Method for the preparation of a water-soluble extract of a vegetable biomass

Abstract

The present invention relates to a method for the preparation of a water-soluble extract based on a vegetable biomass of the Melissa officinalis family, which extract is used as a sulphite substitute in the aforesaid winemaking process, which method comprises one or more of the following steps: a) harvesting the herb, b) drying the herb obtained in step a), c) extracting the dried herb obtained in step b), wherein the aforesaid extraction step c) comprises at least two sub steps, a first sub step comprising a first extraction step with water, wherein the aqueous phase thus obtained functions as the starting material for the second sub step comprising an extraction step with an organic solvent, wherein the aqueous phase is acidified before the second extraction step.

Claims

1. A method for the preparation of a water-soluble extract based on a vegetable biomass of the Melissa officinalis family, which extract is used as a sulphite substitute in a winemaking process, which preparation method comprises the following steps: a) harvesting the herb, b) drying the herb obtained in step a), c) extracting the dried herb obtained in step b), wherein the aforesaid extraction step c) comprises at least two sub: steps, a first sub: step comprising a first extraction step with water, wherein the aqueous phase thus obtained functions as the starting material for the second sub-step comprising an extraction step with an organic solvent, wherein the aqueous phase is acidified before the second extraction step.

2. The method according to claim 1, characterised in that the organic phase obtained in the second extraction step is subjected to an evaporation step subsequent to the aforesaid extraction step with an organic solvent.

3. The method according to claim 2, characterised in that the traction obtained after the aforesaid evaporation step is mixed with water to obtain an herbal extract.

4. The method according to claim 3, characterised in that the herbal extract obtained after mixing with water is stored at a temperature in a range of 0-10 C., during a period of at least 5 hours, wherein the cold aqueous mixture of the herbal extract is filtered to obtain an herbal filtrate, wherein a solid product obtained during the filtration steps is removed.

5. The method according to claim 4, characterised in that the herbal filtrate obtained after the filtration step is subjected to an additional evaporation step, wherein the dried herbal filtrate is mixed with water for a second time and is stored at a temperature in a range of 0-10 C., during a period of at least 5 hours, wherein the cold aqueous mixture of the herbal filtrate is filtered for a second time to obtain a purified herbal filtrate.

6. The method according to claim 5, characterised in that a diluted herbal extract is obtained by diluting the purified herbal filtrate with water for a third time until a FRAP value in a range of 100,000-300,000 micromol Fe.sup.2+/L, calculated on the total volume of the diluted herbal extract is measured.

7. The method according to claim 6, further comprising the addition of one or more additional components to the purified herbal filtrate obtained in claim 6, which components are selected from the group of quinone inhibitors and oxidase inhibitors.

8. The method according to claim 1, characterised in that butanol is used as the organic solvent in the aforesaid extraction step with an organic solvent.

9. The method according to claim 1, characterised in that in addition to Melissa officinalis, the herb in step (a) comprises one or more pants selected from the group consisting of Mentha piperita, Thymus pulegioides, Thymus vulgare and Echinaceae root.

10. The method of claim 8, wherein the butanol is n-butyl alcohol or iso-butyl alcohol or the combination thereof.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a process flow diagram according to an embodiment of the present invention.

(2) FIG. 2 is a chromatogram of the aqueous fraction obtained after extraction with an organic solvent.

(3) FIG. 3 is a chromatogram of the organic fraction obtained after extraction with an organic solvent.

(4) The present invention will be discussed with reference to the enclosed process flow diagram. This process flow diagram covers an embodiment of the present invention.

(5) According to FIG. 1, the herb, especially Melissa officinalis, is harvested as indicated by reference number 1. According to a preferred embodiment the herb is sieved and unwanted parts are removed, such as sand, stones etc. The herb thus harvested 50 is dried in unit 2, for example a band dryer. In unit 3 the dried herb 51 is then extracted with water, at room temperature, i.e. a first extraction step with water. The aqueous extract 52 is acidified in unit 4, for example by the addition of phosphoric acid until a pH of about 1.7 and 3.5 to form an acidified plant extract 53. In unit 5 this acidified plant extract 53 is filtrated and the filtrate 54 thus obtained is sent to an extraction unit 6 for carrying out an extraction step with an organic solvent. The solvent used in unit 6 is for example n-butanol. The organic phase 55 originating from this extraction step with an organic solvent is evaporated in unit 7 under vacuum conditions of about 5 mbar and at a temperature of at most 40 C. This evaporation step aims to remove a substantial part of the organic solvent used in unit 6. The concentrate or concentrated fraction 56 obtained after the evaporation step in unit 7 is brought into contact with a liquid phase, i.e. water, in unit 8. In unit 9 there is a cold storage of the aqueous mixture of herbal extract 57 at a temperature of about 4-5 C., during a period of at least 5 hours. The cold aqueous mixture 58 thus stored is filtered in unit 10. The herbal filtrate 59 is sent to a second evaporation step in unit 11. In unit 11 de remainder part of the organic solvent is removed from filtrate 59. The concentrated fraction 60 obtained after the evaporation step in unit 11 is brought into contact with a liquid phase, i.e. water in unit 12. In unit 13 there is a cold storage of the aqueous mixture 61 at a temperature of about 4-5 C., during a period of at least 5 hours and the cold aqueous mixture is filtered in unit 13 as well. The filtrate 62 thus obtained, i.e. the purified herbal filtrate, is sent to unit 14 for standardisation. Unit 14 may receive several filtrates 62 from different batches. The standardisation in unit 14 includes the dilution with water until a specific FRAP value is reached. The thus diluted and standardised stream 63, i.e. the diluted herbal filtrate, is combined with additives in unit 15. Examples of additives comprise one or more additional components, which components are selected from the group of quinone inhibitor and oxidase inhibitor, especially vitamin C is used as the quinone inhibitor and/or kojic acid is used as the oxidase inhibitor. The thus prepared extract 64 is stored in unit 16. From unit 16 several grades of extracts 65 will be delivered to the end suppliers 17.

(6) Although the process flow diagram has been discussed for only one specific herb Melissa officinalis, the process flow diagram may also include one or more other natural components selected from the group of Mentha piperita, Thymus pulegioides, Thymus vulgare and Echinaceae root. These natural components may be processed simultaneously with Melissa officinalis, but may also be blended with other processed herbs in unit 14.

(7) In addition, in some embodiments the addition of additives, such as one or more additional components, in unit 15 may also take place before the standardisation in unit 14. This means that the dilution with water in unit 14 may take place after the addition of additives. In another embodiment of the present invention the concentrate 56 may undergo only one time the steps of mixing with water, cold storage and filtration. In such an embodiment the herbal filtrate 59 is sent directly to unit 14 for standardisation. As mentioned above, unit 14 may receive several filtrates, for example filtrates that have undergone both the first evaporation in unit 7 and the second evaporation in unit 11 and filtrates that have only undergone a single evaporation step in unit 7.

(8) As mentioned in this application, the extraction step with an organic solvent is preferably carried out with butanol, n-butyl alcohol and/or iso-butyl alcohol. The present inventors found that by using these preferred organic solvents a broad spectrum of anti-oxidant components is extracted. This can be seen with the chromatographic fingerprints of the discarded water fraction after the organic extraction, as shown in FIG. 2. The amount of rosmarinic acid in this water fraction is high. Application tests with white wine showed that the extract with high rosmarinic acid purity had a detrimental effect on the taste of the wine and did not stop brown discoloration during storage of the wine.

(9) The chromatogram of the organic phase obtained after the organic extraction step as shown in FIG. 3 shows much more peaks besides the rosmarinic acid peak. The present inventors assume that the presence of these other components provides the unexpected anti-oxidative properties of the herbal extract obtained according to the present invention.

(10) An example of the method for measurement Ferric reducing abilityantioxidant power (FRAP) will now be discussed.

(11) Preparing Reagents:

(12) 1. Stock solution of 300 mM acetate buffer, pH 3.6 (store at 4.c for maximum 1 month) For 100 ml:

(13) weigh 0.31 g sodium acetate (C.sub.2H.sub.3NaO.sub.2.3H.sub.2O), dissolve in 50 ml distilled water

(14) add 1.6 ml acetic acid (C.sub.2H.sub.4O.sub.2)

(15) add distilled water up to 75 ml

(16) measure the pH, if necessary adjust to 3.6

(17) add distilled water up to 100 ml.

(18) 2. Stock solution of 10 mM 2,4,6-tripyridyl-s-triazine (TPTZ) (store at 4 C. for maximum 1 month) For 10 ml:

(19) weigh 31 mg TPTZ, dissolve in 5 ml of distilled water

(20) add 34 l HCl (36%, d=1.18 kg/l)

(21) add distilled water up to 10 ml.

(22) 3. Stock solution of 20 mM FeC.sub.3.6H.sub.2O (store at 4 C. for maximum 1 month)

(23) For 10 ml:

(24) weigh 54.1 mg FeCl.sub.3.6H.sub.2O, dissolve in 5 ml of distilled water

(25) add distilled water up to 10 ml.

(26) 4. Work solution: FRAP reagent

(27) Mix 25 ml acetate buffer (solution 1), 2.5 ml TPTZ-reagent (solution 2) and 2.5 ml FeCl.sub.3.6H.sub.2O (solution 3).

(28) Place the solution at 37 C. 30 minutes before starting the analysis.

(29) Calibration:

(30) Weigh on the analytical balance 27.8 mg FeSO.sub.4.7H.sub.2O in a measuring flask of 100 ml, note the weight, dissolve in a little distilled water, and add distilled water up to 100 ml

(31) Make dilutions 1/10, 2/10, 4/10, 6/10, 8/10 and 10/10

(32) Add 225 l of FRAP reagent in all needed wells of the 96 well plates

(33) Add to the appropriate well 25 l of the standard dilutions, 25 l of distilled water for the blank, mix by carefully pipetting up and down

(34) Measure the optical density at 593 nm at 0, 5, 10, 15 and 20 minutes, incubate the plate in the spectrophotometer, place the incubator of the spectrophotometer at 37 C.

(35) Samples:

(36) If possible dilute the samples within the range of the calibration curve (100-1000 mol/l Fe.sup.2+)

(37) Add for each sample 225 l of FRAP reagent (37 C.) in a well of the 96 well plates

(38) Add 25 l of the sample to the appropriate well, 25 l of distilled water for the blank, mix by carefully pipetting up and down

(39) Measure the optical density at 593 nm at 0, 5, 10, 15 and 20 minutes, incubate the plate in the spectrophotometer, place the incubator of the spectrophotometer at 37 C.

(40) Determine the FRAP value by recalculation with the use of the calibration curve/