a coarse filtration done by centrifugation of the plant source extract, by a filtration with a first filter having a pore size of about 100 m, and a fine filtration is done using a second filter having a pore size of about 10 m. Then, the filtrate mixed with the acidified polar solvent is evaporated for substantially separating the acidified polar solvent from the filtrate to obtain a semi-solid extract of anthocyanin derivatives.

Claims

1. A method for extracting anthocyanins from a plant source, comprising the steps of: i) separating an homogenized extract of said plant source in a polar solvent different than water for obtaining a residual solid and a filtrate, said filtrate containing residual water mixed with said polar solvent different than water; and ii) substantially separating said polar solvent different than water from said filtrate of step i) while substantially retaining said residual water, said separating being performed to obtain a semi-solid extract of anthocyanins.

2. The method as claimed in claim 1, wherein said polar solvent different than water is acidified.

3. The method as claimed in claim 1, wherein step i) comprises a coarse filtration performed using a step selected from: a centrifugation of said homogenized extract of said plant source, a filtration of said homogenized extract of said plant source with a first filter, or a combination thereof.

4. The method as claimed in claim 1, wherein step ii) is performed by evaporating said filtrate mixed with said polar solvent different than water of step i).

5. The method as claimed in claim 3, wherein said step i) further comprises a fine filtration performed by a second filter.

6. The method as claimed in claim 1, further comprising an anthocyanins purification step iii): iii) performing a solid phase extraction of said semi-solid extract of step ii) with an adsorbent material and a first elution solvent to adsorb said anthocyanins present in said semi-solid extract on said adsorbent material.

7. The method as claimed in claim 6, further comprising the step of: eluting said adsorbed anthocyanins of step iii) from said adsorbent material for recovering said purified anthocyanins.

8. The method of claim 1, wherein said plant source is a source of vaccinium fruit.

9. The method of claim 6, wherein said adsorbent material is selected from the group consisting of C-18 silica gel or C-8 silica gel.

10. The method of claim 1, wherein said homogenized extract of said plant source is obtained by mixing and pulverizing said plant source in said polar solvent different than water.

11. The method of claim 6, wherein step iii) includes washing said adsorbent material with said first elution solvent for eluting sugars and inorganic substances from said semi-solid extract.

12. The method of claim 1, wherein said polar solvent different than water is selected from the group consisting of ethanol, methanol, ethyl acetate, acetone, butanol or combinations thereof.

13. The method of claim 1, wherein said polar solvent different than water further comprises an acid selected from the group consisting of phosphoric acid, HCl or combinations thereof.

14. The method of claim 13, wherein said polar solvent different than water is 95% ethanol acidified with phosphoric acid.

15. The method of claim 1, wherein said homogenized extract is obtained at a temperature from about 6 C. to an ebullition temperature of said polar solvent different than water while agitating at a frequency of rotation between about 200 and to about 3000 RPM.

16. The method of claim 1, further comprising a step prior to step i) of cooling said homogenized extract before said separating.

17. The method of claim 4 wherein said evaporating operates at an evaporation temperature inferior to an ebullition temperature of said polar solvent different than water.

18. The method of claim 6, wherein said first elution solvent is water or demineralized water.

19. The method of claim 6, further comprising a step prior to step iii) of dissolving in water said semi-solid extract and filtrating said semi-solid extract dissolved in water.

20. The method of claim 7, wherein said recovering said purified anthocyanins is performed using an elution solution of about 95% ethanol in water or a gradient from 30% ethanol in water to 95% ethanol in water.

21. A method for extracting and purifying anthocyanins from a plant source, comprising the steps of: i) separating an homogenized extract of said plant source in an acidified polar solvent different than water for obtaining a residual solid, and a filtrate containing residual water mixed with said acidified polar solvent different than water; ii) evaporating under reduced pressure said filtrate mixed with said acidified polar solvent different than water of step i) for substantially separating said acidified polar solvent different than water from said filtrate while substantially retaining said residual water to obtain a semi-solid extract of anthocyanins; iii) performing a solid phase extraction of said semi-solid extract of step ii) with an adsorbent material and a first elution solvent to adsorb said anthocyanins present in said semi-solid extract on said adsorbent material; and iv) eluting said adsorbed anthocyanins of step iii) from said adsorbent material with a third elution solvent for recovering said purified anthocyanins.

22. A method for extracting anthocyanins from a filtrate containing residual water mixed with a polar solvent different than water used for separating an homogenized extract of a plant source into said filtrate and a residual solid, said method comprising the step of substantially separating said polar solvent different than water from said filtrate while substantially retaining said residual water to obtain a semi-solid extract of anthocyanins.

23. A method for extracting anthocyanins from a plant source containing at least about 87% w/w water, comprising the steps of: i) separating an homogenized extract of said plant source in a polar solvent different than water for obtaining a residual solid, and a filtrate containing residual water mixed with said polar solvent different than water; and ii) substantially separating said polar solvent different than water from said filtrate of step i) while substantially retaining said residual water, to obtain a semi-solid extract of anthocyanins.

24. The method of claim 4, wherein said evaporating is performed under reduced pressure.

25. The method of claim 6, wherein a ratio of adsorbent material/extract is from about 1:1 to about 10:1.

26. The method of claim 9, wherein said C-18 adsorbent silica gel has a particle distribution of about 40 to about 63 m.

27. The method of claim 9, wherein said C-18 adsorbent silica gel has an organic charge of about 0.38 mmol/g.

28. The method of claim 9, wherein said C-18 adsorbent silica gel has a carbon charge of about 9.16%.

29. The method of claim 9, wherein a ratio C-18 adsorbent silica gel/extract is about 2.5:1.

30. The method of claim 11, wherein step iii) further comprises washing said adsorbent material with a second elution solvent for eluting waxes and fatty substances from said extract.

31. The method of claim 11, wherein step iii) further comprises washing said adsorbent material with a third elution solvent for eluting neutral flavonoids from said extract.

32. The method of claim 11, wherein said elution first solvent is water, and wherein said second elution solvent is hexane, and wherein said third elution solvent is ethyl acetate.

33. The method of claim 12, wherein said polar solvent different than water further comprises water.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) Further features and advantages of the present disclosure will become apparent from the following detailed description, taken in combination with the appended drawings, in which:

(2) FIG. 1 illustrates an exemplary implementation of the proposed method for extracting and purifying anthocyanin derivatives from wild blueberries as a plant source, particularly showing a sequence of extraction steps, including the plant source preparation, the extraction of the plant source, the solid-liquid mix filtration, residual solid drying, solvent evaporation, extract purification, to obtain a final extract; and

(3) FIG. 2 illustrates an exemplary sequence of purification steps that can be performed from the primary extract as obtained by carrying out the sequence of extraction steps of FIG. 1. The steps include dissolution of the primary extract in solvent, column-based purification, partial drying, solvent evaporation, crystallization (to obtain carbohydrates), and desolvation (to obtain an anthocyanin concentrate).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

(4) There is provided a method for extracting anthocyanin derivatives from a plant source comprising the steps of:

(5) i) separating an homogenized extract of a plant source in a polar solvent for obtaining a residual solid, and a filtrate mixed with the polar solvent, and

(6) ii) substantially separating the polar solvent from the filtrate for obtaining a semi-solid extract of anthocyanin derivatives.

(7) According to an embodiment, the polar solvent may be acidified. Preferably, the polar solvent may be acidified with phosphoric acid, HCl or combinations thereof.

(8) According to another embodiment, step i) may include a coarse filtration which may be done by centrifugation of the plant source. In another embodiment, the coarse filtration may be done with a first filter, or a combination of centrifugation and a first filter.

(9) According to another embodiment, step i) may further include a fine filtration which is performed by a second filter. According to another embodiment, the coarse filtration may be done by a two stage filtration with a first filter having a pore size of about 100 m, and a second filter having a pore size of about 10 m. According to another embodiment, the two methods may be combined.

(10) The homogenized extract may be obtained by performing the homogenization at a temperature between about 6 C. to about the ebullition temperature of the polar acidified solvent. The homogenization may be performed by agitating at between about 200 RPM to about 3000 RPM.

(11) According to an embodiment, the method may also comprise step iii), which is an anthocyanin derivatives purification step:

(12) iii) performing a solid phase extraction of the semi-solid extract of step ii) with an adsorbent material and an elution solvent to adsorb the anthocyanin derivatives present in the semi-solid extract on the adsorbent material;

(13) According to an embodiment, the method may also comprise step iv)

(14) iv) eluting the anthocyanin derivatives of step iii) from the adsorbent material for recovering the anthocyanin derivatives.

(15) According to an embodiment, the homogenized extract is obtained by mixing and pulverizing the plant source in an polar acidified solvent. Preferably, the plant source is from a vaccinium fruit, such as lowbush blueberries.

(16) According to an embodiment, the adsorbent material may be chosen from C-18 silica gel, or C-8 silica gel. Preferably, the adsorbent material is C-18 silica gel.

(17) According to an embodiment, the method for extracting and purifying anthocyanin derivatives from a source of blueberries comprises the step of mixing a source of blueberries with an acidified polar solvent. The polar acidified solvent may be, without limitations, ethanol, methanol, ethyl acetate, water, acetone, butanol, or combinations, containing phosphoric acid, HCl, lactic acid, tartaric acid, citric acid and the like. The ratio of the source of blueberries on acidified solvent may be from about 1:4 to about 1:20 and more preferably, the ratio is about 1:10.

(18) The step of mixing may occur, without limitations, in a reactor, a mixer or the like. The mixture, or the extract, is agitated at room temperature (about 21 C.), and/or pulverized, for a certain amount of time, and more specifically, for about 2 hours.

(19) The solid/liquid mixture obtained at the end of the extraction period is filtered. The first filtration may occur on a press-filter, or on any other type of filter having a porosity of about 100 m.

(20) A residual solid and the filtrate containing the active substances dissolved in the solvent used for extraction are collected.

(21) Solid (i.e. insoluble substances in the solvent) is dried for obtaining the residual solvent and water. This solid contains a non-negligible quantity of proteins and complex sugars that are mostly not soluble and poor in fat and simple sugars.

(22) The liquid filtrate obtained is filtered another time on a cartridge filter, or any other type of filter having a porosity of about 10 m for removing the fine insoluble particles.

(23) The filtrate is afterward evaporated under a reduced pressure. The evaporation temperature is fixed to be slightly inferior to the ebullition temperature of the solvent that needs to be evaporated.

(24) Once the solvent is eliminated from the filtrate (and residual water), a semi-solid extract containing the extractable substances and the anthocyanins is recuperated.

(25) The extract obtained is dissolved in water. The obtained solution may be filtered and is placed at the head of a Solid Phase Extraction column, or SPE column, for allowing the separation of the anthocyanins through solid phase extraction. Alternatively, the extract could be processed using a batch process without a column.

(26) The SPE column may be constituted of a cylindrical tube of a glass material and may have an internal volume of about 1.6 L.

(27) Preferably, the adsorbent material is a C-18 type adsorbent which is introduced in the SPE column after dispersion in ethanol. The C-18 type adsorbent may be a modified silica gel, or any other suitable C-18 type adsorbent.

(28) After the sedimentation of the gel at the bottom of the column, the residual solvent is eluted at the bottom of the SPE column by gravity.

(29) The gel is then conditioned by eluting ethanol (95%) and demineralised water at a volumic flow of about 25 ml/minute in the SPE column, to clean and humidify the gel. The solvent elution may be achieved at atmospheric pressure (by gravity) or under an external pressure (between 0.1 and 1 Bar) according to the desired volumic flow.

(30) The plant source extract dissolved in water is then introduced at the head of the SPE column. Water is slowly eluted and the adsorbed extract lies on the gel for a certain amount of time, preferably for one hour.

(31) A first wash with water is accomplished by circulating water in the SPE column at a volumic flow of about 50 mL/min. This first wash allows the elution of sugars and inorganic substances present in the extract with the elution solvent. The other substances, comprising the anthocyanins, remain adsorbed on the gel.

(32) A second wash is optional. This second wash may be accomplished with a second elution solvent such as hexane for removing waxes and fatty substances (triglycerides) from the extract. The other substances, comprising the anthocyanins, remain adsorbed on the silica gel.

(33) A third wash is also optional. This third wash may be accomplished with a third elution solvent such as ethyl acetate for removing the neutral flavonoids from the extract. The other substances, comprising the anthocyanins, remain adsorbed on the silica gel.

(34) Two successive washes of the gel are then accomplished with ethanol 95% in water: a wash with 1.5 L; and a wash with 0.5 L. This step allows desorption of anthocyanins from the silica gel. The elution volumic flow needed is about 25 ml/min.

(35) For recuperating under a solid from the anthocyanin concentrated in solution in ethanol, the two ethanol fractions collected are combined and evaporated under reduced pressure.

(36) Since between 1 and 5% by weight of residual solvent remains after the first evaporation of the solvent, the residual solvent is then separated from the collected solid by evaporation under high vacuum (<1 mbar).

(37) According to another embodiment, to obtain an anthocyanins concentrate containing a higher anthocyanins concentration (e.g.: higher than 40%) the extract/gel ratio may be between 1:5 and 1:10, instead of 1:2.5. According to another embodiment, there is also a possibility to use a gradient of water/ethanol, for example a gradient from 30% ethanol in water to 95% ethanol in water.

(38) The present invention will be more readily understood by referring to the following examples which are given to illustrate the invention rather than to limit its scope.

Example 1

Preparation of an Anthocyanin Concentrate (>38%)

(39) One embodiment of the method for extracting and purifying anthocyanin derivatives from a source of blueberries comprises the step of mixing 8.5 kg of frozen blueberries (87% water) with 85 L of a polar solvent, i.e. ethanol (95%) containing 0.01% of phosphoric acid (ratio solid/liquid 1:10) in a double wall 100 L reactor. The mixture, or the extract, is agitated with an agitator/pulveriser at 800 rpm for 2 hours at room temperature (about 21 C.). The solid/liquid mixture obtained at the end of the extraction period is filtered on a press-filter having a porosity of about 100 m. A residual solid and the filtrate containing the active substances dissolved in the solvent used for extraction are collected.

(40) Solid (i.e. insoluble substances in the solvent) is dried for obtaining the residual solvent and water. This solid contains a non-negligible quantity of proteins and complex sugars that are mostly not soluble and poor in fat and simple sugars. The solid also has a theoretical energy of 5 kcal/g. Thus, the solid is a good source of energy in nutrition. 300 g of the solid are obtained (3.5% of the initial mass of source of blueberries).

(41) The liquid filtrate obtained is filtered another time on a cartridge filter having a porosity of 10 m for removing the fine insoluble particles. The filtrate is afterward evaporated under a reduced pressure. The evaporation temperature is fixed at 65 C., which is slightly inferior to the ebullition temperature of the solvent that needs to be evaporated. Once the solvent is eliminated from the filtrate (and residual water), a semi-solid extract containing the extractable substances and the anthocyanins is recovered. 800 g of dry extract are obtained, which corresponds to 9.4% of the initial mass of the source of blueberries.

(42) A fraction of the extract obtained, 100 g of extract, is dissolved in 1 L of water. The obtained solution is filtrated and is placed at the head of a Solid Phase Extraction column, or SPE column, for allowing the separation of the anthocyanins through solid phase extraction.

(43) The SPE column is constituted of a cylindrical tube of a glass material and has an internal volume of about 1.6 L.

(44) 400 g of a C-18 type adsorbent (i.e.: 0.8 L) obtained from CiliCycle inc. (Quebec, Canada) are introduced in the SPE column after dispersion in 0.8 L of ethanol (95%). The C-18 type adsorbent is a modified silica gel. The distribution of its particles is 40-63 m, the organic charge is 0.38 mmol/g and the carbon charge is 9.16%.

(45) After the sedimentation of the gel at the bottom of the column, the residual solvent is eluted at the bottom of the SPE column by gravity. It is to be noted that a small volume of liquid needs to be conserved on the gel in the SPE column for avoiding the gel to dry and to provide air gaps in the adsorbent material.

(46) The gel is then conditioned by eluting 0.8 L of ethanol (95%) and 0.8 L of demineralised water at a volumic flow of 25 ml/minute in the SPE column. The solvent elution may be achieved at atmospheric pressure (by gravity) or under an external pressure (between 0.1 and 1 Bar) according to the desired volumic flow.

(47) The blueberry extract dissolved in 1 L of water is then introduced at the head of the SPE column. Water is slowly eluted and the adsorbed extract lies on the gel for 1 hour.

(48) A first wash with water is accomplished by letting circulate 2 L of water in the SPE column at a volumic flow of 25 mL/min. This first wash allows the elution of sugars and inorganic substances (90 g) present in the extract. The other substances, comprising the anthocyanins, remain adsorbed on the gel.

(49) A second wash is optional. This second wash is accomplished with 1 L of hexane for pulling out waxes and fatty substances (triglycerides) from the extract (2 g). The other substances, comprising the anthocyanins, remain adsorbed on the silica gel.

(50) A third wash is also optional. This third wash is accomplished with 1 L of ethyl acetate for removing the neutral flavonoids from the extract (1 g). The other substances, comprising the anthocyanins, remain adsorbed on the silica gel.

(51) Two successive washes of the gel are then accomplished with 95% ethanol in water: a wash with 1.5 L; and a wash with 0.5 L. This step allows desorption of anthocyanins from the silica gel. The elution volumic flow needed is about 25 ml/min.

(52) For recuperating under a solid form the anthocyanin concentrated in solution in ethanol, the two ethanol fractions collected are combined and evaporated under reduced pressure at a temperature slightly inferior to the ebullition temperature of the solvent that needs to be evaporated (65 C.).

(53) Since between 1 and 5% by weight of residual solvent is obtained after the first evaporation of the solvent, the residual solvent is then separated from the collected solid by evaporation at vacuum pressure (>1 mbar). 6.5 g of concentrated anthocyanins are obtained at a concentration higher than 40%. The obtained yield is 0.61% compared to the frozen initial mass of blueberries source. The concentrate is a fine powder of a blue color and is mainly soluble in water.

Example 2

Temperature Variation for Homogenization

(54) TABLE-US-00001 Assay 1 Assay 2 Assay 3 Assay 4 Assay 5 Initial grinding No Yes Yes No No Blueberry weight 300 g 300 g 300 g 300 g 300 g Solvent ethanol ethanol ethanol ethanol ethanol 95% 95% 95% 95% 95% Solvent volume 3000 mL 3000 mL 2940 mL 2940 mL 2940 mL Solid/solvent 1:10 1:10 1:10 1:10 1:10 ratio Agitation 3000 rpm 200 rpm 3000 rpm 3000 rpm 3000 rpm Agitation type homogenizer Screw homogenizer homogenizer homogenizer Temperature 6 C. 78 C. 26 C. 22 C. 24 C. Length 2 h 2 h 2 h 2 h 2 h Acid Added None None HCl, Tartaric Acid, None 0.0004% 0.0004% Filter 25 m 25 m 25 m 25 m 25 m Extracted weight 41.29 g 36.1 g 36.91 g 29.89 g 33.7 g Yield 13.8% 12% 12.3% 10% 11.2%

Example 3

Variation of Solid/Solvent Ratio

(55) TABLE-US-00002 Assay 6 Assay 7 Assay 8 Assay 9 Initial Yes Yes Yes Yes grinding Blueberry 750 g 500 g 375 g 300 g weight Solvent ethanol 95% ethanol 95% ethanol 95% ethanol 95% Solvent 3000 mL 3000 mL 3000 mL 3000 mL volume Solid/ 1:4 1:6 1:8 1:10 solvent ratio Agitation 3000 rpm 3000 rpm 3000 rpm 3000 rpm Agitation homogenizer homogenizer homogenizer homogenizer type Temperature 24 C. 23 C. 25 C. 26 C. Length 2 h 2 h 2 h 2 h Acid Added None None None None Filter 25 m 25 m 25 m 25 m Extracted 81.2 g 54.9 g 42.3 g 34.2 g weight Yield 10.8% 11% 11.3% 11.4%

Example 4

Ethanol/Ethyl Acetate Mix

(56) TABLE-US-00003 Assay 10 Initial grinding No Blueberry weight 375 g Solvent Ethanol/ethyl acetate 80:20 Solvent volume 3000 mL Solid/solvent ratio 1:8 Agitation 3000 rpm Agitation type homogenizer Temperature 25 C. Length 2 h Acid Added None Filter 25 m Extracted weight 42.7 g Yield 11.4%

Example 5

Alcool/Water Mix

(57) TABLE-US-00004 Assay 11 Assay 12 Assay 13 Assay 14 Initial Yes Yes Yes Yes grinding Blueberry 375 g 375 g 375 g 375 g weight Solvent ethanol/ ethanol/ methanol/ ethanol/ water water water water 70:30 50:50 70:30 70:30 Solvent 2900 mL 3000 mL 3000 mL 3000 mL volume Solid/ 1:8 1:8 1:8 1:8 solvent ratio Agitation 3000 rpm 3000 rpm 3000 rpm 3000 rpm Agitation homogenizer homogenizer homogenizer homogenizer type Temperature 25 C. 30 C. 22 C. 28 C. Length 2 h 2 h 2 h 2 h Acid Added None None Phosphoric Phosphoric acid, acid, 0.02% 0.02% Filter 25 m 25 m 25 m 25 m Extracted 42 g 42.4 g 42.9 g 41.5 g weight Yield 11.2% 11.3% 11.4% 11.1%

Example 6

Acetone, Ethyl Aceate, Water Solvent Testing

(58) TABLE-US-00005 Assay 15 Assay 16 Assay 17 Assay 18 Initial Yes Yes Yes Yes grinding Blueberry 300 g 300 g 215 g 300 g weight Solvent acetone ethyl acetone/ acetone/ acetate water water 80:20 85:15 Solvent 1500 mL 1500 mL 1500 mL 1500 mL volume Solid/ 1:5 1:5 1:7 1:5 solvent ratio Agitation 200 rpm 200 rpm 200 rpm 200 rpm Agitation screw screw screw screw type Temperature 25 C. 22 C. 22 C. 25 C. Length 2 h 2 h 2 h 2 h Acid Added None None None None Filter 25 m 25 m 25 m 25 m Extracted 30.1 g 0.7 g 22.8 g 32.5 g weight Yield 10% 0.2% 10.6% 10.8%

Example 7

Temperature, Ethanol and Citric Acid

(59) TABLE-US-00006 Assay 19 Assay 20 Assay 21 Initial grinding Yes Yes Yes Blueberry weight 300 g 300 g 215 g Solvent ethanol 95% ethanol 95% ethanol 95% Solvent volume 3000 mL 3000 mL 3000 mL Solid/solvent ratio 1:10 1:10 1:10 Agitation 3000 rpm 3000 rpm 3000 rpm Agitation type homogenizer homogenizer homogenizer Temperature 22 C. 78 C. 10 C. Length 2 h 2 h 2 h Acid Added citric acid citric acid citric acid 0.0004% 0.0004% 0.0004% Filter 25 m 25 m 25 m Extracted weight 29.1 g 29.6 g 26.27 g Yield 9.9% 9.9% 8.8%

Example 8

Ethanol/Methanol and Phosphoric Acid

(60) TABLE-US-00007 Assay 22 Assay 23 Assay 24 Assay 25 Initial Yes Yes Yes Yes grinding Blueberry 200 g 200 g 200 g 200 g weight Solvent ethanol 95% ethanol 95% ethanol 95% Methanol Solvent 2000 mL 2000 mL 2000 mL 3000 mL volume Solid/ 1:10 1:10 1:10 1:8 solvent ratio Agitation 200 rpm 200 rpm 200 rpm 3000 rpm Agitation screw screw screw homogenizer type Temperature 80 C. 79 C. 79 C. 22 C. Length 2 h 2 h 2 h 2 h Acid Added phosphoric phosphoric phosphoric phosphoric acid acid acid acid 0.01% 0.01% 0.01% 0.02% Filter 25 m 25 m 25 m 25 m Extracted 22.3 g 21.3 g 24.8 g 44.7 weight Yield 11.2% 10.6% 12.4% 11.9%

Example 9

Ethanol 95% and Room Temperature

(61) TABLE-US-00008 Assay 26 Initial grinding No Blueberry weight 8500 g Solvent ethanol 95% Solvent volume 85000 mL Solid/solvent ratio 1:10 Agitation 1200 rpm Agitation type homogenizer Temperature 18 C. Length 2 h Acid Added Phosphoric acid 0.01% Filter Press filter 100 m and 20 m cartridge Extracted weight 454800 g Yield 9.4%

(62) While preferred embodiments have been described above and illustrated in the accompanying drawings, it will be evident to those skilled in the art that modifications may be made without departing from this disclosure. Such modifications are considered as possible variants comprised in the scope of the disclosure.